The 3rd International Conference
on
Nucleic Acid-Protein Chemistry and
Structural Biology for Drug Discovery
Georgia State University
Place: Petit Science Center
(Room 101 PSC, 100 Piedmont Ave. SE,
Atlanta, GA 30303)
Time: September 14-15, 2013
Andrzej Joachimiak, Bi-Cheng Wang,
Binghe Wang, David Wilson,
Sibo Jiang, Wen Zhang
Sponsors: GSU, NSF, CAPA, SeNA
L
S
G
A
Keynote Speech:
Speakers:
Committee Members:
P
(President of Georgia State University)
(Nobel Laureate in Chemistry, 2009)
Organizer & Chair: Zhen Huang
A
Dr. Mark Becker
Dr. Thomas Steitz
(Starting at 7:30 am)
C
Opening Speech:
Margo A. Brinton
Robert T. Batey
Martin Egli
Eric Ennifar
Markus Germann
Ichiro Hirao
Zhen Huang
Li-Wei Hung
Andrzej Joachimiak
Jeffrey Kieft
Paul Langan
Gaohua Liu
David Lynn
Suresh Srivastava
Bi-Cheng Wang
Irene Weber
Loren Williams
David Wilson
Bo Xiao
Wen Zhang
The 3rd International Conference on Nucleic Acid-Protein
Chemistry and Structural Biology for Drug Discovery
Georgia State University
Place: Petit Science Center
(Room 101 PSC, 100 Piedmont Ave. SE, Atlanta, GA 30303)
Time: September 14-15, 2013
(starting at 7:30 am with registration and continental breakfast)
Organizer & Chair: Zhen Huang
Committee Members:
Andrzej Joachimiak, Bi-Cheng Wang, Binghe Wang, David Wilson, Sibo Jiang, Wen Zhang
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THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Science is Art.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
3
The 3rd International Conference on Nucleic Acid-Protein
Chemistry and Structural Biology for Drug Discovery
Georgia State University
Place: Petit Science Center
(Room 101 PSC, 100 Piedmont Ave. SE, Atlanta, GA 30303)
Time: September 13-15, 2013
Organizer & Chair: Zhen Huang
Committee Members:
Andrzej Joachimiak, Bi-Cheng Wang, Binghe Wang,
David Wilson, Sibo Jiang, Wen Zhang
Contents
Program ……………………………………………………………………………… 4
Abstracts ……………………………………………………………………….……. 6
Sponsors …………………………………………………………………………… 48
Georgia State University
4
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Curiosity leads to Discovery.
Georgia State University
4
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Conference Agenda
Friday, Sep. 13, 2013
17:00 – 21:00
Registration, Check-in, Reception/Pizza Dinner, Poster Session, and Social Hours
Saturday, Sep. 14, 2013
Chair: Margo Brinton
7:30 – 8:00 am
Registration & Continental Breakfast
8:00 – 8:15
Opening Speech: Mark Becker (President of Georgia State University)
8:15 – 9:00
Keynote Speech: Thomas Steitz (Nobel Laureate in Chemistry, 2009)
9:00 – 9:30
David Wilson
“One DNA Minor Groove – Many Possibilities: From the Basics of Recognition to
Inhibition of Transcription Factor-DNA Complexes”
9:30 – 10:00
Paul Langan
“Neutron technologies for nucleic acid research and drug design and delivery”
10:00 – 10:30
Speaker Group Photo Time and Coffee Break
10:30 – 11:00
Andrzej Joachimiak
“Structure Determination of transcriptional factors and their complexes with DNA”
11:00 – 11:30
Ichiro Hirao
“Expansion of the genetic alphabet of DNA and its application to aptamer
generation”
11:30 – 12:00
Irene Weber
“HIV Protease: the Challenge of Drug Resistance”
12:00 – 13:00
Sandwich Lunch and Poster Session
13:00 – 13:30
Bi-Cheng Wang
“Exploring the Biophysical/Biochemical Information of Metals in Macromolecules
Using Wavelength-Dependent Data”
13:30 – 14:00
Eric Ennifar
“Thermodynamics of HIV-1 Reverse Transcriptase in action reveals the
mechanism of action of non-nucleoside inhibitors”
14:00 – 14:30
Margo A. Brinton
“Viral 3′ RNA structures interacting with cell proteins regulate the initiation of
flavivirus RNA synthesis”
14:30 – 15:00
Robert T. Batey
“Recognition of cobalamins by riboswitches”
Chair: David Lynn
Chair: Martin Egli
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
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15:00 – 15:30
Coffee Break
15:30 – 16:00
Jeffrey Kieft
“Molecular self-defense: viral RNAs that use structure to inhibit host cell
nucleases”
16:00 – 16:30
Martin Egli
“Structure, Kinetics and Mechanism of 8-oxoG Bypass by Y-Class DNA
Polymerases”
16:30 – 17:00
Zhen Huang
“Chemistry and Structural Biology of Nucleic Acids Functionalized with Selenium”
17:00 – 21:00
Speakers Dinner Banquet and Social Hour
Sunday, Sep. 15, 2013
Chair: Markus Germann
Chair: Loren Williams
7:30 – 8:00 am
Registration & Continental Breakfast
8:00 – 8:30
David Lynn
“Designing Chimeric Biomolecule Self-Assemblies”
8:30 – 9:00
Gaohua Liu
“Applications of Protein NMR in Protein Engineering and Design”
9:00 – 9:30
Suresh Srivastava
“RNA Synthesis in Reverse Direction and Application in Convenient Introduction of
Ligands, Chromophores and Modifications of Synthetic RNA at the 3’- End and
Highly Efficient Synthesis of Long RNA”
9:30 – 10:00
Li-Wei Hung
“Automated Crystallographic Structure Determination in PHENIX”
10:00 – 10:20
Coffee Break
10:20 – 10:50
Loren Williams
“RNA and Protein - a match made in the Hadean”
10:50 – 11:10
Wen Zhang
“Facilitation of DNA Crystallization by Selenium Functionalization”
11:10 – 11:40
Markus W. Germann
“Structural and Dynamic Aspects of DNA Recognition”
11:40 – 12:00
Bo Xiao
“Mannosylated bioreducible nanoparticle-mediated macrophage-specific TNF-α
RNA interference for IBD therapy”
12:00 – 13:30
Box Lunch, Poster Session, Poster Award Announcement, and Closing
Georgia State University
Chair: Eric Ennifar
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THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Understanding the Structural Basis of the Function of Various Factors
in the Steps of Protein Synthesis
Thomas A. Steitz, Yury Polikanov, Matthieu Gagnon, Sai Seetharaman,
Jinzhong Lin, Ivan Lomakin
Department of Molecular Biophysics & Biochemistry and Department of Chemistry,
Yale University, and Howard Hughes Medical Institute, New Haven, Connecticut USA; email:
[email protected]
We have obtained many insights into the structural basis of ribosome function in
protein synthesis from our structural studies of the large ribosomal subunit as well
as the 70S bacterial ribosome, and their complexes with substrates, protein factors
or antibiotics. These have elucidated the mechanism by which this ribozyme
catalyzes peptide bond formation and the specificity and mode of its inhibition by
antibiotics.
During the process of protein synthesis elongation, the 70S ribosome is in various
conformational states bound to various different ligands, and the structures of these
functional states are beginning to emerge. Our structure of the 70S ribosome
complexed with an mRNA, tRNAfmet in the P site and elongation factor P (EF-P),
shows EF-P bound between the P site and the E site and interacting extensively with
the P-site tRNA along its entire length. However, how EF-P facilitates the translation
through short runs of proline is as yet unknown. Our most recent structures of the
70S ribosome bound to either hibernation promoting factor or ribosome
modulation factor show how these factors prevent the initiation of protein synthesis
by blocking tRNA binding or interaction with the Shine-Dalgarno mRNA sequences.
We have also obtained the structure of a complex with a ribosome rescue protein
(yaeJ), which rescues stalled ribosomes by hydrolyzing the peptidyl-tRNA. It binds
to the site used by the release factors, but is positioned by a peptide tail that lies in
the mRNA binding cleft. Protein synthesis by the ribosome can be regulated by
numerous different nascent chain sequences and the binding of a small molecule
ligand, resulting in polypeptide chain arrest. Progress has been made in obtaining a
crystal structure of the 70S ribosome containing an ermC arrested peptidyl-tRNA in
the tunnel along with erythromycin.
Recently, interesting progress has been made on understanding the structural basis
of the function of initiation factors eIF1 and eIF1a from low resolution structures of
eukaryotic 40S subunit complexes with these factors as well as tRNA and mRNA.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Thomas A. Steitz
Department of Molecular Biophysics and Biochemistry, Yale
University, and Howard Hughes Medical Institute, New Haven, CT
Thomas A. Steitz is Sterling Professor of Molecular Biophysics
and Biochemistry and Professor of Chemistry at Yale
University as well as an Investigator of the Howard Hughes
Medical Institute. He received a B.A. degree in chemistry from
Lawrence University and a Ph.D. degree in molecular biology
and biochemistry from Harvard. After postdoctoral research at
the Medical Research Council Laboratory of Molecular Biology
in Cambridge, England he joined the Yale faculty. He is a member of the U.S.
National Academy of Sciences, the American Academy of Arts and Sciences and a
Foreign Member of the Royal Society. He has received a number of awards,
including the Rosenstiel Award for distinguished work in basic biomedical sciences,
the Keio Medical Science Prize, the Gairdner International Award, the Connecticut
Medal of Science and the 2009 Nobel Prize in Chemistry.
For the last three decades, research in the laboratory of Dr. Steitz has focused on
obtaining insights into the molecular mechanisms by which the proteins and nucleic
acids involved in the central dogma of molecular biology carry out gene expression
from replication and recombination of the DNA genome to its transcription into
mRNA followed by the various components associated with the translation of mRNA
into protein. Not only are these processes fundamental to all life forms, but many of
the macromolecules involved in these processes are known, or potential, targets for
therapeutic drugs. In the 1980s, his lab established the structure of the catabolite
gene activator protein and later its DNA complex, the structure of the first DNA
polymerase and the first structure of an aminoacyl tRNA synthetase bound to tRNA.
His lab is now continuing structural studies of all the components of the replisome.
His studies of T7 RNA polymerase captured in many of its functionally important
states - initiation, intermediate, elongation - as well as stages of nucleotide
incorporation and provide the most complete picture of RNA transcription by an
RNA polymerase. Perhaps the most significant insights have been derived from the
atomic structure of the large ribosomal subunit. This structure proved that the
ribosomal RNA is entirely responsible for catalyzing peptide bond formation and
provided insights into how this mammoth RNA assembly is folded and functions as
an enzyme. Most recently, research has focused on the structures of the 70S
ribosome in complex with factors involved in various steps of the protein synthesis
process. The ribosome is probably the major target of antibiotics. The many
structures of the large subunit complexed with various different antibiotics
determined at Yale have identified numerous different antibiotic binding sites near
the site of protein synthesis. This information has been enormously facilitating to
Rib-X Pharmaceuticals, Inc. in the development of new antibiotics effective against
the antibiotic resistant bacteria.
Georgia State University
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THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
One DNA Minor Groove – Many Possibilities: From the Basics of
Recognition to Inhibition of Transcription Factor-DNA Complexes
Ananya Paul, Shuo Wang, Rupesh Nanjunda W. David Wilson
Arvind Kumar, Yun Chai, Chad E. Stephens, Abdelbasset A. Farahat, David W. Boykin
Gregory M. K. Poon†
Department of Chemistry and Center for Diagnostics and Therapeutics,
Georgia State University, Atlanta, Georgia, USA; email: [email protected]
†
Department of Pharmaceutical Sciences,
Washington State University, Pullman, WA 99164-6534, USA
The recent explosion of information about the gene control functions of DNA sequences, local
duplex microstructures, as well as more complex folding patterns of DNA provide us with
exciting new information for use in DNA biotechnology and design of small molecules for
control of DNA function. Compounds that can regulate cell function in a desired fashion, for
example, by inhibition of transcription factor-DNA complexes, are a central goal in chemical
biology and offer advantages in development of new drugs. While there is generally only one
protein per gene and even fewer proteins than can be selectively targeted with small molecules, a
huge number of DNA control sequences and structures, which should be possible small molecule
receptors, have recently been discovered. Our collaborative groups are focused on the design,
preparation and study of relatively simple, cell-permeable compounds to selectively target at least
6-10 base pair DNA sequences or structures. Novel sets compounds have structures and
substituents that can perturb their interactions with DNA and/or combined units that can
recognize relatively long sequences of DNA that contained mixed base pairs. This is in contrast to
classical minor groove binders which are generally relatively small, simple AT specific
compounds. The compounds and methods offer attractive advantages in developing new types of
agents that can enter cells and selectively the control functions of specific genes. Our initial
results show that relatively small sets of related compounds can target DNA through multiple
different binding modes that allow them to inhibit or perhaps enhance a variety of protein-DNA
complexes at low concentrations. The local sequences/structures of DNA and subtle compound
variations provide differences in DNA complexes and allosteric changes in DNA structure that
provide enhanced selectivity in the biological action of the compounds.
Supported by NIH and NSF
Selected Publications:
1.
2.
3.
4.
5.
6.
7.
Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David (2013) "Probing the electrostatics and pharmacological
modulation of sequence-specific binding by the DNA-binding domain of the ETS family transcription factor PU.1:
A binding affinity and kinetics investigation." J. Mol. Biol. 425, 1655-1669.
Nanjunda, R.; Munde, M.; Liu, Y.; Wilson, W. D (2011) “Real-time monitoring of nucleic acid interactions with
biosensor plasmon resonance," in Methods for Studying DNA/Drug Interactions. Chapter 4 (Eds. Y. Tor and M.
Wanunu), CRC Press, Taylor and Francis.
Bashkin, J. K.; Aston, K.; Ramos, J. P.; Koeller, K. J.; Nanjunda, R.; He, G.; Dupureur, C M.; Wilson, W. D.
(2013) "Promoter scanning of the human COX-2 gene with 8-ring polyamides: Unexpected weakening of
polyamide-DNA binding and selectivity by replacing an internal N-Me-pyrrole with β-alanine.” Biochimie 95,
271
Wang, Shuo; Nanjunda, Rupesh; Bashkin, James; Aston, Karl; Wilson, W. David (2012) "Correlation of local
effects of DNA sequence and position of beta-alanine inserts with polyamide-DNA complex binding affinities and
kinetics." Biochemistry 50, 7674-7683.
Wei, D.; Wilson, W. D.; Neidle, S. (2013) "Small-molecule binding to the DNA minor groove is mediated by a
conserved water cluster." J. Am. Chem. Soc. 135, 1369-1377.
Rettig, M.; Germann, M. W.; Ismail, M. A.; Batista-Parra, A.; Munde, M.; Boykin, D. W.; Wilson, W. D. (2012).
“Microscopic rearrangement of bound minor groove binders detected by NMR.” J. Phys. Chem. B. 116, 5620
Liu, Y.; Kumar, A.; DePauw, S.; Nhili, R.; David-Cordonnier, M. H.; Lee, M. P.; Ismail, M. A.; Farahat, A. A.;
Say, M.; Chackal-Catoen, S.; Batista-Parra, A.; Neidle, S.; Boykin, D. W.; Wilson, W. D. (2011) “Water-mediated
binding of agents that target the DNA minor groove.” J. Am. Chem. Soc., 133, 10171-10183.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
W. David Wilson
Prof. W. David Wilson (Ph.D.) is Regents Professor of Chemistry at
Georgia State University. He obtained his Ph.D. and did postdoctoral
research at Purdue University in protein biophysical chemistry. He
established an independent career at GSU in the structure and chemical
biology of nucleic acid interactions with small molecules and proteins,
and methods to control nucleic acid functions, including gene expression.
He has spent sabbatical leaves at the University of Florida (NSF
supported), the University of London Institute of Cancer Research
(NATO Fellowship), the Institut de Recherches sur le Cancer de Lille,
France (INSERM International Senior Scientist Award) and the Indian
Institute of Technology-Bombay (Visiting Faculty Support Award, IITBombay). This research has led to new ideas for understanding the molecular basis of protein
and designed molecule targeting of unique nucleic acid structures; such as the mitochondrial
kinetoplast DNA of parasitic microorganisms, G-quadruplexes in cancer cell telomeres and
genomic promoter sequences, A-tracts and other bent DNAs, and folded RNAs of disease causing
viruses. He has collaborated with the several investigators to study the binding of a variety of
minor groove binders to different DNAs, transcription factor-DNA complexes and how these
complexes can be inhibited by designed compounds. To carry out the various studies on nucleic
acid interactions, protein-DNA complexes and their inhibition, Dr. Wilson has used a wide array
of biophysical methods. His group is particularly well-known for work in microcalorimetry
(DSC and ITC) and biosensor-surface plasmon resonance (SPR). He is frequently invited to
speak at international meetings in these areas, for example, he was the keynote speaker at the
MicroCal and Biacore International Meeting (DIPIA) in Boston (2011) and the TA Instruments
Microcalorimetry conference in New Orleans (2012) and he gave presentations at numerous other
meetings. Dr. Wilson has also recently written several review chapters in these areas and some
are listed above.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Liu, Y.; Kumar, A.; DePauw, S.; Nhili, R.; David-Cordonnier, M. H.; Lee, M. P.; Ismail, M. A.; Farahat, A. A.; Say, M.;
Chackal-Catoen, S.; Batista-Parra, A.; Neidle, S.; Boykin, D. W.; Wilson, W. D. (2011) “Water-mediated binding of
agents that target the DNA minor groove.” J. Am. Chem. Soc., 133, 10171-10183.
Nhili, Raja; Peixoto, Paul; Depauw, Sabine; Flajollet, Sébastien; Dezitter, Xavier; Munde, Manoj; Ismail, Mohamed;
Kumar, Arvind; Farahat, Abdelbasset; Stephens, Chad; Duterque-Coquillaud, Martine; Wilson, W. David; Boykin,
David; David-Cordonnier, Marie-Hélène (2013). "Targeting the DNA binding activity of the human ERG transcription
factor using new heterocyclic dithiophene diamidines." Nucleic Acids Research 4, 125
Nanjunda, R., Wilson W. D. (2012) "Binding to the DNA Minor Groove by Heterocyclic Dications: From AT Specific
Monomers to GC Recognition with Dimers" Current Protocols in Nucleic Acid Chemistry Chapter 8: Unit 8.8, 1-20.
Liu, Yang; Chai, Y.; Kumar, A.; Tidwell, R. R.; Boykin, D. W.; Wilson, W. David (2012) “Designed compounds for
recognition of 10 base pairs of DNA with two AT binding sites.” J. Am. Chem. Soc. 134, 5290-5299.
Nanjunda, R.; Musetti, C.; Kumar, A.; Ismail, M. A.; Farahat, A. A.; Wang, S.; Sissi, C.; Palumbo, M.; Boykin, D.W.;
Wilson, W. D. (2012). “Heterocyclic dications as a new class of telomeric G-quadruplex targeting agents.” Curr. Pharm.
Des. 18, 1934-1947. (Review of DNA quadruplex interactions)
Liu, Y.; Wilson, W. D. (2010) “Quantitative analysis of small molecule-nucleic acid interactions with biosensor surface
and surface plasmon resonance detection.” Methods Mol. Biol., 613, 1-23.
Nanjunda, Rupesh; Owens, Eric A.; Mickelson, Leah; Alyabyev, Sergey; Kilpatrick, Nancy; Wang, Siming; Henary,
Maged; Wilson, W. David (2012) “Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex
DNA.” Bioorganic and Medicinal Chemistry 20, 7002-7011.
Hunt, Rebecca A.; Munde, Manoj; Kumar, Arvind; Ismail, Mohamed A.; Farahat, Abdelbasset A.; Arafa, Reem K.; Say,
Martial; Batista-Parra, Adalgisa; Tevis, Denise; Boykin, David W.; Wilson, W. David (2011) “Induced topological
changes in DNA complexes: Influence of DNA sequences and small molecule structures.” Nucleic Acids Res. 39, 42654274. (Conformational effects of minor groove interactions)
Wang, S.; Munde, M.; Wang, S.; Wilson, W. D. (2011) “Minor groove to major groove, an unusual DNA sequencedependent change in bend directionality by a distamycin dimer.” Biochemistry, 50, 7674-7683. (Induced bending in
DNA by a minor groove binding polyamide)
Georgia State University
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10
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Neutron technologies for nucleic acid research
and drug design and delivery
Paul Langan* Biology and Soft Matter Division, Oak Ridge National Laboratory,
Oak Ridge, Tennessee, USA; email: [email protected]
Neutron scattering is a well-established tool for studying molecular structure, function,
and dynamics that complements information obtained from photons and electrons.
Neutron scattering techniques can probe enormous ranges of length and time scales; from
Ångstroms to microns and from picoseconds to milliseconds. They are therefore ideal for
studying multi-scale phenomena intrinsic to biological processes. Oak Ridge National
Laboratory (ORNL) hosts two neutron scattering research facilities, the Spallation
Neutron Source (SNS) and the High Flux Isotope Reactor (HFIR). In this talk I will
present an overview of these facilities in particular those beam lines used for nucleic acid
research and drug design and delivery. I will then focus on my research interests in
developing neutron crystallography as a method for directly determining hydrogen atom
positions in biological macromolecules. Knowing these positions can provide information
on the protonation states of amino-acid residues and ligands, the identity of solvent
molecules, and the nature of bonds involving hydrogen. Further, neutron crystallography
can be used to identify hydrogen atoms that are exchanged with their isotope deuterium
(deuteration) and the extent of this replacement, thus providing a tool for identifying
isotopically labeled features, for studying solvent accessibility and macromolecular
dynamics, and for identifying minimal protein folding domains. This unique information,
which is often difficult or impossible to obtain using X-ray crystallography, is important
for understanding the structure and function of nucleic acids and is increasingly being to
study protein-drug interaction, with a view to providing new information that might help
in drug design.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Paul Langan
Paul Langan is Director of the Biology and Soft Matter Division of
Oak Ridge National Laboratory (ORNL) and a prestige research
professor of Chemistry at Toledo University. He is Director of the
U.S. Department of Energy (DOE) funded Center for Structural
Molecular Biology (CSMB) at ORNL, he leads a National Institute
of Health (NIH) funded consortium that develops computational
tools for neutron crystallography, and he has been involved in
leading various ORNL and Los Alamos National Laboratory
(LANL) Directed Research and Development (LDRD) funded
projects over the years in the areas of protein crystallography and
proteomics, and cellulosic biofuels.
After receiving a BSc with honors in Physics from Edinburgh University, Paul was
awarded a PhD in Biophysics on the structure of nucleic acids from Keele University in
1990 and was then appointed to consecutive research fellowships by Keele University to
develop neutron diffraction for biology at the Rutherford Appleton laboratory in Oxford.
In 1994 Paul moved to the Institute Laue Langevin (ILL), Grenoble, where he was
crystallography beam-line scientist and also secretary for the ILL Biology College. In
1998 Paul moved to LANL to work on the design and construction of the Protein
Crystallography Station (PCS), for which he received a Distinguished Performance
Award (2002). In April 2011 Paul moved to ORNL as a Senior Scientist and
Distinguished Research and Development staff member to build science programs across
associate directorates that exploit the world-leading neutron capabilities at ORNL, and to
direct the CSMB. After reorganization of the Neutron Science Directorate in October
2011 Paul became Director of the newly formed Biology and Soft Matter Division.
Paul's research interests include the relationship between structure and function in
biology and chemistry, new computational methods and instrumentation for
crystallography, and building and leading multidisciplinary teams to address mission
driven science in the areas renewable energy and the environment. He has published over
140 articles in the fields of biology, chemistry, physics, and material science and has a
software copyright for a crystallography program called nCNS. His most recent impact
has been in applying neutrons to study enzyme mechanism and drug binding, developing
novel technologies and computational methods for neutron macromolecular
crystallography, and in providing a detailed understanding of the cellulose and lignin
components of cellulosic biomass.
Georgia State University
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12
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Structure determination of transcriptional factors
and their complexes with DNA
Andrzej Joachimiak* Midwest Center for Structural Genomics and Structural Biology
Center,Biosciences, Argonne National Laboratory, Argonne, IL 60439, USA;
email: [email protected]
Many structures of transcriptional factors and their DNA complexes have been
determined by us recently. Several examples will be discussed in this talk. One example
is HetR, which is an essential regulator of heterocyst development in cyanobacteria.
Many mutations in HetR render Anabaena incapable of nitrogen fixation. The protein
binds to a DNA palindrome upstream of hetP and other genes. We have determined the
crystal structures of HetR complexed with palindromic DNA targets, 21, 23, and 29 bp at
2.50-, 3.00-, and 3.25-Å resolution, respectively. The highest-resolution structure shows
fine details of specific protein-DNA interactions. The lower-resolution structures with
longer DNA duplexes have similar interaction patterns and show how the flap domains
interact with DNA in a sequence nonspecific fashion. Fifteen of 15 protein-DNA contacts
predicted on the basis of the structure were confirmed by single amino acid mutations
that abolished binding in vitro and complementation in vivo. A striking feature of the
structure is the association of glutamate 71 from each subunit of the HetR dimer with
three successive cytosines in each arm of the palindromic target, a feature that is
conserved among all known heterocyst-forming cyanobacteria sequenced to date.
Kim Y, Ye Z, Joachimiak G, Videau P, Young J, Hurd K, Callahan SM, Gornicki P, Zhao J, Haselkorn R,
Joachimiak A. (2013) “Structures of complexes comprised of Fischerella transcription factor HetR with
Anabaena DNA targets.” Proc. Natl. Acad. Sci. USA. 110, 1716-23.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Andrzej Joachimiak
Andrzej Joachimiak (Ph.D.) is Director of Structural Biology Center and Midwest Center
for Structural Genomics. He is an expert in synchrotron-based X-ray crystallography and
structural biology and has published over 300 publications. At Argonne, he has made
significant contributions to the high-throughput crystallography using synchrotron
radiation and the development of state-of-the-art facilities for macromolecular
crystallography. The development and integration of the novel synchrotron beamlines,
exploitation of the anomalous signal-based phasing methods in the third-generation
environment and integration of hardware and software at the Structural Biology Center
beamlines at the Advanced Photon Source contributed very strongly to the enhanced
efficiency of such facilities worldwide.
Andrzej’s research has also contributed to methods development in protein expression
and purification, crystallization and data collection using synchrotron radiation. The
methods for phasing novel structures using X-ray crystallography have effectively
reduced time and cost of structure determination. The contribution to high-throughput
molecular biology and crystallography has also made a major impact on structural
biology in the U.S. and globally as well. This encouraged the initiation of structural
genomics and structural proteomics projects and also led to the determination of
thousands of novel protein structures.
He has also made highly important contributions to the field of structural genomics. The
Argonne-based Midwest Center for Structural Genomics (MCSG) is a highly successful
program and major component of NIH funded Protein Structure Initiative (PSI). A
number of technologies have been developed in the MCSG, including gene cloning and
protein expression, new vectors for protein expression in bacteria, automated protein
purification techniques, automated protein crystallization, robot-assisted crystal mounting
and automated structure determination. Andrzej’s current research also focuses on
proteins and protein-nucleic acid interactions and includes enzymes, transcription factors
and molecular chaperones. In addition to his duties at Argonne, Andrzej is also a
Professor at the University of Chicago, Adjunct Professor at Northwestern University,
Senior Fellow at the Computation Institute and the Institute for Genomics and Systems
Biology.
Georgia State University
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14
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Expansion of the genetic alphabet of DNA and its
application to aptamer generation
Michiko Kimoto, Ken-ichiro Matsunaga, Rie Yamashige, Ichiro Hirao*
Synthetic Molecular Biology Team,
RIKEN Center for Life Science Technologies, Yokohama, Japan; email: [email protected]
Genetic information flow in the central dogma relies on only the four nucleobase
components, ruled by A−T(U) and G−C pair formations, which in turn constrain the
Darwinian evolution of nucleic acids as functional molecules. Thus, the expansion of the
genetic alphabet by introducing an artificial extra base pair (unnatural base pair) into
DNA could provide a new biotechnology for generating nucleic acids and proteins with
increased functionality. This genetic alphabet expansion can be achieved by the creation
of an unnatural base pair that functions as a third base pair in replication, transcription,
and/or translation, along with the natural base pairs. Recently, we developed an unnatural
base pair between hydrophobic 7-(2-thienyl)imidazo[4,5-b]pyridine (denoted by Ds) and
2-nitro-4-propynylpyrrole (denoted by Px) that exhibits high selectivity and efficiency in
PCR. DNA fragments containing the Ds−Px pair are amplified ~1028-fold by 100 cycles
(10 cycles ☓ 10 times) of PCR, and more than 97% of the Ds−Px pairs survived at the
initial positions in the amplified DNA. We applied the Ds−Px pair PCR system to DNA
aptamer selection by developing a new SELEX system (ExSELEX: genetic alphabet
Expansion SELEX). In ExSELEX, we prepared a DNA library containing the
hydrophobic Ds bases as a fifth base in its random sequence region, and the DNA library
was amplified by PCR involving the Ds−Px pair system. We demonstrated DNA aptamer
selection targeting human vascular endothelial cell growth factor-165 (VEGF-165) and
interferon-γ and obtained DNA aptamers that bind with Kd vales of 0.65 pM and 38 pM,
respectively, which are >100-fold improved over aptamers containing only natural bases.
Our data showed that the increased complexity of genetic information, with only a few of
the fifth hydrophobic bases, could augment nucleic acid functionality through evolution,
thus providing a powerful tool for creating new functional nucleic acids.
Selected Publications:
1.
2.
3.
4.
5.
6.
M. Kimoto, R. Yamashige, K. Matsunaga, S. Yokoyama, I. Hirao, Generation of high-affinity DNA
aptamers using an expanded genetic alphabet. Nat. Biotechnol., 31, 453-457 (2013).
I. Hirao, M. Kimoto, Unnatural base pair systems toward the expansion of the genetic alphabet in
the central dogma. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci., 88, 345-367 (2012).
I. Hirao, M. Kimoto, R. Yamashige, Natural vs artificial creation of base pairs in DNA: origin of
nucleobases from the perspectives of unnatural base pair studies. Acc. Chem. Res.,45, 2055-2065
(2012).
R. Yamashige, M. Kimoto, Y. Takezawa, A. Sato, T. Mitsui, S. Yokoyama, I. Hirao, Highly specific
unnatural base pair systems as a third base pair for PCR amplification. Nucleic Acids Res., 40, 27932806 (2012).
M. Kimoto, R. S. Cox 3rd, I. Hirao, Unnatural base pair systems for sensing and diagnostic
applications. Expert Rev. Mol. Diagn., 11, 321-331 (2011).
M. Kimoto, T. Mitsui, R. Yamashige, A. Sato, S. Yokoyama, I. Hirao, A new unnatural base pair
system between fluorophore and quencher base analogues for nucleic acid-based imaging
technology. J. Am. Chem. Soc., 132, 15418-15426 (2010).
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Ichiro Hirao
Ichiro Hirao (Ph.D.) was born in Shizuoka, Japan, in 1956. He
graduated from Numazu National College of Technology in 1976,
and received his BS (1978) degree from the Faculty of
Engineering, Shizuoka University, and his M.S. (1980) and Ph.D.
(1983) degrees in the chemical synthesis of 2′-5′ oligonucleotides
and their structures from the Faculty of Science, Tokyo Institute of
Technology. In 1984, he joined Dr. Kin-ichiro Miura’s laboratory
in the Faculty of Engineering, The University of Tokyo, as a
research associate, where he discovered extraordinarily
thermostable DNA mini-hairpin structures. In 1992, he became an
associate professor at Tokyo University of Pharmacy and Life Sciences. To expand his
research areas from organic chemistry and structural biology to molecular biology and
evolutional engineering, in 1995, he moved to Dr. Andrew D. Ellington’s laboratory in
the Department of Chemistry, Indiana University. In 1997, to start the unnatural base pair
studies, he returned to Japan and joined Dr Shigeyuki Yokoyama’s project, ERATO,
Japan Science and Technology Agency as a group leader. In 2002, he continued his work
as both a Professor at the Research Center for Advanced Science and Technology, The
University of Tokyo and as a senior visiting scientist at the RIKEN Genomic Sciences
Center. Since 2006, he has been managing the Nucleic Acid Synthetic Biology Research
Team at the Systems and Structural Biology Center, RIKEN, as a team leader. In 2007,
he founded the venture company ‘TagCyx Biotechnologies’ with Dr. Shigeyuki
Yokoyama, to provide unnatural base pair technologies toward the expansion of the
genetic alphabet of DNA. Now, he is taking charge of the Synthetic Molecular Biology
Team at the Center for Life Science Technologies, RIKEN.
7.
8.
9.
10.
11.
12.
13.
Y. Hikida, M. Kimoto, S. Yokoyama, I. Hirao, Site-specific fluorescent probing of RNA molecules by
unnatural base-pair transcription for local structural conformation analysis. Nature Protocols, 5,
1312-1323 (2010).
M. Kimoto, R. Kawai, T. Mitsui, S. Yokoyama, I. Hirao, An unnatural base pair system for efficient
PCR amplification and functionalization of DNA molecules. Nucleic Acids Res., 37, e14 (2009).
M. Kimoto, T. Mitsui, Y. Harada, A. Sato, S. Yokoyama, I. Hirao, Fluorescent probing for RNA
molecules by an unnatural base-pair system. Nucleic Acids Res., 35, 5360-5369 (2007).
I. Hirao, M. Kimoto, T. Mitsui, T. Fujiwara, R. Kawai, A. Sato, Y. Harada, S. Yokoyama, An
unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into
DNA and RNA. Nature Methods, 3, 729-735 (2006).
I. Hirao, Unnatural base pair systems for DNA/RNA-based biotechnology. Curr. Op. Chem. Biol., 10,
622-627 (2006).
I. Hirao, T. Ohtsuki, T. Fujiwara, T. Mitsui, T. Yokogawa, T. Okuni, H. Nakayama, K. Takio, T.
Yabuki, T. Kigawa, K. Kodama, T. Yokogawa, K. Nishikawa, S. Yokoyama, An unnatural base pair
for incorporating amino acid analogs into proteins. Nature Biotechnology, 20, 177-182 (2002).
T. Ohtsuki, M. Kimoto, M. Ishikawa, T. Mitsui, I. Hirao, S. Yokoyama, Unnatural base pairs for
specific transcription. Proc. Natl. Acad. Sci. USA, 98, 4922-4925 (2001).
Georgia State University
15
16
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
HIV Protease: the Challenge of Drug Resistance
Johnson Agniswamy1, Yuan-Fang Wang1, Chen-Hsiang Shen1, Ying Zhang2, Hongmei
Zhang1, Robert W. Harrison3,1, John M. Louis4, Arun K. Ghosh5, Irene T. Weber1,2*
1
Department of Biology,2Department of Chemistry, 3Department of Computer Science, Georgia
State University, Atlanta, Georgia, USA; 4Laboratory of Chemical Physics, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda,
Maryland 20892, USA; 5Department of Chemistry and Department of Medicinal Chemistry,
Purdue University, West Lafayette, IN 47907, USA; email: [email protected]
Our studies of HIV protease address the medical challenge of drug resistance in HIV by
analyzing the structures and activities of drug resistant protease variants, and by guiding
the design of new antiviral inhibitors. Since the crystal structures of HIV protease were
first solved almost 25 years ago, this protein has become a paradigm for structure-guided
drug design. The introduction of protease inhibitors into antiviral therapy in 1995
converted HIV/AIDS from a lethal to a chronic condition. The virus, however, can
mutate rapidly into drug resistant strains. Therefore, new inhibitors have been designed
for resistant variants. We were the first to report the crystal structure of HIV protease in
complex with the potent antiviral inhibitor darunavir, which has proved highly effective
in treating resistant HIV/AIDS. Our studies have elucidated multiple molecular
mechanisms for resistance to protease inhibitors. Individual mutations can act by altering
the interaction with clinical inhibitors or by altering the dimer interface or stability of the
enzyme. Recently, we have characterized the unusual properties of a highly resistant
protease variant bearing 20 mutations that has drastically lower affinity for inhibitors of
several orders of magniture relative to the wild type enzyme. Our crystal structures show
the coordinated effects of multiple mutations in expanding the inhibitor binding site and
providing large conformational variability in the absence of bound inhibitor. This
analysis of drug resistant mutants gives insight into the most effective designs for new
inhibitors to combat resistant HIV infections.
This work is supported by NIH (R01GM062920).
Selected Publications:
1.
2.
3.
4.
5.
6.
7.
Agniswamy, J., Shen, C.-H., Aniana, A., Sayer, J.M., Louis, J.M., Weber, I.T.*, “HIV-1 protease with 20
mutations exhibits extreme resistance to clinical inhibitors through coordinated structural rearrangements”,
Biochemistry, 2012, 51, 2819-2828.
Agniswamy, J., Shen, C.-H., Wang, Y.-F., Ghosh, A.K., Rao, K.V., Xu, C-X., Sayer, J.M., Louis, J.M., Weber,
I.T. “Extreme multidrug resistant HIV-1 protease with 20 mutations is resistant to novel protease inhibitors with
P1-pyrrolidinone or P2-tris-tetrahydrofuran”, J. Med. Chem., 2013, 56, 4017-4027.
Zhang, H., Wang, Y.-F., Shen, C.-H., Agniswamy, J., Rao, K.V., Xu, C.-X., Ghosh, A.K., Harrison, R.W., Weber,
I.T. “Novel P2 tris-tetrahydrofuran group in antiviral compound 1 (GRL-0519) fills the S2 binding pocket of
selected mutants of HIV-1 protease.” J. Med. Chem., 2013, 56, 1074-83.
Lin, L.; Sheng, J.; Huang, Z. Chemical Society Reviews, 2011, 40, 4591.
Shen, C.-H., Tie, Y., Yu, X., Wang, Y.-F., Kovalevsky, A.Y., Harrison, R.W., Weber, I.T. “Capturing the reaction
pathway in near-atomic resolution crystal structures of HIV-1 protease.” Biochemistry, 2012, 51, 7726-32.
Tie, Y., Wang, Y.-F., Boross, P.I., Chiu, T.-Y. Ghosh, A.K., Tozser, J. Louis, J.M., Harrison, R.W., Weber, I.T.
“Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors“ Protein Sci, 2012, 21, 339350.
Ghosh, A.K., Chapsal, B.D., Steffey, M., Agniswamy, J., Wang, Y.-F., Amano, M., Weber, I.T., Mitsuya, H.
“Substituent effects on P2-cyclopentyltetrahydro-furanyl urethanes: Design, synthesis, and X-ray studies of potent
HIV-1 protease inhibitors.” Bioorg Med Chem Lett, 2012, 22, 2308-2311.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Irene T. Weber
Prof. Irene T. Weber (Ph.D.) was born in 1953 and raised in
England. She received her B.S. degree from the University of
Cambridge in 1974, M.S. the University of Cambridge in 1978,
and Ph.D. degree from the University of Oxford in 1978 (under the
supervision of Professor Louise Johnson). In 1994, she joined the
Department of Molecular Biophysics and Biochemistry as a
postdoctoral fellow, in the laboratory of Professor Thomas Steitz
(Nobel Laureate in Chemistry in 2009). She joined the
Macromolecular Structure Group of Dr. Alexander Wlodawer in
1984 as a Guest Worker of the National Bureau of Standards (now
the National Institute of Science and Technology), Gaithersburg, MD, and was promoted
to Physicist in 1986. In 1987, she was recruited as Staff Scientist and Head of the ProteinNucleic Acid Interactions Group, Basic Research Program, NCI-Frederick Cancer
Research Facility, Frederick, MD. Dr. Weber was recruited as an Associate Professor,
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas
Jefferson University, Philadephia, PA in 1991. In 2000, she was recruited to a joint
appointment as Professor in the Department of Biology and the Department of
Chemistry, Georgia State University, Atlanta, GA. She has received several awards, the
Georgia Cancer Coalition Distinguished Cancer Scientist in 2002, and Regents Professor,
Board of Regents, University System of Georgia in 2011. Her current research interests
are in the structures and mechanisms of enzymes, structure-guided design of inhibitors
for drug resistant HIV/AIDS and bacterial infections. Her accomplishments include over
200 peer-reviewed scientific publications in internationally circulated journals, book
chapters and conference proceedings, 3 patents, and numerous presentations at
professional meetings and university seminars. She has organized a number of local and
national scientific conferences. Her scientific research has been supported by federal and
state grants from the National Institutes of Health, the Fogarty International Center, the
Centers for Disease Control and Prevention, the Georgia Cancer Coalition, and private
foundations including the Elsa U. Pardee Foundation, the Cure for Lymphoma
Foundation, and the American Diabetes Association.
Agniswamy, J., Sayer, J.M., Weber, I.T., Louis, J.M. “Terminal interface conformations modulate dimer stability
prior to amino terminal autoprocessing of HIV-1 protease.” Biochemistry, 2012, 51, 1041-50.
9. Louis, J.M., Aniana, A., Weber, I.T., Sayer, J.M. “Insights into the inhibition of autoprocessing of natural variants
and multidrug resistant mutant precursors of HIV-1 protease by clinical inhibitors.” (2011) Proc. Natl. Acad. Sci.,
2011, 108, 9072-7.
10. Louis, J.M., Zhang, Y., Sayer, J.M., Wang, Y.-F., Harrison, R.W., Weber, I.T. “Drug resistance mutation L76V
decreases the dimer stability and rate of autoprocessing of HIV-1 protease by reducing internal hydrophobic contacts.”
Biochemistry , 2011, 50, 4786-95.
11. Ghosh, A.K., Xu, C.-X., Rao, K.V., Baldridge, A., Agniswamy, J., Wang, Y.-F., Weber, I.T., Miguel, S., Amano,
M., Mitsuya, H. “Probing multidrug resistance/protein-ligand interaction with new oxatricyclic designed ligands
in HIV-1 protease inhibitors.” ChemMedChem, 2010, 5, 1850-4.
12. Agniswamy, J., Weber, I.T. “HIV-1 protease: structural perspectives on drug resistance.” Viruses, 2009, 1, 11101136.
8.
Georgia State University
17
18
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Exploring the Biophysical/Biochemical
Information of Metals in Macromolecules Using
Wavelength-Dependent Data
Palani Kandavelu, John Rose, Zheng-Qing Fu, Unmesh Chinte, Hua Zhang, Dayong
Zhou, Lirong Chen, John Chrzas and Bi-Cheng Wang*
SER-CAT and the Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, GA 30602
X-ray crystallography can provide the detailed structural information needed for
understanding catalytic mechanism; but in general, current approaches lack convenient
“pipeline type” means for identifying differences of one or two electrons that
distinguishes the oxidation state of an atom. This study is an attempt to use the
wavelength-dependent properties from X-ray anomalous scattering to monitor the
electronic (e.g. oxidation) states of metals/ions in the crystals.
To investigate whether a typical macromolecular crystallographic beamline can be used
for this purpose, bovine catalase (a homo tetramer of four Fe-containing polypeptide
chains) and human ferrochelatase (two [Fe2S2] clusters plus another metal) were used for
our preliminary method’s development and to identify areas where future
hardware/software improvements are needed. Our current finding indicates that medium
resolution data are sufficient to provide information about the oxidation states of metals
in these crystals. Thus, our “pipeline type” process, once completely developed, should
be useful for a variety of macromolecular systems.
This work is supported in part by SER-CAT at the Advanced Photon Source and the
University of Georgia.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Bi-Cheng Wang
Prof. Bi-Cheng (B.C.) Wang (Ph.D.) received his B.S. degree
(1960) in Chemical Engineering from the National Cheng Kung
University (Taiwan) and Ph.D. Degree (1968) in Chemistry from
the University of Arkansas. In 1968-70 he jointed the California
Institute of Technology as a Research Fellow (under the
supervision of Professor Dick Marsh). From 1971 to 1986 he was
first hired as a Research Chemist and subsequently promoted to
Assistant Chief of the Biocrystallography Laboratory of the
Veterans Administration Medical Center in Pittsburgh and an
Adjunct Professor of Crystallography at the University of
Pittsburgh. In 1986 he became Professor of Crystallography and Biological Sciences at
the University of Pittsburgh. In 1995 he was recruited and offered the position of
Ramsey-GRA Eminent Scholar in Structural Biology and Professor of Biochemistry and
Molecular Biology at the University of Georgia where he remains today.
Prof. Wang possesses broad expertise in protein crystallography. He has been involved
in protein crystallographic research, teaching, and methodology developments for over
forty years, and has gained extensive experience in many aspects of crystallography. He
has also successfully administrated a number of large-scale collaborative projects, and
interacts very well with many researchers from various biological fields. He is the
founding Director of SER-CAT (Southeast Regional Collaborative Access Team,
www.ser-cat.org), which includes the construction and operations of two synchrotron
beamlines at the Advanced Photon Source, Argonne National Laboratory. He served as
the PI and Program Director of the NIH-funded Southeast Collaboratory for Structural
Genomics (SECSG) between 2000-2007. During the structural genomics project, his lab
acquired/developed various approaches for the high-throughput gene-to-structure
process that includes protein expression, purification, nano-crystallization, crystal
salvaging and rescue pathways for structural analyses. In addition, the lab developed
high-throughput structure determination pipelines, such as SGXPro, which is currently
being successfully used at SER-CAT today.
Prof. Wang has received numerous awards and honors, including the Distinguished
Service Award from the American Crystallographic Association in 1999, the SUR
Award from IBM in 2001, the SER-CAT Golden Magnolia Service Award in 2007, the
Lamar Dodd Award for Creative Research from the University of Georgia Research
Foundation in 2008, the A. Lindo Patterson Award from the American Crystallographic
Association in 2008, and was elected as the Fellow of the American Crystallographic
Association in 2011.
Georgia State University
19
20
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Thermodynamics of HIV-1 Reverse Transcriptase
in action reveals the mechanism of action of nonnucleoside inhibitors
Guillaume Bec, Benoit Meyer, Jessica Steger, Katja Fauster, Ronald Micura, Philippe
Dumas, Eric Ennifar* Architecture et Réactivité de l’ARN,
CNRS/Université de Strasbourg, Strasbourg, France; email: [email protected]
HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA
into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic
data regarding RT interactions with its substrates are still lacking. Here we addressed the
mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration
calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic
dissection of the RT polymerization activity showed that most of the driving force for DNA
synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led
to a re-interpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to
preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with
free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerasecompetent orientation, or form high-affinity dead-end complexes, both RT/NNRTI/DNA
complexes being unable to bind the incoming nucleotide substrate.
This work is supported by the ‘Agence Nationale de Recherche sir le SIDA’ (ANRS).
References:
1.
2.
Burnouf*, Ennifar*, Guedich, Puffer, Hoffmann, Bec, Disdier, Baltzinger, Dumas "kinITC: a new
method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry", J Am
Chem Soc, 2012, 134, 559-65.
Bec, Meyer, Gerard, Steger, Fauster, Wolff, Burnouf, Micura, Dumas, Ennifar*, “Thermodynamics of
HIV-1 Reverse Transcriptase in action elucidates the mechanism of action of non-nucleoside
inhibitors”, J Am Chem Soc, 2013, 135, 9743-52.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Eric Ennifar
Eric Ennifar (Ph.D.) was born in 1972 in Strasbourg, France. He
received a Master degree in Biological Crystallography and NMR
(joint Master from Universities of Strasbourg, Grenoble and Paris
Orsay) in 1998, and a Ph.D. in Structural Biology in 2001 from the
University of Strasbourg (under the supervision of Philippe
Dumas). During his Ph.D., he solved the X-ray structure of the
HIV-1 genomic RNA Dimerization Initiation Site, the first crystal
structure of an RNA kissing-loop complex, and the X-ray structure
of the ribosomal S15/mRNA complex. He then moved to the
European Molecular Biology Laboratory in Heidelberg (Germany)
for a postdoctoral fellowship in the group of Dietrich Suck working on X-ray
crystallography of DNA recombinases and resolvases. In 2003, Dr. Ennifar was recruited
as a CNRS junior research scientist in the laboratory of Bernard Ehresmann (Institut de
Biologie Moléculaire et Cellulaire - IBMC, Strasbourg) to work on HIV RNA/ligand
interactions and the HIV-1 Reverse Transcription complex. Since 2007, he his CNRS
Senior Research Scientist at IBMC (director: Eric Westhof) working in the group of
Biophysics and Structural Biology headed by Philippe Dumas. His research is focused on
biophysical studies of RNA/protein complexes, such as (1) the development of rationally
designed novel drugs targeting the viral RNA genome and (2) thermodynamic and
structural basis of HIV replication and of the innate immune response to HIV infection.
His research has been funded by the Agence Nationale de Recherche sur le SIDA
(ANRS), Agence Nationale de la Recherche (ANR) and Sidaction.
Georgia State University
21
22
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Viral 3′ RNA structures interacting with cell proteins
regulate the initiation of flavivirus RNA synthesis
William Davis, Mausumi Basu, Mohamed Emara, Hsuan Liu, Elizabeth Elrod, Jin Zhang,
Marcus Germann, and Margo Brinton*
Department of Biology and Department of Chemistry
Georgia State University, Atlanta, Georgia, USA; email: [email protected]
The 3′ terminal nts of the West Nile virus (WNV) genomic RNA are predicted to form a
small stem loop (SSL) of 16 nts adjacent to the terminal 80 nt SL. These structures are
conserved among divergent flaviviruses. RNase footprinting and nitrocellulose filter
binding assays were used to map one major and one minor binding site for the cell
protein eukaryotic elongation factor 1a (eEF1A) on the 3′SL and one minor binding site
on the SSL. Base substitutions in the major eEF1A binding site or adjacent areas of the 3′
SL were engineered into a WNV infectious clone. Mutant RNAs were also tested in in
vitro binding assays. Mutations that decreased in vitro eEF1A binding to the 3′ SL RNA
also decreased viral minus-strand RNA synthesis in RNA transfected cells. Also, a
mutation that increased the efficiency of eEF1A binding to the 3′ SL RNA increased
minus-strand RNA synthesis in transfected cells. These results indicated that the
interaction between eEF1A and the WNV 3′ SL facilitates viral minus-strand synthesis.
eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent
flaviviruses, including a tick-borne flavivirus indicating that eEF1A is a host factor for all
of the members of the genus Flaviviridae. A previous in vitro study on truncated WNV 3'
RNAs predicted a tertiary interaction between the 5′ side of the 3′ terminal SL and SSL
loop nts. Substitution/deletion of the 3′ G within the loop of the SSL that formed the only
G-C pair in the predicted tertiary interaction in a WNV infectious clone was lethal
suggesting the tertiary interaction was cis-acting but extensive mutagenesis of nts in the
terminal SL did not identify pairing partners. An NMR analysis confirmed the SSL and
SL structures but not the tertiary interaction. The sequence of the SSL loop destabilized
this hairpin. The SSL was previously shown to contain one of the two minor binding sites
for eEF1A and the 3' G within the loop of the SSL was shown to be important for
efficient EF1A binding. The results indicate that interaction with EF1A, the SSL and two
cis-acting base pairs in the terminal SL may facilitate switching between exclusively 3′
terminal to 3'-5' long distance RNA pairing interactions by the genome RNA during the
initiation of minus strand RNA replication. Support: NIH R01 AI45135
Selected Publications:
1.
2.
3.
Davis, W.G., Basu, M., Elrod, E.J., Germann, M.W., and Brinton, M.A. 2013. Identification of cisacting nucleotides and a structural feature in West Nile virus 3'-terminus RNA that facilitate viral
minus strand RNA synthesis. J. Virol. 87(13):7622-36. Epub 2013 May 1.
Emara, M.M., Liu, H., Davis, W.G., and Brinton, M.A. 2008. Mutation of mapped TIA-1/TIAR
binding sites in the 3' terminal stem loop of the West Nile virus minus-strand RNA in an infectious
clone negatively affects genomic RNA amplification. J Virol. 82:10657-10670.
Emara, M.M. and Brinton, M.A. 2007. Interaction of TIA-1/TIAR with West Nile and dengue virus
products in infected cells interferes with stress granule formation and processing body assembly. Proc.
Natl. Acad. Sci. U S A 104:9041-9046. Epub 2007 May 14.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Margo Brinton
Margo Brinton received her BA in Zoology from Duke University
and her PhD in Microbiology from The University of
Pennsylvania. She did postdoctoral work at the University of
Minnesota and subsequently was an Instructor at the University of
Minnesota, a Senior Researcher at Riker Research Laboratories of
3M, an Assistant and then an Associate Professor at the University
of Pennsylvania. She moved to the Department of Biology at
Georgia State University in 1998 and is currently a Regents’
Professor. She is a Fellow of the American Academy of
Microbiology and the 2013 GSU Alumni Distinguished Professor.
Her lab’s research is focused on identifying and functionally characterizing cell proteins
used by RNA viruses, such as West Nile virus, as “transcription factors” for enhancing
viral RNA synthesis; on understanding how viruses alter the cells they infect to create a
more favorable environment for their replication; and on how particular variations in
human or mouse genes affect host susceptibility to virus-induced disease. To foster
exchange of information among scientists in her field, in 1986, Dr. Brinton founded and
continued to organize until 2010 the triennial International Symposium on Positive
Strand RNA Viruses. In 1991, she founded and continues to organize, the biennial
Southeastern Regional Virology Conference.
4.
Davis, W.G., Blackwell, J.L., Shi, P.Y., and Brinton, M.A. 2007. Interaction between the cellular
protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minusstrand RNA synthesis. J. Virol. 81:10172-10187. Epub 2007 Jul 11. (Selected as a Spotlight paper by
the journal editors)
5. Elghonemy S., Davis, W.G., and Brinton, M.A. 2005. The majority of the nucleotides in the top loop
of the genomic 3’ terminal stem loop structure are cis-acting in a West Nile virus infectious clone.
Virology 331: 238-246.
6. Maines, T. R., Young, M., Dinh, N.N.-N. and Brinton, M.A. 2005. Two cellular proteins that interact
with a stem loop in the simian hemorrhagic fever virus 3’ (+) NCR RNA. Virus Res. 109: 109-124.
7. Li, W., Li, Y., Kedersha, N., Swiderick, K, Moreno, T., Anderson, P., and Brinton, M. A. 2002. Cell
Proteins, TIA-1 and TIAR, Interact with the 3' Stem Loop of the West Nile virus Complementary
Minus Strand RNA. J. Virology 76: 11989-12000.
8. Hwang, Y.-K. and Brinton, M.A. 1998. A 68nt sequence within the 3'noncoding region (NCR) of
simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins. J. Virol.,
72:4341-4351.
9. Blackwell, J.L. and Brinton, M.A. 1997. Translation elongation factor -1 alpha interacts with the 3'
stem-loop region of West Nile virus genomic RNA. J. Virol. 71:6433-6444.
10. Shi, P.-Y., Brinton, M.A., Veal, J.M., Zhong, Y.Y., and Wilson, W.D. 1996. Evidence for the
existence of a pseudoknot structure at the 3' terminus of the flavivirus genomic RNA. Biochemistry 35,
4222-4230.
11. Shi, P.-Y., Li, W. And Brinton, M.A. 1996. Cell proteins bind specifically to West Nile virus minusstrand 3' stem-loop RNA. J. Virol. 70, 6278-6287.
Georgia State University
23
24
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Recognition of cobalamins by riboswitches
James E. Johnson, Francis E. Reyes, Jacob T. Polaski and Robert T. Batey*
Department of Chemistry and Biochemistry,
University of Colorado, Boulder, CO 80301; email: [email protected]
Riboswitches are important regulatory mechanisms in bacteria for controlling gene
expression at the mRNA level. One of the most ubiquitous families of riboswitches
specifically recognizes adenosylcobalamin (coenzyme B12) to regulate transcription or
translation of its associated mRNA. Analyses of these RNAs have revealed two distinct
classes based upon the presence of a peripheral domain that is facilitates ligand
recognition. In this work, we identify a subset of B12 riboswitches that preferentially
bind methylcobalamin over adenosylcobalamin, indicating a distinct subclass.To
understand the structural basis for ligand specificity, we employed chemical probing
experiments to observe ligand dependent structural changes in the RNA. Notably, we
observed nearly identical set of structural changes in each class, leading us to conclude
that all members of the B12 riboswitch family adopt a similar fold in response to ligand.
Moreover, we solved structures of members of both classes of cobalamin riboswitches in
complex with their effector molecule. These structures illustrate how cobalamins are
specifically recognized by the RNA and suggests a mechanism where binding of the
ligand results in a structural rearrangement that occludes the putative ribosome binding
site. This mechanism was validated using both in vitro and in vivo approaches.
This work is supported by a grant from the NIH (R01GM073850)
Selected Publications:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Ceres P, Garst AD, Marcano-Velazquez JG and Batey RT (2013) "Modularity of select riboswitch
expression platforms enables facile engineering of novel genetic regulatory devices." ACS Synth
Biol (epub ahead of print)
Stoddard CD, Widmann J, Trausch JJ, Marcano-Velazquez JG, Knight R, and Batey RT (2013)
“Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine
riboswitch.” J Mol Biol 425: 1596-1611.
Fiegland LR, Garst AD, Batey RT, Nesbitt DJ (2012) “Single-molecule studies of the lysine
riboswitch reveal effector dependent conformational dynamics of the aptamer domain.”
Biochemistry 51: 9223-9233.
Johnson JE, Reyes FE, Polaski J, Batey RT (2012) "B12 cofactors directly stabilize an mRNA
regulatory switch." Nature 492: 133-137.
Garst AD, Porter EB, Batey RT (2012). “Insights into the regulatory landscape of the lysine
riboswitch.” J Mol Biol. 423: 17-33.
Trausch J, Reyes FE, Ceres P, Batey RT (2011). “The structure of a tetrahydrofolate-sensing
riboswitch reveals two ligand binding sites in a single aptamer.” Structure 19: 1413-1423.
Vicens Q, Mondragón E, Batey RT (2011). “Molecular sensing by the aptamer domain of the FMN
riboswitch: a general model for ligand binding by conformational selection.” Nucleic Acids
Research 39: 8586-8598.
Daldrop P, Reyes FE, Robinson DA, Hammond CM, Lilley DM, Batey RT, Brenk R (2011). “Novel
ligands for a purine riboswitch discovered by RNA-ligand docking.” Chemistry & Biology 18: 324335.
Stoddard CD, Montange RK, Hennelly SP, Rambo RP, Sanbonmatsu KY, Batey RT (2010). “Free
state conformational sampling of the SAM-I riboswitch aptamer domain.” Structure 18: 787-797.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Robert Batey
Robert Batey is a professor of Chemistry and Biochemistry at the
University of Colorado, Boulder since 2001. He received a B.S. in
Chemistry and in Biological Sciences from the University of
California, Irvine in 1990 and his Ph.D. in Biology from MIT in
1997 working with Prof. Jamie Williamson on understanding
protein recognition of ribosomal RNA and its relationship to
ribosome assembly. Before joining the faculty at CU Boulder, he
worked in the Department of Molecular Biophysics and
Biochemistry at Yale University as a Jane Coffin Childs
postdoctoral fellow with Prof. Jennifer Doudna. There, he worked
on the structure of a ribonucleoprotein complex, the signal recognition particle, involved
in protein translocation across and into cellular membranes. His current work at CU
Boulder includes structure-function analysis of riboswitches, structure of RNA-protein
complexes and development of new techniques for RNA structure determination by Xray crystallography.
10. Edwards AL, Reyes FE, Heroux A, Batey RT (2010). “Structural basis for recognition of Sadenosylhomocysteine by riboswitches.” RNA 16: 2144-2155.
11. Montange RK, Mondragon E, van Tyne D, Garst AD, Ceres P, Batey RT (2010). “Discrimination
between closely related cellular metabolites by the SAM-I riboswitch.” J Mol Biol 396: 761-772.
12. Gilbert, S. D., Reyes, F. E., Edwards, A. L., and Batey, R. T. (2009) “Adaptive ligand binding by the
purine riboswitch in the recognition of guanine and adenine analogs” Structure 17: 857-868.
Georgia State University
25
26
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Molecular self-defense: viral RNAs that use
structure to inhibit host cell nucleases
Erich Chapman, Jenn Rabe, Jeffrey S. Kieft*
Department of Biochemistry and Molecular Genetics & Howard Hughes Medical Institute,
University of Colorado Denver School of Medicine, Aurora, Colorado, USA; email:
[email protected]
The full diversity of RNA function continues to amaze, and novel coding and noncoding RNAs
with unexpected functions are being discovered with increasing frequency. We are interested in
RNAs of viral origin that interact with cellular machinery and manipulate that machinery in a
manner that is important to the virus. In many cases, the ability of the virally-derived RNA to
function is conferred by a specific folded RNA structure, and thus understanding how these
RNAs work depends on determining the characteristics of the structure, how it interacts with its
target, and how this ultimately leads to manipulation of the cellular machinery. By studying this,
we learn not only about the specific RNA of interest, but more about the fundamental rules of the
RNA structure-function relationship as well as how the cellular machinery itself functions.
One recently discovered class of viral RNAs that interests us is includes RNAs that interact
directly with host cell nucleases and inhibit their function. We want to understand how an RNA
can block the enzymatic activity of a protein that has evolved to degrade RNA. What are the
architectures and biophysical characteristics of these RNA? How stable and important is the fold?
What are the key interactions that occur between enzyme and RNA? Can these processes be
disrupted as a therapeutic tool?
In this talk, we will present new discoveries from the Kieft Lab that address these and other
questions about how these RNAs use structure to manipulate host cell enzymes.
This work is supported by NIH (R01GM081346 and GM097333) and The Howard Hughes
Medical Institute.
Selected Publications:
1.
2.
3.
4.
5.
6.
7.
Filbin, M.E. Vollmar, B. S., Shi, D., Gonen, T., & Kieft, J.S. (2013) HCV IRES manipulates the
ribosome to promote the switch from translation to elongation. Nat Struct Mol Biol 20, 150-1582
Keel, A.Y., Jha, B.K., & Kieft, J.S. (2012) Structural architecture of an RNA that competitively
inhibits RNase L. RNA 18, 88-99.
Filbin, M.E. & Kieft, J.S. (2011) HCV IRES domain IIb affects the configuration of coding RNA in
the 40S subunit's decoding groove. RNA 17, 1258-1273.
Zhu, J., Korostelev, A., Costantino, D.A., Donohue, J.P., Noller, H.F., & Kieft, J.S. (2011) Crystal
structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs
bound to the 70S ribosome. Proc. Natl. Acad. Sci. U.S.A. 108, 1839-1844.
Keel, A.Y., Rambo. R.P., Batey, R.T., & Kieft, J.S. (2007) A general method to solve the phase
problem in RNA crystallography. Structure 15, 761-772.
Pfingsten, J.S., Costantino, D.A & Kieft, J.S. (2006) Structural basis for cap-independent ribosome
recruitment and manipulation by a viral IRES. Science, 314, 1450-1454.
Costantino, D.A., Pfingsten, J.S., Rambo, R.P., & Kieft. J.S. (2008) tRNA-mRNA mimicry drives
translation initiation from a viral IRES. Nat. Struct. Mol. Biol. 15, 57-64.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Jeffrey Kieft
Prof. Jeffrey Kieft (Ph.D.): Jeffrey Kieft received his B.S. from
the US Military Academy at West Point then served in Germany
as a tank platoon leader, support platoon leader, and battalion
logistics officer. Upon leaving active duty Jeff earned his Ph.D.
from the University of California, Berkeley under the mentorship
of Ignacio Tinoco, Jr. He did postdoctoral research at Yale
University in the Lab of Jennifer Doudna. In 2001 he was awarded
the Roger Revelle/AAAS Fellowship in Global Stewardship,
working as a member of the White House Office of Science and
Technology Policy for one year before joining the faculty at the
University of Colorado Denver School of Medicine. In 2009 he became an Early Career
Scientist of the Howard Hughes Medical Institute. He has chaired grant review panels for
the American Cancer Society and serves on the editorial board of the new journal
Translation. He has traveled and spoke extensively in churches and other public forums
on the need to rigorously teach evolutionary theory in public schools and its importance
in scientific research. He is an active volunteer and member of the Denver Astronomical
Society, and was nominated by the U.S. Army to be an astronaut. Among the awards he
is most proud of is a mentoring award from the Graduate School at the University of
Colorado Denver.
Georgia State University
27
28
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Structure, Kinetics and Mechanism of 8-oxoG
Bypass by Y-Class DNA Polymerases
Martin Egli*, Amritraj Patra, Livia Müller, F. Peter Guengerich
Department of Biochemistry and Center in Molecular Toxicology,
Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-0146, USA
*e-mail: [email protected];
laboratory URL: http://structbio.vanderbilt.edu/eglim/
DNA damage incurred by a multitude of endogenous and exogenous factors constitutes
an inevitable challenge for the replication machinery and various mechanisms exist to
either remove the resulting lesions or bypass them in a more or less mutation-prone
fashion. Error-prone polymerases are central to trans-lesion synthesis across sites of
damaged DNA. Four so-called Y-class DNA polymerases have been identified in
humans, Pol , Pol , Pol , and REV1, which exhibit different activities and abilities to
replicate past a flurry of individual lesions. Homologs have also been identified and
characterized in other organisms, notably DinB (Pol IV) in Escherichia coli, Dbh in
Sulfolobus acidocaldarius and Dpo4 in Sulfolobus solfataricus. 7,8-Dihydro-8-oxo-2’deoxyguanosine (8-oxoG), found in both lower organisms and eukaryotes, is a major
lesion that is a consequence of oxidative stress. The lesion is of relevance not only
because of its association with cancer, but also in connection with aging, hepatitis, and
infertility. It is far from clear which DNA polymerases bypass 8-oxoG most often in a
cellular context, but given the ubiquitous nature of the lesion it seems likely that more
than one enzyme could encounter the lesion. Replicative polymerases (A- and B-class)
commonly insert dATP opposite template 8-oxoG, with the lesion adopting the preferred
syn conformation. On the other hand Y-class polymerases exhibit a range of efficiencies
and fidelities in terms of 8-oxoG bypass. For example, Dpo4 from S. solfataricus
synthesizes efficiently past 8-oxoG, inserting ≥ 95% dCTP > dATP opposite the lesion.
Unlike its archaeal homolog Dpo4, hsPol bypasses 8-oxoG in an error-prone fashion by
inserting mainly dATP. We have used X-ray crystallography and steady state and
transient state kinetics in conjunction with mass-spectrometry to analyze in vitro bypass
of 8-oxoG by various Y-class polymerases to understand the diverse responses to the
lesion. The talk will summarize structural insights into the mechanism of 8-oxoG bypass
by the Dpo4, hsPol and hsPol enzymes. This work is supported by NIH grants P01
CA160032, R01 ES010375 and P30 ES000267.
References:
1.
2.
3.
4.
R. L. Eoff, M. Egli, F. P. Guengerich: Impact of chemical adducts on translesion synthesis in replicative and
bypass polymerases: from structure to kinetics. In: The Chemical Biology of DNA Damage (N. E. Geacintov, S.
Broyde, Eds.), Wiley-VCH, Weinheim, Germany, 2010, 299-330.
A. Irimia, R. L. Eoff, F. P. Guengerich, M. Egli: Structural and functional elucidation of the mechanism promoting
error-prone synthesis by human DNA polymerase kappa opposite the 7,8-dihydro-8-oxo-2’-deoxyguanosine
adduct. J. Biol. Chem. 2009, 284:22467-22480.
R. L. Eoff, A. Irimia, K. C. Angel, M. Egli, F. P. Guengerich: Hydrogen bonding of 7,8-dihydro-8oxodeoxyguanosine with a charged residue in the little finger domain determines miscoding events in Sulfolobus
solfataricus DNA polymerase Dpo4. J. Biol. Chem. 2007, 282:19831-19843.
H. Zang, A. Irimia, J.-Y. Choi, K. C. Angel, L. V. Loukachevitch, M. Egli, F. P. Guengerich: Efficient and high
fidelity incorporation of dCTP opposite 7,8-dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA
polymerase Dpo4. J. Biol. Chem. 2006, 281:2358-2372.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Martin Egli
Martin Egli studied chemistry at the Swiss Federal Institute of
Technology (ETH) in Zürich, Switzerland. He obtained his doctorate in
organic chemistry and chemical crystallography in 1988 from the
Laboratory for Organic Chemistry at the same institution, working with
Vladimir Prelog and Jack D. Dunitz. After a postdoc with Alexander
Rich at MIT and Habilitation studies at ETH, he was Assistant Professor
in Molecular Pharmacology and Biological Chemistry at Northwestern
University in Chicago. In 2000 he moved to Vanderbilt University where
he is currently Professor of Biochemistry. He was a visiting professor in
the Departments of Chemistry at Seoul National University (SNU,
Korea) and Oxford University. He currently holds an appointment as
WCU Professor in the Department of Biophysics and Chemical Biology at SNU. Ongoing
research in his laboratory includes: (i) Structure/function analysis of native and chemically
modified nucleic acids and etiology of nucleic acid structure. Using chemical synthesis and
structure determination at high resolution, the effects of chemical modifications on the structures
of DNA and RNA are probed and the results correlated with stability and in vitro and in vivo
activity data to direct the design of nucleic acid analogs with improved efficacies for antisense
and RNAi applications. (ii) The KaiABC circadian clock in cyanobacteria. Using a hybrid
structural biology approach including crystallography, EM and small angle scattering in
combination with functional studies in vitro and in vivo, we are dissecting the protein-protein
interactions that form the basis of a temperature-compensated molecular timer. (iii) Chemistry
and biology of carcinogen-DNA adducts. This program examines relationships between structure
and biological processing of various DNA adducts by Y-class trans-lesion polymerases (Dpo4
from S. solfataricus and human Pols iota, kappa and eta). (iv) Structure and function of P450
enzymes in steroid hormone biosynthesis. This project takes advantage of unique variations in the
21A2 (21-hydroxylase) and 17A1 (17α-hydroxylase/17,20-lyase) cytochrome P450 enzymes to
explain their structure/function relationships in detail and thereby establish a better understanding
of the general basis of P450 function. (v) Probing nucleic acid structure with neutron diffraction.
Neutron diffraction offers unique advantages for structural biology because neutrons interact with
nuclei and thus allow a distinction between light elements including H and their various isotopes.
This project will rely on a new instrument at Oak Ridge National Laboratory (Oak Ridge, TN),
the Macromolecular Neutron Diffractometer on the Spallation Neutron Source.
Selected Publications:
1.
2.
3.
4.
5.
6.
7.
8.
A. Patra, M. Paolillo, K. Charisse, M. Manoharan, E. Rozners, M. Egli: 2′-Fluoro RNA shows increased Watson-Crick
H-bonding strength and stacking relative to RNA: evidence from NMR and thermodynamic data. Angew. Chem. Int. Ed.
2012, 51:11863-11866.
M. Egli: The steric hypothesis for DNA replication and fluorine hydrogen bonding revisited in light of structural data.
Acc. Chem. Res. 2012, 45:1237-1246.
R. L. Eoff, C. E. McGrath, L. Maddukuri, S. G. Salamanca-Pinzón, V. E. Marquez, L. J. Marnett, F. P. Guengerich, M.
Egli: Selective modulation of DNA polymerase activity by fixed conformation nucleoside analogues. Angew. Chem. Int.
Ed. 2010, 49:7481-7485.
C. H. Johnson, M. Egli, P. L. Stewart: Structural insights into a circadian oscillator. Science 2008, 322:697-701.
R. Pattanayek, D. R. Williams, S. Pattanayek, T. Mori, C. H. Johnson, P. L. Stewart, M. Egli: Structural model of the
circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation. EMBO J. 2008, 27:17671778.
M. Egli, P. S. Pallan, R. Pattanayek, C. J. Wilds, P. Lubini, G. Minasov, M. Dobler, C. J. Leumann, A. Eschenmoser:
Crystal structure of homo-DNA and nature's choice of pentose over hexose in the genetic system. J. Am. Chem. Soc.
2006, 128:10847-10856.
R. Pattanayek, J. Wang, T. Mori, X. Yu, C. H. Johnson, M. Egli: Visualizing a circadian clock protein: crystal structure
of KaiC and functional insights. Mol. Cell 2004, 15:375-388.
M. Egli, G. Minasov, L. Su, A. Rich: Metal ion coordination and conformational flexibility in a viral RNA pseudoknot at
atomic resolution. Proc. Natl. Acad. Sci. U.S.A. 2002, 99:4302-4307.
Georgia State University
29
30
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Chemistry & Structural Biology of Selenium Nucleic Acids
(SeNA)
Wen Zhang, Sibo Jiang, Jia Sheng, Huiyan Sun, Julianne Caton-Williams, Rob Abdur,
Zhen Huang*
Department of Chemistry & Department of Biology,
Georgia State University, Atlanta, Georgia, USA; email: [email protected]
RNAs play multiple and essential functions in cells and expand dramatically the
complexity of life by serving as genetic information carrier, catalyst, and regulator. RNA
nanotechnology and therapeutics exploration help better understand properties and
behaviors of RNAs in vitro and in vivo. RNA chemical functionalization and structural
study help understanding RNA nanostructures and offer a great opportunity to therapeutic
discovery. 3D structure studies of RNAs and their protein complexes provide novel
insights into these bio-macromolecules. Crystallography is a powerful tool for structure
determination of RNAs and protein-RNA complexes with high resolution. However,
crystallization and phase determination, two major bottle-neck problems, have largely
slowed down structural determination of RNAs and their protein complexes. Our
laboratory has pioneered and developed atom-specific substitution of nucleic acid oxygen
with selenium, which can be used as an atomic probe for structure and function studies of
nucleic acids. As oxygen and selenium are in the same elemental family, the atomspecific substitution by replacing nucleotide oxygen with selenium or tellurium has
revealed novel chemistry, structure, function and mechanism of nucleic acids and their
protein complexes. Our selenium-nucleic acid (SeNA) strategy has demonstrated great
potentials as a general methodology for structure and function studies of RNAs as well as
their protein complexes. Moreover, we find that the Se-derivatized RNAs have virtually
identical structures to the corresponding natives. Furthermore, we found that the Sederivatization can facilitate crystallization, phase determination, and high-resolution
structure determination. This Se derivatization strategy via the atom-specific substitution
will significantly facilitate crystal structure studies of RNAs as well as their protein
complexes. Excitingly, we have recently determined the first RNA/DNA-protein complex
via the nucleic acid Se-derivatization. This work is supported by NIH (R01GM095881
and GM095086) and NSF (MCB-0824837 and CHE-0750235).
Selected Publications:
1.
2.
3.
4.
5.
6.
7.
8.
Jia Sheng, Jianhua Gan, Alexie Soars, Jozef Salon and Zhen Huang*, “Structural Insights of Non-canonical U•U
Pair and Hoogsteen Interaction Probed with Se Atom”, 2013, Nucleic Acids Research, in press.
Jozef Salon, Jianhua Gan, Rob Abdur, Hehua Liu and Zhen Huang*, “Synthesis of 6-Se-Guanosine RNAs for
Structural Study”, Organic Letter, 2013, in press.
Huiyan Sun, Sibo Jiang, Julianne Caton-Williams, Hehua Liu, and Zhen Huang*, “2-Selenouridine Triphosphate
Synthesis and Se-RNA Transcription”, RNA, 2013, 19, 1309–1314.
Wen Zhang, Jia Sheng, Abdalla E. Hassan, and Zhen Huang*, "Synthesis of Novel 2’-Deoxy-5(Methylselenyl)Cytidine and Se-DNAs for Structure and Function Studies", Chemistry-An Asian Journal, 2012, 7,
476-479.
Huiyan Sun, Jia Sheng, Abdalla E. A. Hassan, Sibo Jiang, Jianhua Gan and Zhen Huang*, “Novel RNA Base Pair
with Higher Specificity using Single Selenium Atom”, Nucleic Acids Res., 2012, 40, 5171-5179.
Jia Sheng, Wen Zhang, Abdalla E. A. Hassan, Jianhua Gan, Alexei Soares, Song Geng, Yi Ren, Zhen Huang*,
"Hydrogen Bond Formation between the Naturally Modified Nucleobase and Phosphate Backbone", Nucleic
Acids Research, 2012, 40, 8111-8118.
Lin, L.; Sheng, J.; Huang, Z. Chemical Society Reviews, 2011, 40, 4591.
Sheng, J.; Hassan, A. ; Zhang, W.; Zhou, J.; Xu, B.; Soares, A. S.; Huang, Z. Nucleic Acids Res., 2011, 39, 3962.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Zhen Huang
Prof. Zhen Huang (Ph.D.) was born in 1964 and raised in
Sichuan, China. He received his B.S. from Sichuan University in
1984, M.S. from Peking University in 1987, and Ph.D. from Swiss
Federal Institute of Technology (ETH, Zurich) in 1994 (under the
supervision of Professor Steven Benner). In 1994, he joined the
Department of Genetics at Harvard Medical School as a research
fellow, in Laboratory of Professor Jack Szostak (Nobel Laureate in
Medicine in 2009). He was hired in 1998 by Brooklyn College,
City University of New York, as assistant professor and was later
promoted to associate professor with tenure. In 2004, Dr. Huang
was recruited to Chemistry Department, Georgia State University, is Professor of
Chemistry and Chemical Biology, and is also University Distinguished Professor
Awardee of Georgia State University. He has received several awards, including Georgia
Distinguished Cancer Scientists Award, from The State of Georgia (GCC). He is
also very active in community services: he has served as editors and guest editors for
several journals and books, and is the first President of Chinese-American Chemistry &
Chemical Biology Professors Association (CAPA; also one of the three Co-Founders).
He has pioneered and developed selenium and tellurium derivatizations of nucleic acids
for structure and function studies of nucleic acids, protein-nucleic acid complexes, and
nucleic acid-small molecular ligands (such as anticancer drugs). His current research
interests are in selenium and tellurium derivatizations of DNAs and RNAs for X-ray
crystallographic studies of nucleic acids and protein complexes (especially for Cancer
Research), synthesis of analogs of nucleosides and nucleotides for structure, function
and anticancer studies, development of RNA microchip technology for direct detection
and quantitation of gene expression profile for Cancer Early Detection, nanomaterialassisted novel RNA microchip, modified nucleic acid-based nano-medicine, nucleic acidbased cancer diagnosis, in vitro selection, evolution and characterization of ligandbinding and catalytic RNAs and DNAs. His research has been funded by federal
agencies, including NIH, NSF, DOD and CDC, state funding agencies, the distinguished
cancer scholar award, and private fundings (such as industries). He has received several
US and European patents, and many US and international patents are pending.
9. Caton-Williams, J.; Lin, L.; Smith, M.; Huang, Z. Chemical Communications, 2011, 47, 8142.
10. Abdalla E. A. Hassan, Jia Sheng, Wen Zhang, and Zhen Huang*, "High Fidelity of Base Paring by 2Selenothymidine in DNA", Journal of American Chemical Society, 2010, 132, 2120-2121.
11. Jozef Salon, Jia Sheng, Jianhua Gan, Zhen Huang*, "Synthesis and crystal structure of 2’-Se-modified guanosine
containing DNA", J. Org. Chem., 2010, 75, 637-641.
12. Jia Sheng, Abdalla Hassan, Zhen Huang*, "Synthesis of the First Tellurium-Derivatized Oligonucleotides for
Structural and Functional Studies", Chemistry - A European Journal, 2009, 15, 10210-10216.
13. Julianne Caton-Williams, Zhen Huang*, "Synthesis and DNA Polymerase Incorporation of Colored 4-Selenothymidine
Triphosphate with a Single Atom Substitution", Angewandte Chemie, 2008, 47, 1723-1725.
14. Jozef Salon, Jiansheng Jiang, Jia Sheng, Oksana Gerlits, Zhen Huang*, "Derivatization of DNAs with Selenium at
6-Position of Guanine for Function and Crystal Structure Studies", Nucleic Acids Res, 2008, 36, 7009-7018.
15. Jiang, Sheng, Carrasco, Huang*, “Selenium Derivatization of Nucleic Acids for Crystallography”, Nucleic Acids
Res, 2007, 35, 477-485.
16. Salon, Sheng, Jiang, Chen, Caton-Williams, Huang*, “Oxygen Replacement with Selenium at the Thymidine 4position for the Se-Base-Paring and Crystal Structure Studies”, Journal of Am. Chem. Soc., 2007, 129, 4862-4863.
Georgia State University
31
32
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Designing Chimeric Biomolecule Self-Assemblies
Jillian E. Smith, Jay T. Goodwin, David G. Lynn*
Department of Chemistry & Department of Biology,
Emory University, Atlanta, Georgia, USA; email: [email protected]
Dynamic chemical networks derived from both nucleic acids and amino acids selfassemble into supramolecular structures. Self-assembly is driven by diverse forces,
including entropic, metal-coordination, and hydrogen-bonding interactions, and the
resulting complexes find uses in a variety of contexts (material science, medicine, and
nano-scale electronic devices). Notable among these are systems directed by the
remarkably iconoclastic self-recognition landscape for guanine and its derivatives.
Supramolecular systems have been developed that utilize non-Watson-Crick guanineguanine hydrogen-bonding networks to form G-ribbon or G-quartet-based assemblies.
Similarly, great advances in design and characterization of peptide-based self-assemblies
have allowed researchers to create a vast chemical landscape to facilitate chemical
reactions and build novel biomaterials. Peptides also have a remarkable ability to
organize into a richly diverse landscape of supramolecular assemblies, driven by many of
the same forces that shepherd guanine supramolecular structures. In this presentation, we
will focus on design lessons learned from both guanine-containing nano-architectures and
cross-β peptide assemblies to develop a chimeric molecular network. Our efforts to
better understand the fundamental molecular interactions of these chimeric constructs
will aid our design strategies for creating dynamic combinatorial networks that can
respond to environmental changes, store and process information, and resulting in
emerging new functions.
This work is supported by NSF, DOE, NIH, and HHMI.
Selected Publications:
1.
2.
3.
4.
Liu, P.; Ni, R.; Mehta, A.K.; Childers, W.S.; Lakdawala, A.; Pingali, S.V.; Thiyagarajan, P.; Lynn,
D.G. 2008. Nucleobase-Directed Amyloid Nanotube Assembly, J. Am. Chem. Soc. 130: 16867-16869.
Childers, W. S.; Anthony, N. R.; Mehta, A. K.; Berland, K. M.; Lynn, D.G. 2012 Phase Networks of
Cross-β Peptide Assemblies. Langmuir, 28, 6386-6395. DOI: 10.1021/la300143j
Childers, W.S; Mehta, A.K.; Bui, T.Q.; Liang, Y.; and Lynn, D.G. 2013 Towards Intelligent Materials,
Molecular Self-Assembly: Advances and Applications, Ed. A. Li, Pan Stanford Ltd
Goodwin, J.T.; Mehta, A.K.; Lynn, D.G. 2012 Analog and Digital Chemical Evolution, Accounts of
Chemical Research special edited edition, 45:2189-2199 DOI: 10.1021/ar300214w
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
David Lynn
ACADEMIC POSITION:
Asa Griggs Candler Professor of Chemistry and Biology at Emory University
EDUCATION:
He received his AB degree in chemistry from the University of North Carolina-Chapel
Hill and his PhD in organic/biological chemistry from Duke University. In addition, he
was awarded a National Institutes of Health (NIH) fellowship at Columbia University.
MEMBERSHIPS/AWARDS:
Dr Lynn received the Camille and Henry Dreyfus Teacher-Scholar Award, was awarded
a Sloan Research Fellowship, and was elected chair of the Gordon Conference on
Bioorganic Chemistry. He has served on NIH scientific advisory boards ranging from
genetics to bioorganic and natural products and is on the advisory boards for Amyloid:
The Journal of Protein Folding Disorders and Current Organic Synthesis. He is currently
a HHMI Professor and a Fellow of the American Association for the Advancement of
Science.
RESEARCH INTERESTS:
The David G Lynn Group at Emory University works to understand the structures and
forces that enable supramolecular self-assembly, how chemical information can be stored
and translated into new molecular entities, and how the forces of evolution can be
harnessed in new structures with new function. Such knowledge offers tremendous
promise for discoveries in fields as diverse as drug design and genome engineering,
pathogenesis and genome evolution, functional nanoscale materials and the origins of
living systems.
Georgia State University
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THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Applications of Protein NMR in
Protein Engineering and Design
Gaohua Liu1.2, Nobuyasu Koga3, Rie Tatsumi-Koga3, Rong Xiao1,2, Thomas B. Acton1,2,
Gregory Kornhaber1,2, David Baker3 & Gaetano T. Montelione1,2,4
1
Northeast Structural Genomics Consortium; 2Rutgers, The State University of New Jersey,
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and
Biochemistry, Piscataway, New Jersey 08854, USA; 3University of Washington, Department of
Biochemistry and Howard Hughes Medical Institute, Seattle, Washington 98195, USA;
4
Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway, New
Jersey 08854, USA; e-mail:[email protected]
Structural characterization of designed proteins is a critical step in validating
computational design methodology. Many of the groups involved in computational
protein design have limited resources for 3D structure determination, and structural
genomics platforms are ideally suited for collaborative projects aimed at accelerating the
field. Here we describe a synergy example of structural genomics platform with
designing ideal protein structures stabilized by completely consistent local and non-local
interactions. The design approach is based on a set of rules relating secondary structure
patterns to protein tertiary motifs, which make possible the design of strongly funnelled
protein folding energy landscapes. Guided by these rules, we designed sequences
predicted to fold into ideal protein structures consisting of alpha-helices, beta-strands and
minimal loops. Designs for five different topologies were found to be monomeric and
very stable in solution. The solution structures of all designs were determined in a blind
fashion by using solution-state NMR spectroscopy at Northeast Structural Genomics
Consortium and found to be nearly identical to the computational models. These results
illuminate how the folding funnels of natural proteins arise and provide the foundation
for engineering a new range of functional proteins free from natural evolution. These
NMR experimental structures also provide unique valuable information on how to
improve the protein strategies and how to design more complicated topologies. The NMR
data are available from http://psvs-1_4-dev.nesg.org/ideal_proteins/. This work was
supported also by the National Institutes of General Medical Science Protein Structure
Initiative (PSI:Biology) program, grant U54 GM094597.
Reference:
1.
Koga N, Tatsumi-Koga R, Liu G, Xiao R, Acton TB, Montelione GT, Baker D. Nature. 2012 Nov
8;491 (7423):222-7. Principles for designing ideal protein structures.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Gaohua Liu
Dr. Gaohua Liu was born in 1972 in Jiangxi, China. He received
his B.S. degree from Nanjing University in 1991 and Ph.D. degree
from Nanjing University (Nanjing, China) in 1996 (under the
supervision of Professor Wenxia Tang). He did postdoctoral
researches in the Center of Magnetic Resonance at Florence
University in Italy with Professors Ivano Bertini and Claudio
Luchinat from 1996 to 2000; and in the department of Structural
Biology at St. Jude Children’s Research Hospital in Memphis with
Dr. Jie Zheng from 2000 to 2002. He has been hired by Northeast
Structural Genomics Consortium (NESG) since 2002, first in Prof.
Thomas Szypersky’s lab at SUNY, Buffalo, and later in Prof. Gaetano Montelion’s lab at
Rutgers University where he is currently working as a Research Assistant Professor. Dr.
Liu’s research activities are focus on three-dimensional solution structures of proteins by
NMR, he has solved and deposited nearly 100 protein NMR structures into the Protein
Data Bank and contributed to more than 50 publications. His current research interests
are in structural genomics and structural biology by NMR on protein and protein
complexes.
Selected Publications:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Koga N, Tatsumi-Koga R, Liu G, Xiao R, Acton TB, Montelione GT, Baker D. Nature. 2012 Nov 8;491
(7423):222-7. Principles for designing ideal protein structures.
Thompson JM, Sgourakis NG, Liu G, Rossi P, Tang Y, Mills JL, Szyperski T, Montelione GT, Baker D.,
Proc Natl Acad Sci U S A. 2012 Jun 19;109(25):9875-80. Accurate protein structure modeling using sparse
NMR data and homologous structure information.
Rosato A, Aramini JM, Arrowsmith C, Bagaria A, Baker D, Cavalli A, Doreleijers JF, Eletsky A, Giachetti
A, Guerry P, Gutmanas A, Güntert P, He Y, Herrmann T, Huang YJ, Jaravine V, Jonker HR, Kennedy MA,
Lange OF, Liu G, Malliavin TE, Mani R, Mao B, Montelione GT, Nilges M, Rossi P, van der Schot G,
Schwalbe H, Szyperski TA, Vendruscolo M, Vernon R, Vranken WF, Vries Sd, Vuister GW, Wu B, Yang
Y, Bonvin AM. Structure. 2012 Feb 8;20(2):227-36. Blind testing of routine, fully automated determination
of protein structures from NMR data.
Liu, G.; Huang, Y.J.; Xiao, R.; Wang, D.; Acton, T.B.; Montelione, G.T. PROTEINS: Struct. Funct.
Bioinformatics 2010, 78:2170-5. Solution NMR structure of the ARID domain of human AT-rich interactive
domain-containing protein 3A: a human cancer protein interaction network target.
Raman, S.; Lange, O.F.; Rossi, P.; Tyka, M.; Wang, X.; Aramini, J.; Liu, G.; Ramelot, T.A.; Eletsky, A.;
Szyperski, T.; Kennedy, M.A.; Prestegard, J.; Montelione, G.T.; Baker, D. Science 2010, 327:1014-8. NMR
structure determination for larger proteins using backbone-only data.
Liu, G.; Huang, Y.J.; Xiao, R.; Wang, D.; Acton, T.B.; Montelione, G.T. PROTEINS: Struct. Funct.
Bioinformatics 2010, 78:1326-30. NMR structure of F-actin-binding domain of Arg/Abl2 from Homo
sapiens.
Raman S, Huang YJ, Mao B, Rossi P, Aramini JM, Liu G, Montelione GT, Baker D. J Am Chem Soc. 2010,
132(1):202-7. Accurate automated protein NMR structure determination using unassigned NOESY data.
Shen, Y.; Lange, O.; Delaglio, F.; Rossi, P.; Aramini, J.M.; Liu, G.,Eletsky, A.; Wu, Y.; Singarapu, K.K.;
Lemak, A.; Ignatchenko, A.; Arrowsmith, C.H.; Szyperski, T.; Montelione, G.T.; Baker, D.; Bax, A. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105:4685-90. Consistent blind protein structure generation from NMR
chemical shift data.
Liu, G.; Shen, Y.; Atreya, H.S.; Parish, D.; Shao, Y.; Sukumaran, D.K.; Xiao, R.; Yee, A.; Acton, T.B.;
Arrowsmith, C.H.; Montelione, G.T.; Szyperski, T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102: 10487-10492.
NMR data collection and analysis protocol for high-throughput protein structure determination.
Georgia State University
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36
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
RNA Synthesis in Reverse Direction and Application in Convenient
Introduction of Ligands, Chromophores and Modifications of Synthetic
RNA at the 3’- End and Highly Efficient Synthesis of Long RNA
Suresh C. Srivastava, ChemGenes Corporation, 33 Industrial Way, Wilmington,
MA 01887; [email protected]
We have synthesized and studied the coupling efficiency of 3’- DMT -5’- CED
phosphoramidites. The coupling efficiency per step surpasses 99% in the reverse
direction synthesis methodology, leading to high purity RNA in a large number of
homopolymers, 20-21 mers and long chain oligonucleotides. The data clearly indicate a
drastic improvement of coupling efficiency per step of the new monomers during oligo
synthesis using the reverse RNA monomers (for 5’3’- direction) as compared to
standard 3’- CED phosphoramidites in synthesis in 3’5’ direction, i.e., the conventional
method. Besides the improvement in coupling efficiency, which results in very high
quality of oligonucleotides, these synthons provide method for synthesis of ribonucleic
acid oligomers with modification or labeling of 3’- end of an oligonucleotide. The
synthesis of 3’- end modified RNA requiring lipophilic, long chain ligands or
chromophores fluorophores and quenchers can be performed using the new synthons
along with the ligand –chromphore phosphoramidite as last base coupling at the 3’- end
terminal. Our data, as captured in Figures 11 and 12, show that 5’3’- direction
synthesis has very distinct advantage compared to conventional method. In addition, we
observed almost complete absence of M+1 in reverse RNA synthesis methodology
consistently even when the last amidite was a macromolecule and this resulted in very
high purity of HPLC purified and 3’- modified oligonucleotides. This method of RNA
synthesis is expected to be very useful and a practical method of choice.
We have further extended the reverse RNA synthesis technology in smooth synthesis of
long RNA’s of high purity. It has been possible to easily obtain long RNA’s of well over
100-mer with this technology. Various details and analytical data will be presented.
Selected Publications (Patents or Patent Applications in Nucleic Acid Field (Selected 7 out of 14):
1. Suresh C. Srivastava, Satya P. Bajpai and Sant K. Srivastav: Nucleosides and Oligonucleotides
for reversal of Cytotoxic and mutagenic damage of DNA.
2. Appl. US 20120149888; Published 07-14-2012: Suresh C. Srivastava, Divya Pandey, Naveen P.
Srivastava & Alok Srivastava: Synthesis of Ara-2’-Omethyl nucleosides, corresponding
phosphoramidites and oligonucleotides incorporating novel modifications for biological applications
in therapeutics, diagnostics, G- tetrad forming oligonucleotides and aptamers.
3. PCT. Prov. 61/795851: Dithiolane Based thio modifiers for labeling and strong immobilization
of bio-molecules on solid surfaces.
4. US20120058476; Published 03-08-2012: Andrei Laikhter, Suresh C. Srivastava, Naveen P.
Srivastava. Labeling of oligonucleotides with reporter moieties using cycloaddition reaction.
5. US7956169; published 06-07-2011: Synthesis of novel azo-dyes as fluoresce quenchers and their use
in oligonucleotide synthesis. US 20110040082; Published 02-17-2011: Suresh C. Srivastava, Satya P.
Bajpai, Kwok- Hung Sit: Modification of antimetabolite gemcitabine for incorporation in CpG
oligonucleotides.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Suresh C. Srivastava
Dr. Suresh C. Srivastava is founder & president of ChemGenes
Corp. USA. Dr. Srivastava received his Ph.D. in organic chemistry
from Lucknow University, India in 1968. Dr. Srivastava continued
research in synthetic organic chemistry for additional two years at
Central Drug Research Institute, Lucknow India. He moved to
United States in year 1970 as Cancer Research Scientist at Roswell
Park Memorial Institute, Buffalo, New York and carried out
research and development in anticancer therapeutics research.
Susequently in year 1972, Dr. Srivastava moved to Research
Triangle Institute, Raleigh, North Carolina, USA as scientist and carried out synthetic
efforts in total synthesis of a terpenoid molecule called a Strigol. After one year Dr.
Srivastava took research scientist position at Purdue University, Lafayette, Indiana , USA
and stayed there till year 1976. At Purdue University, Deparment of Chemistry as well as
Department of Medicinal Chemistry and carried out research in antibiotics, Mitomycin;
synthetic and mechanistic aspect in organo palladium chemistry. Subsequently Dr.
Srivastava moved to Boston Biomedical Research Institute as Staff Scientist and stayed
there till year 1981 and carried out extensive chemistry of nucleosides and synthesized a
number of oligonucleotides, utilizing phosphodiester and phosphotriester methodologies.
In the year 1981, Dr. Srivastava founded ChemGenes Corporation, a biotechnology
company. The company has been in operation since then. Currently located in
Wilmington, Massachusetts, has been a strong partner to researchers engaged in the field
of DNA/RNA synthesis for almost 30 years.
ChemGenes, the industry leader in Oligonucleotide Reagent manufacturing, high quality
phosphoramidites and solid Supports in the market. ChemGenes facilities setup for
therapeutic grade phosphoramidites and DNA/RNA synthesis products suitable for GMP
grade oligonucleotide manufacturing. ChemGenes’ product lines include
phosphoramidites for DNA and RNA synthesis, antisense phosphoramidites, modified
bases for DNA and RNA modification. In addition, ChemGenes produces a variety of
modified phosphoramidites for the introduction of chromophores and ligands. The
availability of high quality solid supports, prepacked disposable columns of various pore
sizes, loadings, low volume columns, ancillary reagents in configurations suitable for
each synthesizer, and DNA purification cartridges has vastly increased growth of
synthetic oligonucleotides rapidly.
6.
7.
Method Of The Synthesis of 2’,3’- AND 3’,5’-Cyclic Phosphate Mono and Oligonucleotides:
Andrei Laikhter, Suresh C. Srivastava and Naveen P. Srivastava.
WO 2011103468; Published 12-23-2010;Suresh C. Srivastava, Divya Pandey, Naveen P.
Srivastava & Satya P. Bajpai.: RNA synthesis –Phosphoramidites for synthetic RNA in the reverse
direction and application in convenient introd. of ligands, chromophores & modifications of
synthetic RNA at the 3’-End.
Georgia State University
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38
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Automated Crystallographic Structure
Determination in PHENIX
Li-Wei Hunga, Paul D. Adamsb,c, Pavel V. Afonineb, Gábor Bunkóczic, Lindsay Deise, Nathaniel Echolsb,
Bradley Hintzee, Jeffrey J. Headde, Swati Jain e, Gary J. Kaprale, Ralf W. Grosse-Kunstleveb, Airlie J.
McCoyd, Nigel W. Moriartyb, Robert Oeffnerd, Randy J. Readd, David C. Richardsone, Jane S. Richardsone,
Thomas C. Terwilligerea, Christopher Williamse, and Peter H. Zwartb
a
Los Alamos National Laboratory, Los Alamos, NM 87545, USA, bLawrence Berkeley National
Laboratory, Berkeley, CA 94720, USA, cDepartment of Bioengineering, UC Berkeley, CA 94720, USA,
d
Department of Haematology, University of Cambridge, Cambridge, England, eDepartment of
Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
Macromolecular X-ray crystallography is a critical tool in the study of biological processes at a
molecular level. Significant time and effort are often required to achieve structural solutions of
many macromolecular structures because of the need for manual interpretation of complex
numerical data using many different software packages, and the repeated use of interactive threedimensional graphics. The PHENIX software package has been developed to provide a
comprehensive system for macromolecular crystallographic structure solution with an emphasis
on automation. This has required the development of new algorithms that minimize or eliminate
subjective input in favor of built-in expert-systems knowledge, the automation of procedures that
are traditionally performed by hand, and the development of a computational framework that
allows a tight integration between the algorithms. The application of automated methods is
particularly appropriate in the field of structural proteomics, where high throughput is desired.
Automation also encourages researchers to test unconventional structural determination
strategies, and enables aggressive exploration of parameter spaces for difficult cases. Features in
PHENIX for the automation of experimental phasing with subsequent model building, molecular
replacement, structure refinement, completion, and validation as well as examples of running
PHENIX from both the command line and graphical user interface will be presented.
This work is supported by NIH (Grant No. P01GM063210) and the Phenix Industrial
Consortium
Selected Publications:
1.
2.
3.
4.
Terwilliger TC, Read RJ, Adams PD, Brunger AT, Afonine PV, Grosse-Kunstleve RW, Hung LW. " Improved
crystallographic models through iterated local density-guided model deformation and reciprocal-space
refinement." Acta Crystallogr D Biol Crystallogr., Jul;68(Pt 7):861-70 (2012)
Adams PD, Afonine PV, Bunkóczi G, Chen VB, Echols N, Headd JJ, Hung LW, Jain S, Kapral GJ, Grosse
Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner RD, Read RJ, Richardson DC, Richardson JS, Terwilliger TC,
Zwart PH. “The Phenix software for automated determination of macromolecular structures.” Methods. Jul 29.
(2011)
Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, GrosseKunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC,
Zwart PH "PHENIX: a comprehensive Python-based system for macromolecular structure solution.", Acta
Crystallogr D Biol Crystallogr., 66:213-21 (2010)
Terwilliger TC, Adams PD, Read RJ, McCoy AJ, Moriarty NW, Grosse-Kunstleve RW, Afonine PV, Zwart PH,
Hung LW "Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol
wizard." Acta Crystallogr D Biol Crystallogr. 65:582-601(2009)
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Li-Wei Hung
Dr. Li-Wei Hung received his Ph. D. in Biophysics from the
University of California, Berkeley in 1997. He has been one of the
PHENIX developers since the inception of the project in 2001. Dr.
Hung developed de novo structure determination pipelines in early
part of the PHENIX project, and has developed automated ligand
identification algorithm in PHENIX to automatically interpret
unknown electron densities in the process of structure completion.
Dr. Hung's work in structural biology encompasses membrane
protein structure, RNA crystallography, small-angle X-ray
scattering, computational protein modeling, and crystallographic
methods development. Dr. Hung's research interests in biological sciences have been
focused on structures of ABC transporters and multidrug resistant proteins. Dr. Hung
received the Federation of European Biochemical Societies (FEBS) Special Young
Investigator’s Award in 1999 for his work in structural studies of the first ABC
transporter protein determined by X-ray crystallography. He has been a Staff Member of
Los Alamos National Laboratory (LANL), and the team leader of LANL's highthroughput crystallization and X-ray data collection facilities since 2001. Dr. Hung has
authored over 50 papers in peer-reviewed journals including lead authorship in Nature
and the Proceedings of the National Academy of Sciences.
5.
6.
7.
Terwilliger TC, Grosse-Kunstleve RW, Afonine PV, Moriarty NW, Zwart PH, Hung LW, Read RJ, Adams PD.
“Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard.”
Acta Crystallogr D Jan;64(Pt 1):61-9.(2008)
Zwart PH, Afonine PV, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, McKee E, Moriarty NW, Read
RJ, Sacchettini JC, Sauter NK, Storoni LC, Terwilliger TC, Adams PD. “Automated structure solution with the
PHENIX suite.” Methods Mol Biol. ;426:419-35 (2008)
Terwilliger TC, Grosse-Kunstleve RW, Afonine PV, Adams PD, Moriarty NW, Zwart P, Read RJ, Turk D, Hung
LW. “Interpretation of ensembles created by multiple iterative rebuilding of macromolecular models.” Acta
Crystallogr D Biol Crystallogr., 63:597-610. (2007)
Georgia State University
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40
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
RNA and Protein - a match made in the Hadean
Loren Dean Williams
The Center for Ribosomal Origins and Evolution
School of Chemistry and Biochemistry
Georgia Institute of Technology, Atlanta, GA, 30332
Biological systems record historical information, as seen in the growth rings of trees. On
a molecular level, records are detailed and extensive, connecting us to the pre-history of
biology (the origin of life). The most ancient macromolecules in biology are found in the
ribosome, which is the RNA-protein complex responsible for the synthesis of all coded
protein in all living organisms. The catalytic core of the ribosome a deeply-frozen
molecular fossil that is older than modern biology. The origins and early development of
the ribosome, billions of years ago, remain firmly imprinted in the biochemistry of extant
life. The ribosome tells us part of the story of the origin of life and of the earliest
biochemistry. The information contained within the ribosome guides our laboratory in
experimentally recapitulating critical chemical and biochemical steps in the origin and
early evolution of life.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Loren Dean Williams
Loren Williams was born in Seattle, Washington. In 1981 he
received his B.Sc. in Chemistry from the University of
Washington. In 1985 he received his Ph.D. in Physical Chemistry
from Duke University. He was an American Cancer Society
Postdoctoral Fellow first at Duke then at Harvard. From 1988 to
1992 he was an NIH Postdoctoral Fellow in the laboratory of Alex
Rich in the Department of Biology at MIT. He joined the School of
Chemistry and Biochemistry at Georgia Tech in 1992. Currently he
is director of a NASA Astrobiology Institute funded center
focusing on the transition from nucleic acid-based life to proteinbased life. This transition was made by the macromolecular machine responsible for the
synthesis of proteins, called the ribosome. The collective scientific goal of the Georgia
Tech Astrobiology Center is to chemically rewind the "tape of life" to before the last
universal common ancestor (LUCA) of all living organisms.
Georgia State University
41
42
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Facilitation of DNA Crystallization by Selenium
Functionalization
Wen Zhang and Zhen Huang
Department of Chemistry, Georgia State University,
Atlanta, Georgia, USA; email: [email protected]
The elucidation of DNA and RNA structures by X-ray crystallography contribute to the
understanding of molecular mechanism of DNA and RNA functions. Beside the phase
determination, crystallization is the other long-standing challenge in nucleic acid X-ray
crystallography. Our lab has developed the novel approach to systematically synthesize
Se-DNA and Se-RNA (SeNA), in which the selenium element is able to provide the
rational power to solve phase problem. More interestingly, our unique selenium
mutagenesis offered the unique solution to facilitate crystallization and promote highquality structure determination. We have experimentally and computationally
investigated the mechanistic insight of the DNA crystallization facilitated by the Semodification. We have discovered that the intramolecular and intermolecular stacking
interactions mediated by the Se-functionalization have significantly increased DNA
duplex stability and reduced DNA flexibility and molecular dynamics, which may play
critical roles in enhancing molecular packing and DNA nucleation in crystallization. The
combination of these factors may broaden the crystallization conditions and facilitate the
growth and quality of crystals. Our novel discoveries suggest that in addition to phase
determination, the Se-derivatization has great potential for crystallization in DNA and
DNA-ligand structure study.
This work is supported by NIH (R01GM095881).
Selected Publications:
1. Wen Zhang, Zhen Huang, “DNA Crystallization Facilitated by Selenium-nucleobase Stacking”,
submitted
2. Wen Zhang, Jia Sheng, Abdalla E. A. Hassan and Zhen Huang, “Synthesis of Novel 2’-Deoxy-5(Methylselenyl)Cytidine and Se-DNAs for Structure and Function Studies”, Chemistry-An Asian Journal,
2012, 7, 476–479.
3. Wen Zhang and Zhen Huang, “Synthesis of the 5’-Se-Thymidine Phosphoramidite and Convenient
Labeling of Oligonucleotides”, Organic Letters 2011, 13, 2000-2003.
4. Wen Zhang and Zhen Huang, “Sulfur, Selenium and Tellurium Derivatized Nucleic Acids”, Medicinal
Chemistry of Nucleic Acids (John Wiley & Sons, Inc.), 2011, 101-141.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Wen Zhang
Dr. Wen Zhang is currently a postdoc associate at department of
chemistry, Georgia State University. He received his B.S. degree
from Tianjin University, China in Pharmaceutical Science at 2006
(under the supervision of Professor Jinfeng Wang) and Ph.D.
degree from Georgia State University in Chemistry at 2012 (under
the supervision of Professor Zhen Huang). He has turned postdoc
associate in Professor Zhen Huang’s lab since 2012 to carry out his
postdoctoral research. Dr. Zhang’s research deals with the organic
synthesis, structural biology and molecular biology of selenium
modified nucleosides and nucleic acids. His current research
interest focuses on the Se-DNA and Se-RNA design and synthesis for nucleic acidprotein complex X-ray structural determination and development of novel nucleic acid
therapeutics for disease treatment. Dr. Zhang has published more than 10 scientific
papers in international peer-reviewed journals, and received several patents based on the
novel selenium-DNA research.
Georgia State University
43
44
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Structural and Dynamic Aspects of DNA Recognition
Chris Johnson, Michael Rettig Alex Spring, David Wilson, Markus W. Germann*
Department of Chemistry & Department of Biology,
Georgia State University, Atlanta, Georgia, USA; email: [email protected]
DNA exhibits a remarkably polymorphism that ranges from single to multistranded forms
including parallel stranded segments. These structures have different base paring
schemes and present different determinants for recognition. In addition, it has become
apparent that the microstructural variations and subtle conformational features present are
essential for specific DNA recognition. The extensive structural repertoire presents both
a challenge and an opportunity for specifically targeting DNA. Moreover, DNA is
intrinsically flexible and dynamic which is important for recognition by DNA servicing
proteins and also for the design of DNA binding ligands. Our laboratories have studied
the effects of DNA mismatches or alpha anomeric lesions on the recognition by
MSH2/MSH6 and endonculease IV proteins. This work showes that both preformed
structural features, in the case of alpha anomeric damage, as well as enhanced flexibility
are utilized to localize DNA damage. Minor groove specific ligands such as netropsin,
also bind to a preformed binding site that require only minor adjustment, or, depending
on the sequence and its malleability may significantly adapt the minor groove topology
and DNA bending. Our work sheds light on the dynamic behavior of the bound netropsin
molecule. Specifically, we show that the ligand is rapidly flipping between two
orientations while in close association with the DNA. The ligand reorientation is
believed to contribute favorably to the binding thermodynamics.
Selected Publications:
1.
2.
3.
4.
5.
6.
Rettig, M., Germann, M.W., Wang, S. & Wilson, W. D.: “Molecular basis for sequence-dependent
induced DNA bending” ChemBioChem (2013) DOI: 10.1002/cbic.201200706
Rettig, M., Germann, M. W., Ismail, M. A. Batista-Parra, A., Munde, M., Boykin, D. W., Wilson, W.
D.: “Microscopic Rearrangement of Bound Minor Groove Binders Detected by NMR” Journal of
Physical Chemistry B. (2012), 116, 5620-5627. DOI: 10.1021/jp301143e. PMID: 22530735
Johnson, C. N., Spring, A. M., Desai, S., Cunningham, R. P. & Germann, M. W.:“DNA sequence
context conceals  anomeric lesions.” J. Mol. Biol. (2012) 416, 425-437. DOI:
10.1016/j.jmb.2011.12.051
Johnson, C. N., Spring, A. M., Shaw, B. R & Germann, M. W.: Structural Basis of the RNase H1
Activity on Stereo Regular Borano Phosphonate DNA/RNA Hybrids. Biochemistry, 50, 3903-3912,
(2011). DOI: 10.1021/bi200083
Mazurek, A., Johnson C. N., Germann, M. W. & Fishel, R.: "The Role of Nearest Neighbor Sequence
Context in Mismatch Recognition by hMSH2-hMSH6". PNAS (2009) 106, 4177-4182.
Aramini, J. M., Cleaver, S. H., Pon, R. T., Cunningham, R. P. & Germann M. W.: Solution Structure
of a DNA Duplex Containing a
-Anomeric Adenosine: Insights into Substrate Recognition by
Endonuclease IV. J. Mol. Biol. (2004), 338, 77-91. DOI: 10.1016/j.jmb.2004.02.035
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Markus W. Germann
Markus W. Germann was born in 1959 in Moutier, Switzerland.
Following an apprenticeship with Ciba-Geigy in Basel, he earned
his MS in chemical engineering from the polytechnic in
Winterthur. He obtained his PhD in nucleic acid biochemistry
under the direction of Professor Hans van de Sande in 1989 from
the University of Calgary, Canada. Subsequently he did post
doctoral work on NMR structure determination with Professor
Hans Vogel at the University of Calgary and joined BrukerSpectrospin in Switzerland as an NMR application specialist in
1991. He returned to academia in 1993 where he joined the faculty
at Thomas Jefferson University in Philadelphia as an assistant Professor. He was
promoted to associate Professor in 2001 and in the same year he moved to Georgia State
University as a Georgia State Distinguished Cancer Scientist (Georgia Cancer
Coalition). He was he was promoted to Professor in 2006. His research interests include
DNA damage and repair, macromolecular structures and dynamics and design of antiviral
agents.
Georgia State University
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THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Mannosylated bioreducible nanoparticle-mediated
macrophage-specific TNF-α RNA interference for
IBD therapy
Bo Xiao1*, Hamed Laroui1, Saravanan Ayyadurai1, Emilie Viennois1,2, Moiz A.
Charania1, Yuchen Zhang1, Didier Merlin1,2
1
Department of Biology and Chemistry, Center for Diagnostics and Therapeutics,
Georgia State University, Atlanta, 30302, USA; 2Atlanta Veterans Affairs Medical
Center, Decatur, 30033, USA; email: [email protected]
The application of RNA interference (RNAi) for inflammatory bowel disease (IBD)
therapy has been limited by the lack of non-cytotoxic, efficient and targetable small
interfering RNA (siRNA) carriers. TNF-α is the major pro-inflammatory cytokine mainly
secreted by macrophages during IBD. Here, a mannosylated bioreducible cationic
polymer (PPM) was synthesized and further spontaneously assembled nanoparticles
(NPs) assisted by sodium triphosphate (TPP). The TPP-PPM/siRNA NPs exhibited high
uniformity (polydispersity index = 0.004), a small particle size (211−275 nm), excellent
bioreducibility, and enhanced cellular uptake. Additionally, the generated NPs had
negative cytotoxicity compared to control NPs fabricated by branched polyethylenimine
(bPEI, 25 kDa) or Oligofectamine (OF) and siRNA. In vitro gene silencing experiments
revealed that TPP-PPM/TNF-α siRNA NPs with a weight ratio of 40:1 showed the most
efficient inhibition of the expression and secretion of TNF-α (approximately 69.9%,
which was comparable to the 71.4% obtained using OF/siRNA NPs), and its RNAi
efficiency was highly inhibited in the presence of mannose (20 mM). Finally, TPPPPM/siRNA NPs showed potential therapeutic effects on colitis tissues, remarkably
reducing TNF-α level. Collectively, these results suggest that non-toxic TPP-PPM/siRNA
NPs can be exploited as efficient, macrophage-targeted carriers for IBD therapy.
This work was supported by grants from the Department of Veterans Affairs and
the National Institutes of Health of Diabetes and Digestive and Kidney by the grant
RO1-DK-071594 (to D.M).
Selected Publications:
1.
2.
3.
4.
Bo Xiao*, Hamed Laroui, Saravanan Ayyadurai, Emilie Viennois, Moiz A. Charania, Yuchen Zhang,
Didier Merlin. Mannosylated bioreducible nanoparticle-mediated macrophage-specific TNF-α RNA
interference for IBD therapy. Biomaterials, 2013, 34, 7471-7482.
Yuchen Zhang, Emilie Viennois, Bo Xiao, Mark T. Baker, Stephen Yang, Ijeoma Okoro, Yutao Yan*.
Knockout of Ste20-Like Proline/Alanine-Rich Kinase (SPAK) Attenuates Intestinal Inflammation in
Mice. Am. J. Pathol., 2013, 182, 1617-1628.
Bo Xiao*, Didier Merlin. Oral colon-specific therapeutic approaches toward treatment of inflammatory
bowel disease. Expert Opin. Drug Deliv., 2012, 9, 1393-1407.
Bo Xiao, Xiaoyu Wang, Zhiye Qiu, Jun Ma, Lei Zhou, Ying Wan*, Shengmin Zhang*. Synthesis of a
safe dual functionally modified chitosan derivative for efficient liver-targeting gene transfer. J. Biomed.
Mater. Res. A., 2013, 101A, 1888-1897.
Georgia State University
THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Bo Xiao
Dr. Bo Xiao was born in 1984 and raised in Hubei, China. He
received his B.S. degree from Southwest University (China) in
2006, M.S. and Ph.D. from Huazhong University of Science and
Technology (HUST) in respective 2008 and 2011 (under the
supervision of Professor Ying Wan and Professor Shengmin
Zhang). In 2011, he joined Dr. Didier Merlin’s lab in Department
of Biology at Georgia State University as a Postdoctoral Research
Associate. He has received several awards, including Excellent
Thesis Scholarship (HUST) and American Heart Association
(AHA) Award. His current research interests are in synthesis of
novel environment-sensitive polymers, targeted siRNA/plasmid/drug delivery for
inflammatory bowel disease, colon cancer and atherosclerosis therapy, as well as
inflammation and cancer imaging. He has applied several Chinese and international
patents.
5. Moiz A. Charania*, Saravanan Ayyadurai, Sarah A. Ingersoll, Bo Xiao, Emilie Viennois, Yutao Yan,
6.
7.
8.
Hamed Laroui, Shanthi V. Sitaraman, Didier Merlin. Intestinal epithelial CD98 synthesis specifically
modulates expression of colonic microRNAs during colitis. Am. J. Physiol. Gastrointest. Liver Physiol.,
2012, 302, G1282-1291.
Bo Xiao, Ying Wan, Xiaoyu Wang, Haomin Liu, Zhiye Qiu, Shengmin Zhang*. Synthesis and
characterization of N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride for potential
application in gene delivery. Colloids Surf. B: Biointerfaces, 2012, 91, 168-174.
Bo Xiao, Ying Wan, Maoqi Zhao, Yiqun Liu, Shengmin Zhang*. Preparation and characterization of
antimicrobial chitosan-N-arginine with different degrees of substitution. Carbohydr. Polym., 2011, 83,
144-150.
Ying Wan*, Bo Xiao, Siqi Dalai, Xiaoying Cao, Quan Wu. Development of polycaprolactone/chitosan
blend porous scaffolds. J. Mater. Sci.: Mater. Med., 2009, 20, 719-724.
Georgia State University
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THE 3RD INTERNATIONAL CONFERENCE ON NUCLEIC ACID-PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY FOR DRUG DISCOVERY
Conference Sponsors:
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Lab Scientific Group
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Professors Association)
JAN SCIENTIFIC
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ChemGenes
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MiTeGen
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C
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P
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SeNA
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Center for Diagnostics and Therapeutics (CDT)
Chemistry Department
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Molecular Basis of Disease (MBD)
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Georgia State University