Non-enzymatic Copying of Nucleic Acid Templates
Jack W. Szostak
HHMI, MGH, HMS
Schematic Model of a Protocell
Mansy et al., Nature, 2008
Nucleic Acid Replication Needed to Code for Heritable Functions
Enzymatic vs. Chemical Replication
Rate and Fidelity must exceed
critical thresholds
O
O
-
O
P
-
O
O
O
-
O
P
O
N
-
P O
O
N
N
O
N
H
NH2
O
OH OH
‘Modern’ substrates
Very polar
O
C H3
N
N
N
OP O
O
N
N
N
H
NH2
O
OH OH
Prebiotic model substrates
Less polar
More membrane permeable
Sugars: ‘oligomers’ of formaldehyde
HO
OH
O
H
OH
OH
H
C
O
ribose
formaldehyde
Purines: ‘oligomers’ of cyanide
H
H
H
N
N
N
H
N
H
N
H
C
N
Cytosine = Cyanoacetylene + Urea
cytosine
O
NH2
H20
HN
O
N
HN
N
H
O
N
cyanoacetylene
uracil
NH2
O
NH2
New approach to pyrimidine synthesis HO
O
OH
OH
NH2
N
OH
N
H
O
NH2
N
N
HO
O
O
OH
OH
NH2
N
O
Polymerization of ImpNs on Clay
Ferris JP, Hill AR Jr, Liu R, Orgel LE. Nature. 1996, 381(6577):59-61.
Polymerization of ImpNs in an Ice Eutectic Phase
27 µ
Kanavarioti A, Monnard PA, Deamer DW.
Astrobiology. 2001;1(3):271-81.
RNA: spontaneous primer-extension
O
N
CH3
N
N
NH
O
P
N
O
O
N
NH2
O-
OH
OH
5'-r-GCUGCCAGUG
3'-d-CGACGGTCAC-C-CCTTGAG
MgCl2
5'-r-GCUGCCAGUG-GGG
3'-d-CGACGGTCAC-C-CCTTGAG
Challenges for Chemical Replication of RNA
rate
fidelity - stalling effect
regiospecificity
monomer concentration, purity
monomer hydrolysis, cyclization
reactivation chemistry
Mg2+ concentration
high Tm
rapid strand reannealing
primer-free copying
It seems likely that informational
replication will be achieved in the
next decade, and that it will throw
new light on the origins of life.
Leslie Orgel, 1992
Phosphoramidate-linked Nucleic Acids
2'-NP-GNA
3'-NP-DNA
2'-NP-DNA
N
N
O
O
P
O
O
O
NH
NH
O
N
O
N
O-
P
O
O
NH
O-
N
P
O
NH
N
O-
O
P
O
O
NH
N
O-
P
O
O
O
NH
O-
O
N
O-
P
O
O
NH
O
N
O
O
NH
O
O-
P
O
P
O
NH
O-
O
P
O
O-
Phosphoramidate-linked Nucleic Acids
2'-NP-TNA
3'-NP-MNA
N
O
O O
N
O
N
HN
O
P
N
O-
O
P
OH
O
O O
N
O
N
HN
O
P
N
O-
O
O O
P
OH
O
N
O
HN
O
P
O
N
OO
P
O
O-
2’-NP-GNA is structurally simple, but…
O
2'-NP-GNA
N
NH
O
O
N
N
P
O
O-
NH
O
P
N
N
O-
NH2
O
NH
O
P
N
O-
O
NH
O
P
O
O-
O
Activated GNA monomers cyclize rapidly
O
NH
O
N
N
P
N
O
O
ONH2
-  polymerization could be
very fast given optimal
positioning of nucleophile
and electrophile Template-directed non-enzymatic synthesis: advantages of 2’-NH2, 2’,3’-dideoxyribo-monomers
O
N
2'-NP-DNA
O
N
N
O
N
P
O
N
O
O
ONH2
NH
O
P
O
NH
O-
N
O
NH
O
P
O
N
OO
NH
O
P
O
O-
N
NH2
Efficient copying of a C15 DNA
Template with 2ʹ′-NH2 ImpdG O
N
NH
O
N
N
P
O
N
O
ONH2
N
NH2
Efficient copying of a C15 DNA
Template with 2ʹ′-NH2 ImpdG Time (hr)
+15
Primer
0 1
3
6
12 24
A - U
NH2
N
N
N
O
HN
N
N
O
D - Up
NH2
N
N
N
O
HN
N
N
NH2
O
Copying D4 and Up4 Templates with 2’-NH2-ImpddUp and 2’-NH2-ImpddD O
NH
O
N
N
N
P
O
O
ONH2
O
Copying D4 and Up4 Templates with 2’-NH2-ImpddUp and 2’-NH2-ImpddD NH2
N
N
O
N
N
P
O
N
O
ONH2
N
NH2
Copying mixed sequence RNA Templates
5’Cy3GCGTAGACTGACTGCNH2!
3’-CGCAUCUGACUGACGDDDCCC !
******!
p p p!
5’Cy3GCGTAGACTGACTGCUUUGGGNH2!
3’-CGCAUCUGACUGACGDDDCCC !
+ G Up
-G -Up!
min: 0 60 180 0 180 180!
+6
+3
Schrum et al., JACS, 2009
Copying mixed sequence RNA Templates
Cy3 5’
CCGCCGCCGA!
Copying mixed sequence RNA Templates
Cy3 5’
p
GCDCDDCUGDCCDDC!
How to solve both rate and fidelity problems?
-  different nucleobase(s)
-  different backbones (conformational constraint) -  oligonucleotide substrates
-  catalysis
D - Up
NH2
N
N
N
O
HN
N
N
NH2
O
A – 2-thio-U
NH2
N
N
N
O
HN
N
N
S
Template-directed non-enzymatic synthesis: 3’-amino, 2’-3’ dideoxyribo-nucleotides
3'-NP-DNA
N
O
O
NH
O
P
O-
N
O
O
NH
O
N
O-
P
O
O
NH
O
P
O
O-
Template-directed non-enzymatic synthesis: 3’-amino, 2’-3’ dideoxyribo-nucleotides
NH2
N
O
N
N
N
P
O
O
O
ONH2
- 3’-5’ linkages formed, as in RNA
- monomers do cyclize, but slowly
- duplex has very high Tm
Conformationally constrained phosphoramidate nucleic acids
2’-NP-TNA
2’-NP-MoNA Importance of Constrained Template Geometry
5’Cy3GCGTAGACTGACTGCNH2!
3’-CGCATCTGACTGACGCCCCA !
****!
5’Cy3GCGTAGACTGACTGCGGGGNH2!
3’-CGCATCTGACTGACGCCCCA !
Schrum et al., JACS, 2009
O
ONH2
2’-NP-TNA: a conformationally constrained backbone
O
2'-NP-TNA
N
N
N
O
O
O O
N
N
P
N
O
O
O
P
NH2
N
O-
O O
HN
O
P
N
O-
O O
HN
O
P
O
O-
N
NH2
N
N
P
O-
O-
HN
NH
N
O
O
NH
Structure of TNA
RNA
duplex
TNA
duplex
Ebert et al. (2008) JACS 130: 15105
O
ONH2
3’-NP-MoNA: a constrained backbone
O
O3'-NP-MoNA
N
O
N
NH2
NH
N
N
N
NH2
O
N
O
N
P
N
O
N
O
O-
P
N
O
N
O
P
OH
O
N
O
N
O
P
O
O-
N
O
NH
OH
O
NH
NH2
Replicating Nucleic Acids
inside Replicating Vesicles
Template Copying in Vesicles
DNA primer and template encapsulated in 2:1
myristoleic acid:monomyristolein vesicles.
Template Copying in Vesicles
+15
Primer
DNA primer and template encapsulated in 2:1
myristoleic acid:monomyristolein vesicles.
DNA Template Copying in a “Prebiotic”
Vesicle Composition
Time:
+15
Primer
DNA Template Copying in a Modern
Vesicle Composition
Time:
+15
Primer
Are we any closer to
complete cycles of replication?
Back to RNA…
How important is monomer homogeneity?
DNA library
PT7
random r/d library
T7RNAP - Y689F
enriched r/d shuffled library
T7RNAP - Y689F
selection
PCR
RT
aptamers that bind ATP, GTP
How important is regiospecificity?
-  some 2’-5’ heterogeneity may still
allow evolution of ribozymes
- 2’-5’ linkages will lower the Tm
- perhaps 2’-5’ vs. 3’-5’ heterogeneity was essential for RNA to work as the primordial genetic material
Nucleic Acid Replication:
Monomers, Oligos, or a Mix?
Could the primordial replicase be a nuclease?
terminal mismatch
exonuclease
primer-extension
continues
Could the primordial replicase be a nuclease?
overhang
exo- or endo-nuclease
chemical ligation
Is there a path to the chemical replication of RNA?
"   Monomer purity may not matter as much as we thought
"   Incomplete regiospecificity may solve the Tm issue
"   2-thio-U may enhance both rate
and fidelity
Acknowledgements
Nucleic Acids
Vesicles
S. Zhang
Itay Budin
Craig Blain
Ting Zhu Support
B. Heuberger
Raphael Bruckner
HHMI
Na Zhang
Sheref Mansy
NASA
Matt Powner
K. Adamala
NSF
A. Ricardo
Irene Chen
MGH
J. Schrum
Michael Sacerdote
Harvard
Sylvia Tobé
Shelly Fujikawa
Jesse Chen Martin Hanczyc
Geron:
M. Krishnamurthy
Janet Iwasa
Sergei Gryaznov
Doug Treco