NPTEL – Nanotechnology - Nanobiotechnology
Self-assembly of oligonucleotides
Dr. K. Uma Maheswari
Professor, School of Chemical & Biotechnology
SASTRA University
Joint Initiative of IITs and IISc – Funded by MHRD
Page 1 of 16
NPTEL – Nanotechnology - Nanobiotechnology
Table of Contents
1 INTRODUCTION TO DNA NANOTECHNOLOGY ............................................................................3
1.1 STRUCTURE OF DNA........................................................................................................................................... 4
2 SELF-ASSEMBLY OF DNA NANOSTRUCTURES....................................................................... 11
2.1 BRANCH FORMATION ........................................................................................................................................ 12
2.2 BRANCH MIGRATION .......................................................................................................................................... 13
2.3 NON-STANDARD BASE PAIRING ..................................................................................................................... 15
2.4 PERSISTENCE LENGTH ..................................................................................................................................... 15
3 PROMISES AND CHALLENGES OF DNA NANOTECHNOLOGY ......................................... 15
4 REFERENCE .............................................................................................................................................. 16
5 ADDITIONAL READING ......................................................................................................................... 16
Joint Initiative of IITs and IISc – Funded by MHRD
Page 2 of 16
NPTEL – Nanotechnology - Nanobiotechnology
Module Objective
At the end of the module, the learner would be able to understand the factors influencing the
design of oligonucleotide-based self-assembled structures for novel applications. The learner
also will be also familiar with some oligonucleotide based self-assembled structures and their
potential applications.
Preface
The famous economist Kenneth Boulding once said, “DNA was the first threedimensional Xerox machine”. However, newer insights about the self-assembling
characteristics of this wonderful molecule have lead to a paradigm shift in the
applications for DNA. This module gives an insight into the fundamentals of various
types of oligonucleotide structures and their potential applications in many fields.
Lecture 1 provides an overview of the principles that have led to the development of
DNA nanotechnology and the factors that contribute to the self-assembly process
involving oligonucleotides. Lectures 2 and 3 introduce the learner to some passive and
active structures that have been developed using oligonucleotides. Lecture 4 provides
a glimpse of a few potential applications of such structures in different fields.
In this introductory lecture, we will look into the key structural aspects of DNA and
the unique properties that drive it’s self-assembly. This will provide the basis for
understanding the structures and devices that we will discuss in future sessions.
1 Introduction to DNA nanotechnology
Deoxyribonucleic acid (DNA) is generally associated with the nucleus and is said to
carry information about the proteins that are required to sustain life and functions.
Hence it is often referred to as the ‘blueprint of life’. The recipe for synthesizing a
protein is encoded in the form nucleotide sequences in a gene. An interesting aspect
about DNA is that its information is passed on from generation to generation and
hence is also referred to as the hereditary carrier of information. The information
stored in the genes of a cell is converted into proteins by complicated protein making
machineries known as ribosomes. The transfer of information from the DNA to the
ribosomes is carried out by another nucleic acid known as ribonucleic acid (RNA). As
in the case of proteins, DNA and RNA have unique recognition properties that enable
them to spontaneously assemble into structures with great precision and
reproducibility. This characteristic has generated huge amount of interest among the
research community to utilize such structures for novel applications which nature
never intended for these molecules. The high degree of orderliness, structural
robustness and the ability to self-assemble to complicate and intricate structures have
Joint Initiative of IITs and IISc – Funded by MHRD
Page 3 of 16
NPTEL – Nanotechnology - Nanobiotechnology
opened the gates to a new world of DNA nanotechnology. In order to appreciate and
understand the self-assembling properties of DNA, one needs to understand its
structure.
1.1 Structure of DNA
Both DNA and RNA can be chemically defined as polymers of nucleotides. One
nucleotide comprises of a nucleoside and a triphosphate residue. A nucleoside in turn
comprises of a nitrogenous base and a pentose sugar. One of the key differences in the
structure of DNA and RNA arises in the types of nitrogenous bases and the pentose
sugar present in them. While RNA is made of ribose sugar (and hence the name
‘ribo’nucleic acid), DNA is made of deoxyribose sugar (hence the name
‘deoxyribo’nucleic acid). Figure 1 gives the structure of ribose and deoxy ribose.
Fig. 1: Structure of ribose sugar and deoxyribose sugar
There are two types of nitrogenous bases found in nucleic acids – purines and
pyrimidines. The two purine bases found in both DNA and RNA are adenine and
guanine (denoted by A and G). The pyrimidines found in DNA are cytosine and
thymine (denoted by C and T) while in RNA cytosine and uracil (denoted by C and U)
are present. The structures of the various purines and pyrimidines found in nucleic
acids are given in Figure 2.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 4 of 16
NPTEL – Nanotechnology - Nanobiotechnology
NH2
6
O
5
1N
H
N7
1
8
2
4
N
N
8
2
H2N
9
3
3
Adenine
N9
H
4
Guanine
O
5
HN
1
N 3
H
3N
O
4
2
4
2
N
H
6
O
N7
5
6
HN
O
Thymine
HN
1
5
6
Uracil
Fig. 2: Structure of nitrogenous bases present in nucleic acids
The bases are always substituted in the first carbon of the sugar (ribose or deoxy
ribose) to form the corresponding nucleosides namely adenosine, guanosine, cytidine,
thymidine and uridine. The structures of nucleosides found in DNA are given in
Figure 3.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 5 of 16
NPTEL – Nanotechnology - Nanobiotechnology
NH2
6
N7
5
N
1
8
2
N9
N3 4
1'
O
HO
4'
2'
OH
3'
5'
HO
Adenosine
O
6
N7
5
N
1
H2N
8
2
N9
N3 4
H
1'
O
HO
4'
2'
OH
3'
5'
HO
Guanosine
Joint Initiative of IITs and IISc – Funded by MHRD
Page 6 of 16
NPTEL – Nanotechnology - Nanobiotechnology
Fig. 3: Structure of nucleosides present in DNA
The fifth carbon of the sugar is phosphorylated to form the corresponding nucleotide.
Figure 4 gives the structure of a nucleotide.
Fig. 4: Structure of adenosine monophosphate (nucleotide)
One nucleotide links to the next nucleotide through the third carbon of the sugar to
form the oligonucleotides via formation of phosphodiester links. The structure of an
oligonucleotide is depicted in Figure 5.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 7 of 16
NPTEL – Nanotechnology - Nanobiotechnology
NH2
N
A
N
HO
N
N
5`
O
H3` 2`
H
H
H
O
H
O P OO
5`
O
N
G
N
O
H 2`H
H 3`
H
O
H
Phosphodiester
bond
O P OO
N
NH
NH2
NH2
N
A
N
N
N
5`
O
H3` 2`
H
H
H
O
H
O P OO
5`
O
N
G
N
NH
NH2
N
O
H 2`H
H 3`
H
O
H
NH2
N
O P O-
C
N
O
O
O
H
H
H
O
H
O
H
O P O-
T
O
N
NH
O
O
H
H
O
H
H
NH2
H
N
O P ON
O
O
O
H
H
H
O
H
H
O P OOFig. 5: Structure of an oligonucleotide
An interesting property of DNA is that it does not exist as a single strand but is found
as a duplex. Why? The nitrogenous bases are relatively hydrophobic when compared
Joint Initiative of IITs and IISc – Funded by MHRD
Page 8 of 16
NPTEL – Nanotechnology - Nanobiotechnology
with the highly polar sugar and phosphate residues. Therefore association with another
strand enables the nitrogenous bases to minimize contact with water environment by
turning towards the interior while the phosphate and sugar residues tend to maximize
their contact with the aqueous environment owing to their polarity.
How does the DNA structure maintain its integrity? Well, the answer lies in the
associative forces that exist between the two strands! The nitrogenous bases are
stabilized through hydrogen bonds between them. A fascinating aspect in this structure
is that adenosine always tends to pair with thymidine via two hydrogen bonds while
guanosine tends to pair with cytidine through three hydrogen bonds. Such specific
pairing is referred to complementary base pairing. The process of association of two
strands by complementary base pairing is also known as ‘hybridization’. Obviously,
if the nucleotide sequence of one strand is known, then the sequence of the second
strand could be predicted. In other words, both strands are complementary to each
other. This pairing is also known as the Watson & Crick pairing after the two
scientists who unraveled the structure of DNA and awarded the Nobel prize for the
same. Figure 6 depicts the complementary base pairing that exists between purines and
pyrimidines in DNA.
Fig. 6: Complementary pairing between A-T and G-C
Joint Initiative of IITs and IISc – Funded by MHRD
Page 9 of 16
NPTEL – Nanotechnology - Nanobiotechnology
Another unique aspect is that the two strands are anti-parallel to each other. In other
words the first strand has a phosphate in the 5’ position of the sugar and terminates
with a free 3’ position in the sugar while the complementary strand will start with a
free 3’ position and end with a 5” phosphate. One of the proposed reasons for having
two complementary strands is to enable more efficient replication process.
The secondary structure of DNA however is not a parallel bilayer of the two strands
but adopts a characteristic double helical structure. This helical structure is mainly
attributed to the asymmetry in the size and associative forces between the nitrogenous
bases, which makes it difficult to be stacked. Hence, a twist is introduced in each
strand as they are being stacked upon one another leading to formation of the double
helix. The double helical structure of the most common form of DNA encountered in
biological systems is shown in the following figure. As the double helical structure is
formed, major and minor grooves are also observed. Figure 7 represents the structure
of a DNA double helix.
3.4 nm
Fig. 7: Structure of B-DNA
Depending upon the packing and twist and handedness of the double helix, three
forms of DNA has been identified. These are the B-DNA (most common), A-DNA
and Z-DNA. The A and Z forms are only formed under special conditions. B-DNA is
a right-handed double helix with 10 nucleotides per turn. In the case of A-DNA, which
Joint Initiative of IITs and IISc – Funded by MHRD
Page 10 of 16
NPTEL – Nanotechnology - Nanobiotechnology
is found in abnormal conditions such as in partially denatured conditions and in cases
where a DNA-RNA hybrid pairing is formed, the right handed double helix is wide
and contains about 11 nucleotides per turn. The Z-DNA is a left handed double helix
with 12 nucleotides per turn and is usually formed in high salt conditions in sequences
containing a methylated cytosine. The following animation (Figure 8) gives a glimpse
of the various structural aspects of DNA discussed thus far.
Fig. 8: Animation of key elements of DNA structure
Note: Can be viewed only in Acrobat Reader 9.0 and above
The double stranded DNA structure is very robust due to the large number of
hydrogen bonds that are formed between the two strands. Any factor that is capable of
disrupting the hydrogen bonds can destabilize the DNA structure, which is then said to
be denatured. Strong alkalis and high temperatures can contribute to the destruction of
the double helical structure by disrupting the hydrogen bonds.
2 Self-assembly of DNA nanostructures
The unique complementarity of the bases coupled with the predictability of the
structures formed using only four different nucleotides has given DNA-based selfassembly an edge over peptide based self-assembled structures. Another aspect that
has contributed to the evolution of DNA nanotechnology is the ability to cut DNA
Joint Initiative of IITs and IISc – Funded by MHRD
Page 11 of 16
NPTEL – Nanotechnology - Nanobiotechnology
sequences at specific points using enzymes known as ‘nucleases’. There are two types
of nucleases – endonucleases and exonucleases. While endonuclease will lyse
phosphodiester bonds in the interior of a sequence, exonucleases will be active at the
terminals only. A large number of endonucleases are available with different site
specificities. These enzymes are referred to as ‘molecular scissors’. Also available are
a category of enzymes known as ‘ligases’ which can catalyse the formation of
phosphodiester bonds between the nucleotides thereby enabling fusion of different
nucleotide sequences. Ligases are popularly referred to as ‘molecular glue’. The
nucleases are active in both 5’to 3’ and 3’to 5’ directions in a DNA double helix while
the ligases are direction specific and can act only along the 3’ to 5’ direction. These
enzymes have given the ability to manipulate sequences according to one’s desire to
achieve different structures.
The development of techniques for rapid synthesis of custom-made oligonucleotide
sequences has further accelerated the field of DNA nanotechnology. It has also been
found that these self-assembled structures can be altered both by varying the selfassembling conditions as well as the sequence of the oligonucleotide. These properties
have been exploited to develop novel DNA-based structures and devices. As in the
case of proteins, very long sequences of nucleotides are not preferred but rather short
nucleotide sequences (oligonucleotides) typically about 15-50 base pairs are generally
used to generate the self-assembled structures. In the following section, let us discuss
about a few key properties that have been exploited in DNA nanotechnology.
2.1 Branch formation
Two complementary DNA strands can be paired through hydrogen bonds forming
either a ‘blunt’ end or a ‘sticky’ end. A blunt end is one in which all bases in one
strand have a complementary base pair in the other strand and hence will not have any
unpaired bases to form further associated structures. A sticky end will have unpaired
bases, which will be available for interaction with other complementary sequences.
Thus designing sequences that share partial complementarity will result in formation
of a sticky end, which can be paired with yet another sequence. This can lead to
formation of branched structures with multiple branches as shown in the Figure 9.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 12 of 16
NPTEL – Nanotechnology - Nanobiotechnology
Fig. 9: Branched structure formation using oligonucleotide strands
Interestingly, such branches are also found in natural systems during the formation of
the replication fork in cell division. Similar property can be exploited for developing
robust DNA-based self-assembled structures.
2.2 Branch migration
In biological systems, a phenomenon known as cross-over was first reported by Prof.
Holliday when studying a kind of genetic recombination in yeast cells. The formation
of a four-way junction now known as the ‘Holliday junction’ formed by four DNA
strands was proposed to explain this genetic phenomenon. During the formation of
this junction it was found that a single strand breaks and migrates to form a
complementary base pairing with part of another strand that constituted the Holliday
junction. This phenomenon resulted in development of new characteristics for the next
generation of yeast thus effectively introducing genetic heterogeneity in the offspring.
This phenomenon is referred to as ‘cross-over’. The same concept has now been
adopted successfully in DNA nanotechnology to create newer structures with different
affinities to various oligonucleotide strands. Such structures with cross-over junctions
are referred to as the cross-over motifs. Double cross-over and triple cross-over motifs
have been achieved using oligonucleotide sequences which have been effectively used
to develop complicated structures and patterns. The double cross-over motifs are
denoted as DX and the triple cross-over motifs are denoted as TX. Figure 10
represents a few types of cross-over junctions tat can be formed using oligonucleotide
sequences.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 13 of 16
NPTEL – Nanotechnology - Nanobiotechnology
Fig. 10: Cross-over motifs using oligonucleotides
Apart from the number of cross-overs seen in a structure (double, triple etc.), the
double and triple cross-over motifs can be further classified as parallel or anti-parallel
depending on the orientation of the stationary segments of the strand that have not
crossed over. The parallel cross-over motifs are indicated by P and the anti-parallel
ones by A. Thus DP refers to a double cross-over motif with a parallel orientation. In
addition, the motifs are indicated as either E or O depending on the number of turns
between each cross-over point. E represents even number of turns while O represents
odd number of turns. Thus DAE represents a double cross-over motif with antiparallel orientation having even number of turns between its cross-over points. The
presence of a major or minor groove at the cross-over point especially in the DPO
systems can be denoted by a fourth letter W or N representing major and minor
grooves respectively. Such cross-over motifs can be designed with sticky ends to
ensure further hybridization to achieve unique assemblies. Induction of cross-over or
branch migration between strands in double strands or multiple strands of DNA can be
achieved by introduction of localized temperature fluctuations that will disrupt
existing hydrogen bonds and shift the position of the segment to a new position.
Alternately, design of sequences with partial complementarity can also be used to
obtain cross-over motifs.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 14 of 16
NPTEL – Nanotechnology - Nanobiotechnology
2.3 Non-standard base pairing
Apart from the standard Watson-Crick pairing, it has also been observed that under
special conditions, non-standard or unnatural base pairing also exists. For example, in
the telomeres, four guanine residues pair by hydrogen bonds in the presence of
potassium ions to form a structure known as the G-tetrad or G-quartet. Similarly,
adenine residues have been found to associate via hydrogen bonds at acidic pH.
Cytosine associations known as i-motif have also been reported. Such unusual base
pairing can be exploited for changing conformations of self-assembled
oligonucleotides by simply changing the environment in which the self-assembly
occurs. Figure 11 represents a G-quartet structure.
Fig. 11: Structure of a G-quartet
2.4 Persistence length
Persistence length of a molecule provides a measure of its ability to withstand stress
due to random collision of molecules when placed in a solution without structural
deformation. The average length of the molecule at which it tends to deform/bend due
to collision of other molecules with it is called as the persistence length. The
persistence length of a molecule is an important measure of the stiffness or rigidity of
the nanostructures formed from it. In this regard, a single stranded DNA has very low
persistence length of about 1 nm indicating its flexible nature. But a double stranded
DNA has a persistence length of 50 nm indicating that it can form highly rigid and
robust structures, which can be invaluable in development of DNA-based structures.
3 Promises and challenges of DNA nanotechnology
One of the major challenges in DNA nanotechnology remains the problem of errors
during base pairing. When many strands are introduced, it is likely that partial
Joint Initiative of IITs and IISc – Funded by MHRD
Page 15 of 16
NPTEL – Nanotechnology - Nanobiotechnology
complementarity can give rise to unexpected variants in the structure. Also, the speed
at which conformation changes are effected in a DNA-based structure is in the order
of a few seconds, which is comparatively slow. The expenses involved in
synthesizing, purifying and characterization of the strands are quite high making the
end device expensive. Another major impediment is the poor thermal stability of the
structures.
But in spite of these challenges, DNA nanotechnology is making rapid inroads due to
the numerous merits that far outweigh its challenges. The prospect of obtaining
intricate structures not possible by other molecules or methods using DNA is a major
attraction of this emerging field. One of the prime requirements in the nanotechnology
era is a device that has a small size, high degree of reproducibility in structure and
capable of high-speed operations. The DNA self-assembled structures can satisfy these
requirements and hence has attracted immense attention in recent years. Moving away
from the conventional double helical DNA structure to obtain non-linear complex
structures with excellent reproducibility can be exploited in the field of
nanoelectronics, nanoarchitechtonics and nanomedicine. The possibilities are immense
and the future of DNA nanotechnology definitely looks promising.
4 Reference
Encyclopedia of Nanoscience & Nanotechnology, Volumes 2 & 9. Edited by: H.S.
Nalwa, American Scientific Publishers, 2005
5 Additional Reading
1. First blueprint, now bricks: DNA as construction material on the nanoscale,
Sethuramasundaram Pitchiaya and Yamuna Krishnan, Chem. Soc. Rev., 2006,
35, 1111–1121
2. Design of DNA origami, Paul W.K. Rothemund, Proceedings of the 2005
IEEE/ACM International conference on Computer-aided design, 2005 (can be
freely downloaded at:
http://authors.library.caltech.edu/8913/1/ROTiccad05.pdf)
Joint Initiative of IITs and IISc – Funded by MHRD
Page 16 of 16