DNA is an essential biomolecule which is responsible for encoding the complex information necessary for life. The specific pairing of dA with dT and dC with dG in duplex DNA (Figure 1.1) and during polymerase-mediated replication is the basis of the genetic alphabet, ultimately leading to the basis of genetic code.1 However, there is no reason to limit the genetic alphabets and hence the information stored in them to only two base pairs. It was a logical thought among the scientific community that an expanded genetic alphabet would not only enable the encoding of additional information for both in vitro and in vivo applications but also enable a wide variety of biotechnology applications. Expansion of the genetic alphabet to include a third base pair, formed between two identical or different unnatural nucleotides, referred to as self-pairs and hetero pairs, respectively, would expand the informational and functional potential of DNA such as site directed oligonucleotide labeling and in vitro selections with oligonucleotides bearing increased chemical diversity.2 Thus, not only the design and synthesis of efficient new base pair/pairs is an exciting research area but also the application of these artificial base-pairs to drive the synthesis of unnatural proteins is currently an attractive field of research with a hope to translate an expanded genetic alphabet into an expanded genetic code creating a synthetic organism one day with ability to encode proteins with new physico-chemical properties.3
Adenine (A) : Thymine (T) Guanine (G) : Cytosine (T)
N N N
N N
O HO HO
O OH
OH N N
O
O
N N N
O
N N O HO HO
O OH
OH N
N N
O H H
H
H H
H
H H
1.1 1.2 1.3 1.4
Figure 1.1: Presentation of hydrogen bonding between the DNA bases. The idea of expansion of genetic alphabet for generating DNA and RNA with enhanced functional abilities was pioneered by Alex Rich4 in 1962 to propose the concept of orthogonal base pairing between iso-G and iso-C and inspired Prof. Steven A. Benner in the late 1980’s to expand the genetic alphabet from four to six letters.5
Benner’s early research work focused on the development of new base pairs based on hydrogen bonding patterns orthogonal to those in canonical Watson-Crick base pairs.6 Following Benner’s work, many researchers have contributed to the field of expansion of genetic alphabets. As for example, a modified version of Rich’s proposed iso-G/iso-C base pair has been demonstrated in RNA by Dervan in 1993.7 As a result of tremendous research efforts, a large number of non-natural nucleosides capable of showing H-bonding/π-stacking interaction properties have been developed and their biophysical properties in the context of DNA have vigorously been investigated. As for example, a number of base analogues with orthogonal H-bonding complementarities8 in relation to the natural Watson-Crick H-bonding have been exploited to examine the importance of hydrogen bonding interactions in the stabilization of nucleic acids structure, in the study of interbiomolecular interactions,9
a-b and in the base recognition ability of enzymes.9c-g Several modified nucleosides with reporter functionalities have also been synthesized for monitoring the local microenvironmental change around the nucleic acids associated with interbiomolecular interactions.10 Latter on in 1994, creation of non-H-bonding unnatural nucleobase surrogates by Kool et al. has opened a new dimension in the design of hydrophobic unnatural DNA base analogues.11 Thus, they have explored the possible aromatic stacking, hydrophobic or CH-π interactions between the bases and shown that these attractive forces are good enough to stabilize a DNA duplex and are well recognized by DNA polymerases. Triggered by Kool’s work, much efforts have been put forth to develop non-natural, stable, hydrophobic base pairs of orthogonal recognition properties towards expanding the genetic alphabets.11 Recently, the design of unnatural DNA base pairs with tuned charge transfer/photophysical properties is a rapidly growing research field towards the development of nucleic acid based diagnostics and sensing materials.12 While the development of bases with improved charge transfer characteristic would lead to oligonucleotides with novel electronic properties,12 the fluorescent nucleobases could offer opportunity for in vivo imaging as well as for the development of nucleic acid based sensors.13 Toward this end, several unnatural nucleobases have been designed for the development of functional nucleic acids.10 h,14 However, the rational design of non-hydrogen bonding base pairs
remains a challenge. In most of the design of non-hydrogen bonding base pairs, researchers have concentrated mainly on the factors like, π-stacking, hydrophobicity, steric shape mimicry and in few cases the dipole moment, etc., in the stabilization of DNA duplex.11 `
Thus, the efforts toward developing a third base pair have focused on the design of nucleobase analogues to pair via orthogonal hydrogen bonding (H-bonding, Figure 1.2), based on the work of the Benner group and more recently, on predominantly non-H-bonding (Figure 1.3) analogues that pair via hydrophobic interactions, based on the work of the Kool group.
Figure 1.2: Presentation of H-bonding base pairs among unnatural nucleosides.
Figure 1.3: Presentation of non H-bonding base pairs among nucleoside base analogues.
However, the unnatural base pairs so far have been reported have several shortcomings, including tautomerization of iso-G and poor recognition of iso-C by RNA polymerases. These shortcomings pose difficulties for mRNA preparation.
Since, all modern molecular biology techniques require the amplification of DNA by PCR, therefore, it would be worthwhile to design such unnatural pair for which both PCR amplification and transcription by RNA polymerase would be efficient.
O OH
HO N
O OH HO
O OH HO
F
N N N
O OH HO F
1.9 1.10
1.11 1.12
CH3
H H3C
O
dBEN 5MP
dF: dQ
Self pair Hydrophobic interaction
dT analogue dA Analogue
Hetero pairs-nucleoside shape mimics O
OH HO
N N O
NH N N N
NH2
O OH HO NH2
dC Analogue dG Analogue diso-C : diso-G
O
O OH HO
N N
NH2
NH NH N N
O
O OH HO NH2
dT Analogue dA Analogue dκκκ : dXκ
O
1.5 1.6 1.7 1.8
Increasing diversity of these modified DNA and RNA molecules promises their enhanced widespread potential applications in biomedical sciences such as drug candidates. Novel, hydrophobic base pairs have been thus developed recently, but their use in transcription is still under investigation. Thus, there is a need to develop conceptually new and novel base analogues which will be recognized by DNA polymerases for both replication and transcription process with high efficiency.