4.19.2. Why Unnatural Nucleotide Bases?

 The natural bases only differ in their organic structure and functional group only belongs to two different heterocyclic families. Basically they do not differ much in their properties in wider sence like in stacking ability, size, sensitivity in protonation in neutral pH etc. They only absorb light in ultraviolet region and not responsive to the visible light or can not be detected by highly sensitive fluorescence detection techniques as the intrinsic fluorescence of the naturally occurring nucleotide bases in DNA and RNA is extremely weak. These bases exhibit very short fluorescent decay times, generally in the range of a few picoseconds, and do not provide much structural information since signals are normally averaged over all bases in the oligonucleotide sequence. Although the major nucleotide bases are essentially non-fluorescent, there do exist a few naturally occurring fluorescent bases, such as wyosine (Yt) which is found in the anticodon region of tRNAPhe. The rarity of such naturally occurring fluorescent bases, however, has limited their use in fluorescence studies. Thus, in contrast to proteins, which may contain one or more naturally occurring tryptophan or tyrosine residues that can be exploited for fluorescence measurements, RNA and DNA molecules in general lack naturally occurring intrinsic fluorescent reporters.
Thus, the lack of naturally occurring fluorescent bases has spurred the development of new nucleosides which will be the probe for DNA analysis. These nucleosides can be so design as to get optimized fluorescence properties (i.e. higher quantum yields and longer lifetimes) and that can be incorporated into oligonucleotides using standard automated synthetic methods. The fluorescence properties, from DNA and RNA molecules, thus can be observed without any competing background signals mainly by two ways: (a) incorporation of unnatural nucleotide base analogs into the oligonucleotide sequence and/or (b) incorporation of fluorophore in to the natural bases i.e. by synthesizing labeled bases.

4.19.3. Designing Criteria for Modification of Nucleosides

For probing DNA structure, the ideal fluorescent nucleoside:

1. Should have bright fluorescence, which is sensitive to its environment, and a large Stokes shift;
2. Should be amenable to phosphoramidite preparation for incorporation into  oligo-nucleotides by solid-phase synthesis;
3. Should not disrupt duplex formation and should mimic one of the regular nucleosides; In general, maintaining Watson-Crick and Hoogsteen base pairing hydrogen bonding is an important aspect in the design and synthesis of fluorescent nucleotide base analogs.
4. Should behave as a regular nucleoside in its interaction with proteins and enzymes; and
5. Should be capable of being converted to the triphosphate and be incorporated into DNA with high efficiency by current commercial polymerases.

Thus, considering the steric hypothesis, shape complementarity and hydrophobic stabilization several base analogues for DNA replication have been reported. Researchers are hopeful to translate an expanded genetic alphabet into an expanded genetic code. Provocatively, this effort may lead to the assembly of such a system within a living cell, potentially creating a semi-synthetic organism and life with increased diversity.
4.19.4. Design of Unnatural Base Pairs
Therefore, the major efforts are focused on the development of a stable third base-pair that would be efficiently replicated with high fidelity. Recent efforts have resulted in designing and construction of a number of such base-pairs that are stable within the DNA duplex. However, to date, because of the challenging problem of enzymatic replication of such base pairs, only very few of these artificial base-pairs have been efficiently and selectively replicated. The base-pairs formed are of following categories: (a) unnatural hydrogen-bonding pattern as well as upon the shape complementarity, (b) hydrophobic forces, (c) metal-bridged base-pairing,(d) and even covalent cross-linking.

4.19.4.1 Artificial Base-Pairs Based on Hydrogen Bonding

4.19.4.1.1. Modification of Heterocyclic (Purine or Pyrimidine) Bases

Specific base-pairing between nucleic acids is essential to the accurate duplication and expression of genetic information. It is made possible, in part, by unique hydrogen bonding complementarity, which depends critically on the tautomeric states of the bases. Tautomerization reverses the polarity by interconverting H-bond donors and acceptors, and the different electronegativities of oxygen and nitrogen exocyclic substituents are consistent with observed equilibrium constants of ca 104 in favour of the amino tautomers of A and C, and the keto forms of G and T. Modification of these groups though, perturbs the equilibrium, it is made possible by suitable design and with the help of synthetic chemistry. Thus, the compounds, which are mutagenic in vivo, can function as analogues of both A, G, C and T, depending on the opportunities for H-bonding available in a given environment. The concept of hydrogen-bonding patterns and shape complementarity was pioneered by Benner (e.g., isoguanosine, iso-G; isocytidine, iso-C; Figure 4.28). Further development has led to the introduction of other donor−acceptor (D−A) purine−pyrimidine pairs and finally to a generalization of the Watson−Crick nucleobases pairs.
A number of nucleotide base analog probes are now available, some even commercially, for incorporation into oligonucleotides for biophysical and biochemical studies. These nucleotide base probes share common chemical features, like faithful mimicry of native base structure and optimization of fluorescence quantum yields and lifetimes. Each base analog also exhibits some unique fluorescence, structural or chemical properties, which should be considered when deciding on the optimal probe for use in studies of a particular nucleic acid system. We are summarizing below some of the nucleotide base analogs available to date.