Enzymic recognition of the base pair between isocytidine and isoguanosine

Abstract

The ability of various polymerases to catalyze the template-directed formation of a base pair between isoguanine (iso-G) and isocytosine (iso-C) in duplex oligonucleotides has been investigated. A new procedure was developed for preparing derivatives of deoxyisoguanosine suitable for incorporation into DNA using an automated DNA synthesizer. T7 RNA polymerase, AMV reverse transcriptase, and the Klenow fragment of DNA polymerase all incorporated iso-G opposite iso-C in a template. T4 DNA polymerase did not. Several polymerases also incorporated iso-G opposite T, presumably through pairing with a minor tautomeric form of iso-G complementary to T. In a template, iso-G directs the incorporation of both iso-C and T when Klenow fragment is the catalyst and only U when T7 RNA polymerase is the catalyst. Further, derivatives of iso-C were found to undergo significant amounts of deamination under alkaline conditions used for base deprotection after automated oligonucleotide synthesis. Both the deamination reaction of iso-C and the ambivalent tautomeric forms of iso-G make it unlikely that the (iso-C).(iso-G) base pair was a part of information storage molecules also containing the A.T and G.C base pairs found in primitive forms of life that emerged on planet earth several billion years ago. Nevertheless, the extra letters in the genetic alphabet can serve useful roles in a contemporary laboratory setting.

Stacking Effects on Local Structure in RNA: Changes in the Structure of Tandem GA Pairs when Flanking GC Pairs Are Replaced by isoG−isoC Pairs

“…7,9,16 Different shapes of GA pairs in turn provide different molecular features, e.g., van der Waals and electrostatic surface, and hydrogen bonding donors and acceptors, for higher order RNA folding and molecular recognition. [13][14][15][17][18][19] The Watson-Crick like iGiC pair, with the amino and carbonyl groups transposed relative to the Watson-Crick GC pair, provides an expanded alphabet [20][21][22][23][24][25][26] for further understanding interactions that shape nucleic acid structure (Figure 1). 7,[25][26][27][28][29][30][31][32] Here, thermodynamic studies of tandem GA pairs flanked by iGiC pairs are reported along with NMR structures of the RNA duplexes (GCiGGAiCGCA) 2 and (GGiCGAiGCCA) 2 .…”

Neighbouring bases in template influence base-pairing of isoguanine

“…Benner and co-workers, in their search for ' new letters of the genetic alphabet ', have found that iG can form a pair with another odd base, isocytosine (iC), and also with thymine (or uracil) [13][14][15][16]. They proposed that iC pairs with iG in its keto form, whereas thymine (or uracil) pairs with iG in its enol form ( Figure 1).…”

“…Of course, the ambiguity of pairing caused by tautomerism limits the use of iG as a ' letter in the genetic alphabet '; on the other hand, this fact makes it possible to study the influence of various factors on the tautomeric equilibrium of iG and its coding properties. Benner and co-workers [15,16] observed significant differences in the acceptance by various polymerases of iG : iC versus iG : T (or iG : U) pairs, studied in manifold combinations, i.e. in the template or in NTP, as riboor deoxyribo-derivatives.…”

Effects of 8-hydroxy-GTP and 2-hydroxy-ATP on in vitro transcription

“…Thus, the effects of 8-OH-GTP observed in this study are also expected to be relevant for prokaryotic and eukaryotic RNA pols. Switzer et al reported that T7 RNA pol incorporated 2-OH-ATP opposite an unnatural base, isocytosine [32]. They did not examine whether 2-OH-ATP was inserted opposite T, although they found that T7 RNA pol incorporated UTP opposite 2-OH-Ade in DNA.…”

“…They did not examine whether 2-OH-ATP was inserted opposite T, although they found that T7 RNA pol incorporated UTP opposite 2-OH-Ade in DNA. Since the corresponding DNA precursor, 2-hydroxy-dATP, is inserted opposite T and other bases in DNA templates by prokaryotic and eukaryotic DNA pols [32][33][34], it seems valid to speculate that 2-OH-ATP is also incorporated into RNA by prokaryotic and eukaryotic RNA pols. In addition, T7 RNA pol has been used as a model enzyme in the transcription experiments using DNA lesions, including 8-OH-Gua [28,[35][36][37][38], and similar results may be obtained when prokaryotic and eukaryotic RNA pols are employed in the transcription experiments with 8-OH-GTP and 2-OH-ATP.…”