between the G–sC, H–sC and H-C base pairs. Here steric clash may have prevented hydrogen bonding between the thioketo group of C and the amino group of G. If so sub- stitution of H for G or C for sC would eliminate clash without any loss of base pairing strength. The nA–sT couple provides yet a third example of steric clash. In this case the analogous clash-free couples of A–sT and A–T were much more stable than nA–sT even though they only possessed two hydrogen bonds (10). It is likely that altered electronic and stacking properties of the respective base pairs also have an important role in determining their relative stabilities. The three examples demonstrate that Table 4. Apparent melting temperatures of 12 bp DNA hybrids substituted with weakly pairing analogs of G and C X sG and MePa G sG sG G H and sCb G G H H Y C C MeP MeP c sC sC C a50–XCAXACTTCTCC 30–YGTYTGAAGAGG b50–XCAXACTTCTCC 30–TYGTYTGAAGAGGTACAACTGCACAG Tm (8C) 52 41 34 32 54 45 44 45 Tm – 11 18 20 – 9 10 9 Nucleic Acids Research, 2008, Vol. 36, No. 22 7005 elimination of clash does not necessarily lead to a more stable base pair. Specificity of base pairing by G/C analogs The short single-stranded primer extension products described previously were used to evaluate whether other- wise promising G/C analogs discriminated against incor- rect bases. For this analysis the modiﬁed DNAs were incubated under stringent conditions with four 12-mer DNA probes each of which presented a diﬀerent base to the analog of interest. Reactions were quenched by adding an unlabeled competitor oligonucleotide to sequester free probe followed by rapid cooling in an ice bath. Hybrid formation was detected by gel mobility shift and the ratio of mismatched to matched hybrid was used as a proxy for speciﬁcity. Using this methodology we previously showed that nA and sT are comparable to A and T in their dis- crimination of mismatched bases (11). In the current study speciﬁc pairing was observed for NitroC and sC (Figure 3A) but not for any of the G analogs (data not shown). The low yield of perfect-match hybrid in the sC panel may be attributable to strand displacement of radio- labeled target from the duplex by unmodiﬁed competitor oligonucleotide. Pairing properties of the hypoxanthine analogs were further investigated using probes that hybridized under more stringent conditions. The naturally high binding aﬃ- nity of LNA and its preferential stabilization of the NitrocH-C base pair were suﬃcient to impart speciﬁcity to this analog when using 8-mer LNA probes (Figure 3B). By comparison, some of the same LNA probes formed Figure 3. Speciﬁcity of base pairing by selected base analogs. Analogs of (A) cytosine and (B) guanine were evaluated using probe-target hybrids 2 and 3, respectively (Figure 1B). Radiolabeled targets substituted with the indicated analog of G or C were prepared by primer extension and hybridized under stringent conditions to probes that presented each of the regular bases to a centrally positioned C or G analog. Reactions were quenched by sequestering free probe with unlabeled competitor oligonucleotide after which probe-target hybrid was resolved by gel mobility shift assay. DNA targets substituted with NitrocH or H also contained nA and sT while DNA substituted with NitroC or sC did not. The hybridization temperature is indicated next to each reaction series; apparent Tm’s of the corresponding perfect-match hybrids were 458C (sC), 478C (NitroC), 438C (H) and 638C (NitrocH).