Substitution None nA + sT G Analogs cG NitrocH IcH H cH sG C Analogs NitroC sC 7004 Nucleic Acids Research, 2008, Vol. 36, No. 22 Table 2. Apparent melting temperatures of the HP25 hairpin substi- tuted with analogs of G or C Table 3. Apparent melting temperatures of hybrids substituted in one strand with analogs of G or C Tm (8C) Tm/analog Substitution Tm (8C) Tm/analog 76 40 68 64 61 55 50 46 57 35 0.57 0.86 1.1 1.5 1.9 2.1 1.4 2.9 enhance stability of the probe-target hybrid. Probes with a locked nucleic acid (LNA) backbone hybridize with high aﬃnity to complementary sequences (45). Since enhanced stability was unnecessary when hybridizing to hairpins substituted with nA and sT, the probe to these hairpins contained a DNA backbone. Strand invasion of the mod- iﬁed hairpins by either probe is always a possibility and would result in underestimation of the true Tm. The apparent melting temperatures listed in Table 2 provide a ranking of base pairing strength for the G/C analogs in the absence of nA and sT. Among the G ana- logs, the sG–C couple with its inherent steric clash decreased the Tm by 308C. The H–C base pair, with only two hydrogen bonds, lowered the Tm by 218C. Relative to this base pair the cH–C couple was more destabilizing while the IcH–C and NitrocH–C couples were less desta- bilizing. Only two C analogs were successfully incorpo- rated into the hairpin. Based on the melting analysis, the NitroC–cG base pair reduced the Tm by 198C while the sC–cG couple, with its associated steric clash, reduced it by 418C. With the exception of NitrocH and NitroC all of the other testable analogs [IcH, H, cH, sG and sC] eliminated secondary structure in the HP25 hairpin when used in conjunction with nA and sT. Hybridization of the 25-mer DNA probe to these hairpins was complete at 108C. By comparison, HP25 substituted with nA + sT or with nA + sT + cG had Tm’s of 408C and 268C, respec- tively. Hairpin substituted with NitrocH + nA + sT had a Tm of 188C. The NitroC analog was unimpressive as a candidate for destabilizing DNA since it did not contri- bute to decreased hybrid stability when used in conjunc- tion with nA and sT. Hairpins substituted with nA + sT + NitroC or nA + sT + NitroC + cG had Tm’s of 488C and 448C, respectively. The results obtained with hairpins were extended by determining apparent melting temperatures of short Watson–Crick hybrids in which one strand was substi- tuted with an analog of G or C. Each modiﬁed strand was prepared by primer extension of a radiolabeled primer and then puriﬁed by electrophoresis in a 7 M urea–12% polyacrylamide gel. The modiﬁed strands – 3.5 4.5 9 – 2.5 3 5.5 6 6.5 7.5 +9 – – 9 15 16 17 18 C analogs (12 bp DNA–DNA hybrid 1) C 54 NitroC 47 sC 45 MefP 36 G analogs (12 bp DNA–DNA hybrid 2) G 58 NitrocG 53 cG 52 H 47 NitrocH 46 IcH 45 cH 43 G analogs (8 bp LNA–DNA hybrid 3) NitrocG 69 G 60 cG 60 NitrocH 51 cH 45 H 44 IcH 43 sG 42 were hybridized to a complementary 12-mer DNA or 8-mer LNA probe to form the hybrids depicted in Figure 1B. Apparent melting temperatures were deter- mined using a variation of the gel mobility shift assay described earlier. The Tm’s listed in Table 3 conﬁrm the relative base pairing strengths of the C analogs and further demonstrate that MefP paired much more weakly to G than did sC or NitroC. The hypoxanthine bases were nearly equivalent to one another in destabilizing the 12 bp DNA hybrid. However, this was not the case for the 8-bp LNA–DNA hybrid where NitrocH exhibited much greater pairing strength than H, cH and IcH. Interestingly, NitrocG also exhibited enhanced pairing strength when one strand of the duplex had an LNA backbone. sG, which was only tested in the LNA–DNA hybrid, was the most destabilizing of the G analogs. Hybridization of oligonucleotide probes to DNA substi- tuted with sG or sC will probably require probes that are themselves modiﬁed to eliminate steric clash with the thio- keto group of these base analogs. For example, sG–MeP and H–sC lack the steric clash of sG–C and G–sC and might be more stable. Indirect evidence suggested that this was the case for sG–MeP, which was found to be iso- stable with A–T (46). We evaluated base pair stability by determining Tm’s of a 12 bp DNA hybrid in which two G–C base pairs were replaced by sG–C, sG–MeP, G–MeP, G–sC, H–sC or H–C couples. The results in Table 4 show that sG–MeP and G–MeP, which had similar pairing strengths, were much less stable than sG–C. This suggests that hydrogen bonding between the thioketo group of sG and the amino group of C stabilized the sG–C base pair even in the presence of steric clash. Substitution of MeP for C or G for sG removed steric clash but also eliminated a hydrogen bond thus leading to further destabilization. In contrast, virtually no diﬀerence in stability was observed