free probe was sequestered by adding a competitor oligo- nucleotide to 10 mM and the reaction was quenched in an ice bath. Competitor oligonucleotides contained regular bases and were identical in backbone and complementary in sequence to the probe. Each reaction set was then sub- jected to gel mobility shift analysis as described above to construct a melting curve. A similar protocol was followed for the determination of speciﬁcity. In this case the single- stranded target was hybridized to one perfect-match and three mismatch probes at a ﬁxed temperature under strin- gent conditions. These reactions were usually quenched by adding a competitor oligonucleotide that was complemen- tary to the perfect-match probe. Tiling experiments con- sisted of hybridizing 5 mM probe to radiolabeled target for 5–10 min at the indicated temperature. No competitor oli- gonucleotide was added to the tiling reactions prior to electrophoretic analysis. RESULTS Selection and evaluation of base analogs Candidate bases (Figure 1A) were synthesized as dNTPs since eﬃcient enzymatic incorporation into DNA was deemed an important attribute. Primer extension was used to prepare single-stranded and hairpin DNAs that contained each of the analogs either alone or in combina- tion with nA and sT (Figure 1B). Apparent melting temperatures of the modiﬁed DNA products were deter- mined by gel mobility shift assay and allowed us to ascer- tain the stability and speciﬁcity of nonnatural G-C base pairs. A 25 bp DNA hairpin (HP25) was used extensively in these studies and its Tm provided a convenient way to measure hybrid stability in the presence of an analog of G or C. Tiled sets of 12-mer DNA and 8-mer LNA probes were hybridized to the modiﬁed hairpins as a test of whether a given set of bases rendered the target structure-free. The analogs investigated here were known or expected to pair very weakly with their standard base complements. cH, IcH and NitrocH lack the C2 exocyclic amino group of G and pair with C through two hydrogen bonds. By analogy with the corresponding cG derivatives, cH should be less stable than H (the free base of inosine) while IcH and NitrocH should be more stable (12,19–21). The 7-deaza substitution in these analogs has the advantage of eliminating self-association and reducing the likelihood of mispairing to G (22). We have previously shown that DNA substituted with nA, sT and H is pseudo- complementary but does not exhibit speciﬁcity in its pair- ing due to degeneracy of H (11,23). P [the free base of zebularine; (24)] and MefP (25) are analogs of C that lack the C4 exocyclic amino group. Pairing of these bases to G is limited to two hydrogen bonds. A third analog of C, NitroC (16,26), can form three hydrogen bonds with G. However, the strongly electron withdraw- ing nitro group should weaken the central hydrogen bond. In combination with nA and sT any one of these analogs should reduce the secondary structure of DNA pro- vided that the respective dNTPs are utilized by DNA polymerase. Nucleic Acids Research, 2008, Vol. 36, No. 22 7001 Also of interest were sG (27–30) and sC (31). The sG–C and G–sC base pairs are weakened by steric clash between an amino group on one base and a thioketo group on the other base (Figure 1C). In this respect they bear similarity to the nA–sT mismatch. While either of these couples would be expected to work in concert with the nA–sT couple to prevent intramolecular pairing in DNA, success- ful hybridization might require the use of modiﬁed probes that hydrogen bond to sG or sC without clash. For instance, analogs of P or H might be developed for stable and speciﬁc pairing to sG or sC, respectively. Several groups have reported that dsGTP is incorporated into DNA using regular templates even though the sG–C pair is weak and distorted (32–36). The properties of dsCTP have not been described and its uptake into DNA might require the presence of an ‘adaptor’ base in the template DNA. Enzymatic incorporation of dGTP and dCTP analogs Each analog was substituted for dGTP or dCTP in primer extension reactions catalyzed by Sequenase. This engi- neered version of T7 DNA polymerase lacks proofreading function and is noted for its ability to utilize a variety of base analogs (37). Many of the analogs prematurely terminated synthesis if the template contained a stable hairpin element. For instance, when dsGTP was substi- tuted for dGTP, Sequenase was unable to synthesize a full length 75 mer that contained an embedded 25 bp hair- pin (Figure 2). Synthesis terminated midway into the hair- pin element after addition of two consecutive sG residues. We postulate that synthesis was blocked because the 30-end of the nascent DNA product was displaced from the template strand by the homologous strand of the hair- pin element. In this situation strand displacement would be thermodynamically driven since two weak sG–C base pairs would be replaced by two much stronger G–C base pairs. We cannot, however, exclude the possibility that two consecutive sG’s at the primer terminus might fray the duplex such that Sequenase, which does not have 30-exonuclease activity, would not be able to add the next dNTP. We reduced this block to synthesis by switching to f29 DNA polymerase (DNAP), an enzyme which has potent strand displacement activity and can readily extend a primer through a double-stranded DNA. The catalytic center of this enzyme resides within a ‘tunnel’ that accom- modates the template and nascent product strands (38) and conceivably reduces fraying by stabilizing the nascent hybrid. The non-template strand of a dsDNA template is excluded from the active site thus also preventing strand displacement as described for Sequenase. Consequently, f29 DNAP could utilize weakly pairing dNTPs to synthe- size both single-stranded and hairpin products. The uptake of such dNTPs could be further enhanced by using the N62D mutant of f29 DNAP (39). This mutation severely depresses the proofreading function of wild-type enzyme without impairing its ability to carry out strand displacement synthesis. By nearly eliminating 30–50 exonu- clease activity, possible hydrolysis of weak pairing bases from the 30-end of the nascent DNA was minimized.