than the other analogs and was readily incorporated into DNA where it exhibited good speciﬁcity of pairing. In contrast to these base analogs, NitrocH proved to be a promising candidate for use as a pseudo-complementary base. As expected, the electron-withdrawing nitro group strengthened base pairing such that NitrocH–C was more stable than H–C. Consequently, the dNTP of NitrocH was less prone than H to inhibit primer extension through G/C-rich secondary structure features. When incorpo- rated into DNA along with nA and sT, NitrocH was nearly as eﬀective as H in eliminating secondary structure. Furthermore, long primer extension products substituted with nA, sT and NitrocH could be puriﬁed from template DNA by electrophoresis through a formamide agarose gel (data not shown). Importantly, NitrocH was less degen- erate in its base pairing than H and exhibited good discrimination in the presence of LNA probes. A contri- buting factor to the performance of LNA probes was the unusually high stability of NitrocH-C base pairs in LNA– DNA hybrids. This phenomenon was also observed with 7-nitro-7-deazaguanine, which exhibited much greater base pairing strength than G in those hybrids. Since LNA–DNA duplexes are less hydrated than DNA duplexes (49), the polar nitro group may preferentially stabilize the mixed hybrid by attracting water molecules to the major groove. Of course, LNA–DNA hybrids diﬀer in many respects from dsDNA and other factors such as base stacking, hydrogen bonding and steric constraints could account for the diﬀerent responses of the two hybrids to 7-nitro-7-deazapurine bases. Since unmodiﬁed DNA probes were unable to discrimi- nate between matched and mismatched NitrocH bases, probes that contain G clamp or LNA C residues were also evaluated. Although both modiﬁcations exhibited a high aﬃnity for NitrocH, indiscriminate pairing was still observed. A more speciﬁc version of NitrocH is clearly needed but how to design such an analog is not obvious. Among the analogs of C that we evaluated, sC has potential for use as a pseudo-complementary base. It forms a very weak pair with G due to steric clash yet does not accommodate mismatching to A, T or C. Eﬃcient incorporation of dsCTP into DNA is made pos- sible by the use of ‘adaptor’ templates in which G has been replaced by H. DNA substituted with nA, sT and sC is structure-free but does not form stable hybrids with short unmodiﬁed probes. The challenge is to ﬁnd an analog of G that speciﬁcally pairs to sC by forming three hydrogen bonds without steric clash. Such an analog could be substituted for G in probes. While inosine can be utilized as a template base in primer extension reactions, it is too indiscriminate to be used in probes. We are actively pur- suing the design and evaluation of novel heterocycles capable of pairing to sC and in parallel are characterizing the ﬁdelity of converting regular DNA into pseudo- complementary DNA by using dNTPs of nA, sT and sC in asymmetric PCR. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. Nucleic Acids Research, 2008, Vol. 36, No. 22 7007 ACKNOWLEDGEMENTS Dr Margarita Salas (Madrid, Spain) generously provided us with the N62D mutant of f29 DNA polymerase. We thank Dr Jeﬀrey Sampson (Agilent Technologies) for his enthusiastic support. We are grateful to aﬃliates of Trilink Biotechnologies including Dr Richard Hogrefe, Dr Gerald Zon, Dr Natasha Paul and Dr David Combs for support and helpful discussions, and Dr Inna Koukhareva and Stephanie Perry for assistance in the synthesis of dNTPs. FUNDING The National Institutes of Health (NIGMS Grant 74564 to H.G.); Agilent Technologies. Funding for open access charge: National Institutes of Health (GM74564). 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