7006 Nucleic Acids Research, 2008, Vol. 36, No. 22 mismatched hybrids when the DNA target contained H, cH or IcH. Attempts to enhance the speciﬁcity of NitrocH by utilizing 12-mer DNA probes substituted with stabiliz- ing analogs of C were unsuccessful. For example, even though the tricyclic aminoethyl-phenoxazine analog of C [(47); the so-called ‘G-clamp’] formed a very stable base pair with NitrocH and increased hybrid stability by 128C, mispairing of the standard bases to NitrocH was still observed under stringent conditions. Hybridization of tiled probes to modified hairpins The HP25 hairpin provided an opportunity to test whether highly structured DNA substituted with nA, sT and H or NitrocH could hybridize to 8-mer LNA probes under non-denaturing conditions. The probes were tiled in two base increments across the 30 arm of the hairpin stem. Each probe-hairpin combination was incubated for 5–10 min at 308C in the presence of 5 mM probe and limit- ing hairpin target. Gel mobility shift analysis of the reac- tions showed that unmodiﬁed hairpin hybridized with an average eﬃciency of only 3.5% to the probe set. Introduc- tion of nA and sT into the hairpin increased hybridization eﬃciency to 34%, underscoring the need to destabilize G-C pairing as well. NitrocH was emphasized in this study since it had the best uptake and pairing properties of the analogs studied. For comparison, we also prepared hairpins that were substituted with H. In the presence of nA and sT, both G analogs promoted robust hybridiza- tion with the LNA probes (Figure 4A). Identical results were obtained using a parallel set of tiled 12-mer DNA Figure 4. Hybridization of tiled 8-mer LNA probes to (A) the HP25 hairpin at 308C and (B) the H18 hairpin at 458C. Each hairpin was substituted with nA, sT and NitrocH or H. Probes to HP25 were tiled across the 30 arm of the hairpin stem while probes to HP18 were tiled across both arms of the hairpin stem. Gel mobility shift assay was used to determine the fraction of hairpin that was hybridized to each probe. probes (data not shown). The ready accessibility of the hairpin sequences indicates that the modiﬁed bases had eliminated secondary structure. The ability of NitrocH to destabilize a more highly G/C rich duplex was tested by hybridizing a tiled set of 8-mer LNA probes to both arms of an 18 bp hairpin (HP18; Figure 1B) that was also substituted with nA and sT. With a G/C content of 78% and an estimated Tm of 968C the unmodiﬁed HP18 control hairpin hybridized poorly to the probes. When the same set of probes was hybridized to hairpin substituted with nA, sT and NitrocH at 308C, the average hybridization level was only 33%, suggesting the presence of residual secondary structure in the hairpin target. This hypothesis was conﬁrmed by demonstrating that the extent of hybridization increased to 50% when the temperature was raised to 458C (Figure 4B). At this temperature NitrocH and H were nearly interchangeable. Interestingly, G-rich probes/hybri- dized to a greater extent than C-rich probes, possibly reﬂecting a decrease in hybrid stability that was propor- tional to the number of NitrocH–C or H–C base pairs in the duplex. Signiﬁcant probe-to-probe variation was also observed, with one C-rich probe giving <10% hybrid. A more uniform hybridization proﬁle was obtained by using a parallel set of tiled 12-mer DNA probes that contained LNA C residues (data not shown). DISCUSSION We have previously shown that pseudo-complementary DNA can be generated from regular DNA by carrying out primer extension with the dNTPs of nA, sT, H and cytosine (11). DNA composed of these bases can be synthesized by DNA polymerases and is nearly struc- ture-free since nA–sT is a mismatch and H–C is a very weak base pair. Hybridization of this DNA to unmodiﬁed probes is driven by the formation of nA–T, A–sT, G–C and H–C base pairs. Unfortunately, H exhibits degenerate pairing and cannot be used in hybridization assays where speciﬁcity is required. In this study we have evaluated the uptake and base pairing properties of several highly desta- bilizing analogs of G and C that might function similarly to H but with greater speciﬁcity. Several of the base analogs evaluated in this study have anti-viral or anti-cancer activity. When incorporated into DNA, P is potent inhibitor of cytosine methyltransferase (14). Our results suggest that this is probably a rare event since P/MeP did not form a stable base pair with G and was a poor substrate for primer extension. The anti-viral activity of MefP type bases does not depend upon their incorporation into DNA (15). This is consistent with our observations that MefP paired weakly to G and was a marginal substrate for primer extension. The anti-cancer agent dsGTP was readily incorporated into DNA as long as the template lacked secondary structure. sG paired weakly to C and mispaired to other bases. It has been shown that mis-incorporation of bases opposite sG contributes to the cytotoxicity of this agent (48). NitroC exhibits anti-viral activity by binding tightly to viral poly- merases (16). In our study this base was less destabilizing