Published online 5 November 2008 Nucleic Acids Research, 2008, Vol. 36, No. 22 6999–7008 doi:10.1093/nar/gkn797 Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine Georges Lahoud 1 , Victor Timoshchuk 2 , Alexandre Lebedev2, Khalil Arar3, Ya-Ming Hou1 and Howard Gamper1,* 1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, 2TriLink BioTechnologies, 9955 Mesa Rim Road, San Diego, CA 92121, USA and 3Sigma-Proligo, 1 Rue Delaunay, Paris 75011, France Received August 11, 2008; Revised October 5, 2008; Accepted October 10, 2008 ABSTRACT A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were character- ized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly struc- ture-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC. INTRODUCTION Secondary structure in single-stranded DNA and RNA is a signiﬁcant impediment to the use of short oligonucleotide probes in hybridization assays (1–5). Under conditions where oligonucleotides form stable hybrids, long single- stranded nucleic acid targets are highly structured and many sequences are inaccessible to short probes. This phe- nomenon becomes increasingly restrictive as the length of a probe is reduced (6–9). If oligonucleotides <20 nt in length could readily access any sequence in a given nucleic acid target without loss of speciﬁcity, the detection of allelic variants by hybridization would beneﬁt. For example, the availability of structure-free nucleic acid samples would signiﬁcantly improve the sensitivity, resolution and relia- bility of high density oligonucleotide microarrays that are used to detect single nucleotide polymorphisms in whole genome association studies. Pseudo-complementary DNA targets would be ideally suited for use with short oligonucleotide probes since they are by deﬁnition structure-free molecules that can partic- ipate in hybridization reactions. This strategy requires the development of two complementary sets of bases, with one set used in the target DNA and the other set used in the probes. Importantly, the set of bases used in the target DNA should not interact with one another thus leading to a structure-free target. The bases present in the probe can form complementary pairs with each other since sec- ondary structure is less of an issue. In one format the probes contain regular bases and the target DNA contains modiﬁed bases. Alternatively, both the target and probe molecules can be prepared with a mixture of regular and modiﬁed bases. Regardless of which strategy is employed, practical application of the technology requires that pseudo-complementary DNA be readily prepared by primer extension or asymmetric PCR from the sample of interest. In either case the template DNA must contain regular or modiﬁed bases that support uptake of pseudo- complementary base analogs into the DNA product. There is an ongoing need for analogs of G and C that can be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate pseudo-complementary DNA. nA and sT *To whom correspondence should be addressed. Tel: +1 215 503 9798; Fax: +1 215 503 4954; Email: howard.gamper@jefferson.edu 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.