Nucleic Acids Research Advance Access originally published online on November 5, 2008
Nucleic Acids Research 2008 36(22):6999-7008; doi:10.1093/nar/gkn797
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2008, Vol. 36, No. 22 6999-7008
© 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.
Chemistry and Synthetic Biology |
Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine
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
*To whom correspondence should be addressed. Tel: +1 215 503 9798; Fax: +1 215 503 4954; Email: howard.gamper{at}jefferson.edu
Received August 11, 2008. Revised October 5, 2008. Accepted October 10, 2008.
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 characterized 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 structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.