Nucleic Acids Research Advance Access originally published online on April 29, 2008
Nucleic Acids Research 2008 36(10):3409-3419; doi:10.1093/nar/gkn209
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Nucleic Acids Research, 2008, Vol. 36, No. 10 3409-3419
© 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 |
Enzymatic synthesis of structure-free DNA with pseudo-complementary properties
1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, 2TriLink BioTechnologies, 9955 Mesa Rim Road, San Diego, CA 92121, USA, 3Instituto de Biologia Molecular ‘Eladio Viñuela’ (CSIC), Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Campus Universidad Autonóma, Canto Blanco, 28049, Madrid, Spain and 4Sigma-Proligo, 1 Rue Delaunay, Paris 75011, France
*To whom correspondence should be addressed. Tel: +215 503 9798; Fax: +215 503 4954; Email: howard.gamper{at}jefferson.edu
Received March 16, 2008. Revised April 7, 2008. Accepted April 8, 2008.
Long single-stranded DNAs and RNAs possess considerable secondary structure under conditions that support stable hybrid formation with oligonucleotides. Consequently, different oligomeric probes can hybridize to the same target with efficiencies that vary by several orders of magnitude. The ability to enzymatically generate structure-free single-stranded copies of any nucleic acid without impairing Watson–Crick base pairing to short probes would eliminate this problem and significantly improve the performance of many oligonucleotide-based applications. Synthetic nucleic acids that exhibit these properties are defined as pseudo-complementary. Previously, we described a pseudo-complementary A-T couple consisting of 2-aminoadenine (nA) and 2-thiothymine (sT) bases. The nA-sT couple is a mismatch even though nA-T and A-sT are stable base pairs. Here we show that 7-alkyl-7-deazaguanine and N4-alkylcytosine (where alkyl = methyl or ethyl) can be used in conjunction with nA and sT to render DNA largely structure-free and pseudo-complementary. The deoxynucleoside triphosphates (dNTPs) of these bases are incorporated into DNA by selected mesophilic and thermophilic DNA polymerases and the resulting primer extension products hybridize with good specificity and stability to oligonucleotide probes composed of the standard bases. Further optimization and characterization of the synthesis and properties of pseudo-complementary DNA should lead to an ideal target for use with oligonucleotide probes that are <25 nt in length.
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