Unrestricted hybridization of oligonucleotides to structure-free DNA.
The existence of secondary structure in long single-stranded DNA and RNA is a serious obstacle to the practical use of short oligonucleotide probes (<20-mers). Here, we show that replication of a highly structured DNA in the presence of a unique set of dNTP analogues leads to synthesis of daughter DNA with a significantly reduced level of secondary structure. This replicated DNA, composed of 2-aminoadenine, 2-thiothymine, 7-deazaguanine, and cytosine bases, was readily accessible to tiled 8-mer LNA and 15-mer DNA probes, whereas an unmodified version of the same DNA was inaccessible. Importantly, while the base analogues enhanced probe-target stability, they did not significantly reduce the specificity of base pairing. The availability of structure-free DNA targets should facilitate the use of short oligonucleotide probes and promote development of generic oligonucleotide microarrays.
Gamper
Howard B
HB
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. howard.gamper@jefferson.edu
Arar
Khalil
K
Gewirtz
Alan
A
Hou
Ya-Ming
YM
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry; D004261:DNA Replication; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*
D015342:DNA Probes; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D004247:DNA