Book of Abstracts: Albany 2007
June 19-23 2007
Binary Probes for Selective Nucleic Acid Recognition
Numerous techniques for DNA/RNA analysis rely on the ability of the probe to recognize nucleic acid sequences specifically by forming duplexes. The formation of at least 15-20 nucleotide hybrids between the probe and the analyte is required to uniquely define a specific fragment in a nucleic acid the size of a genome. Hybrids of such length are too stable to be sensitive to a base miss-pairing; since a single mismatch unit results in a relatively small energetic penalty (1). It was shown that two-component probes can improve selectivity of nucleic acid recognition (2, 3). Each fragment of such probes binds to a relatively short (7-10 nucleotide) analyte fragment. This makes the hybrid to be extremely sensitive to a single base substitution even at mild conditions.
Three embodiments of the binary probes for nucleic acid analysis will be presented. First example is binary malachite green aptamer probe, which is made purely of RNA and can reliably discriminate 41 out of 42 possible single nucleotide substitutions in 14-mer DNA analyte at near physiological conditions. Second, binary DNA probe uses molecular beacon as a fluorescent reporter. It distinguishes single nucleotide substitutions at any position of 20 nucleotide DNA analytes at room temperature. And third, binary deoxyribozyme probe not only selectively recognizes 20 mer oligodeoxyribonucleotides at mild conditions, but also improves sensitivity by catalytic amplification of the positive signal.
This work was supported by the NIH, NHGRI R21 HG004060.
References and Footnotes
Dmitry M. Kolpashchikov
Division of Experimental Therapeutics