Modern DNA technologies utilize the classic base-pairing principle to rationally design primers and probes to capture and hybridize to their complementary nucleic acids. Though in theory hybridization happens only between perfectly complementary sequences, cross-hybridization occurs if the sequence of one single-stranded DNA (ssDNA) is closely but not completely complementary. Consequently, methods have been exploited to differentiate even a single-base mutation in a DNA sequence to enhance the accuracy of hybridization-based assays. For example, enzymatic reactions target to single-base mutations, while thermal- and salt-induced stringency wash selectively separate partially complementary sequences; both of which either generate extra chemical waste or consumes a large portion of assay time. We report an alternative method of using light to control DNA hybridization stringency by using an optically-responsive azobenzene-modified DNA, which achieves the same degree of hybridization accuracy with single-base resolution. We show that light induces a differential melting such that perfectly matched double-stranded DNA (dsDNA) exhibits a slower melting behavior than that of a dsDNA with one single-base mutation. We further study light-controlled stringency in DNA assays with the goal of making it faster, simpler and more suited to environments where traditional chemical and thermal methods are difficult to use.

Using light to control DNA hybridization stringency for improved DNA assays

248th National Meeting (2014)

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