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UltraFast PhotoInduced double duplex DNA invasion into a 400-mer dsDNA target
Siddhant Sethi1, Hailili Zumila1, Yasuha Watanabe1
1Biofunctional Medical Engineering Research Area, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa, Japan.
Background:
Natural DNA restriction enzymes bind duplex DNA with high affinity at multiple sites; however, for some of the artificial chemical-based restriction moieties, invasion of the double-strand for efficient cleavage is an obstacle. We have previously reported photo-induced double-duplex invasion (pDDI) using 3-cyanovinylcarbazole (K)-containing probes for both the target strands that photo-crosslink with pyrimidine bases in a sequence-specific manner on both the strands, stabilizing the opened double-strand for cleavage. The drawback of the pDDI was low efficiency due to inter-probe cross-linking, solved by the inclusion of 5-cyano-uridine at -1 position on the complimentary strand with respect to K in both probes. Although this led to reduced inter-probe cross-linking, the pDDI efficiency was still low.
Results:
Here, we report that inter-probe cross-linking and intra-probe cross-linking of a single probe is also leading to reduced pDDI efficiency. We addressed this problem by designing DDI probes to inhibit both inter-probe and intra-probe cross-linking.
Conclusion:
Based on the new design of pDDI probe with 5-cyano uridine led to a drastic increase in the efficiency of pDDI in (400-mer) double-stranded DNA with only 1 s of photo-irradiation.
