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An autonomous fueled machine that replicates catalytic nucleic acid templates for the amplified optical analysis of DNA

Abstract

Here we describe a protocol for the amplified detection of a target DNA using a DNA/FokI-based replicating cutting machine. The protocol is based on the design of a sensing hairpin oligonucleotide that is opened upon hybridization with the analyte DNA. The endonuclease FokI binds to the double-stranded complex and cleaves it to a “cutter” unit. The “cutter” unit reacts with a fuel oligonucleotide to generate and amplify the signal. The fuel molecule is an oligonucleotide in a hairpin configuration with a fluorophore/quencher pair attached to the 5′ and 3′ ends. Formation of the duplex between the cutter and the fuel leads to the scission of the duplex by FokI, leading to a second, replicated “cutter”, a fluorescent waste product, and to the regeneration of the original “cutter” unit. The autonomous replication of the “cutter” unit, as a result of the primary recognition of the analyte DNA, leads to the amplified fluorescent detection of the analyte DNA with a sensitivity limit of 1 × 10−14 M. The operation of the machine and the sensing process are monitored by the fluorescence generated by the waste product. Here we apply the protocol, which takes about 2 h to complete, to analyze a Tay-Sachs genetic disorder mutant DNA.

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Figure 1: Scheme of the amplified detection of DNA using the FokI/DNA complex as a biocatalytic template for the scission of the fuel substrate (3), yielding the fluorescent waste product, (4) (F = FAM; Q = TAMRA).
Figure 2
Figure 3: Time-dependent changes in fluorescence intensities (λem = 520 nm) upon analyzing different concentrations of the mutant DNA (1): (a) 1 × 10−6 M; (b) 1 × 10−11 M; (c) analysis of the normal gene (1a) (5′- GGGCCATAGGATATACGGTTCAGG-3′, 1 × 10−6 M); (d) analysis of a foreign DNA (5′-CTCCCGTCTAATGCGCTTCCCTGTTTT-3′, 1 × 10−6 M).
Figure 4: Nondenaturating PAGE analysis of the mutant DNA (1) by the hairpin-sensing nucleic acid (2) by the FokI-induced mechanical scission of the fuel (3).

References

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Correspondence to Itamar Willner.

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Weizmann, Y., Cheglakov, Z., Pavlov, V. et al. An autonomous fueled machine that replicates catalytic nucleic acid templates for the amplified optical analysis of DNA. Nat Protoc 1, 554–558 (2006). https://doi.org/10.1038/nprot.2006.78

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