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Kinetically Modulated Specificity against Single-base Mutants in Nucleic Acid Recycling Circuitry Using Destabilization Motif

Abstract

Signal amplification in nucleic acid sensing improves detection sensitivity, but difficulties remain in sustaining specificity over time particularly under excess amount of single-base mutants. Here, we report a simple, self-refining target recycling circuitry, which cumulates differentiation between on and off targets by the 2-step cyclic interaction with the sensing probe. In the reaction, analyte recylces only if the protective strand of the Sensing Probe is removed. The dissociation kinetics of such interaction was modulated by reacting with different lenghts of assistant strands.When shorter assistant strands are used, the destabliization motif of the Sensing Probe has to spontaneously dissociate before another assistant strand approaches and fully displaces it. This sets up a high kinetic barrier sensitive to the subtle reaction energy differences imposed by the single-base mutants, and substantially improved specificity. As a proof of concept, a microRNA 21 DNA analogue, was chosen as our target analyte; together with its 14 point mutants (substitution, insertion, or deletion) for specificity measurements. The experimental results corroborate that our system amplifies signal comparable to traditional one-layer recycling approach but with negligible system leakage. With the use of shortened assistant strands, up to 100 fold increase in discrimination factor against the single-base mutants is observed. Specificity is sustainable or even increased over long period measurement (i.e. 4 days). More importantly, target differentiation is successfully demonstrated even in excess amount of spurious analogs (100X) and low target frequency mixture (i.e. 0.1%), which mimic the lean conditions practically encountered. Explicit mechanisms of the system’s specificity are elucidated through analytical calculation and free energy level diagrams. The modularity of destabilization motif herein promises detection of different nucleic acid based targets and integration to other signal amplification approaches for specificity enhancement.

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Supplementary files

Publication details

The article was received on 27 Dec 2016, accepted on 10 Jun 2017 and first published on 12 Jun 2017


Article type: Paper
DOI: 10.1039/C6AN02731H
Citation: Analyst, 2017, Accepted Manuscript
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    Kinetically Modulated Specificity against Single-base Mutants in Nucleic Acid Recycling Circuitry Using Destabilization Motif

    T. W. Fan and I. Hsing, Analyst, 2017, Accepted Manuscript , DOI: 10.1039/C6AN02731H

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