Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2

A method of detecting target polynucleotides in a sample, comprising the steps of:

• (a) providing a sample comprising polynucleotides;
• (b) providing a composition comprising at least one DNA polymerase enzyme, at least one enhancer, and at least one DNA polymerase inhibitor, wherein;
• i) the enhancer is a polynucleotide comprising a sequence that is complementary to a target polynucleotide sequence;
• ii) the DNA polymerase inhibitor is a polynucleotide comprising a conserved region and a variable region, wherein the conserved region is recognized and bound by the DNA polymerase enzyme, and the variable region is complementary to a portion of the enhancer;
• iii) complementary sequences of the variable region of the inhibitor and enhancer form a duplexed inhibitory DNA complex which inhibits DNA polymerase activity;
• iv) the composition comprises an amount of inhibitory complex that has been determined to have the fastest response and/or highest signal-to-noise ratio, by using first derivative of a titration curve of inhibitory complex v polymerase activity and/or by using first derivative of a titration curve of ratios of enhancer:
• inhibitor;
• (c) contacting the sample comprising nucleic acid with the composition of (b), wherein target polynucleotide binding to:
• (i) the enhancer sequence region of the duplex in (b) displaces the inhibitor, thereby releasing and activating the DNA polymerase;
• (d) providing a signaling nanostructure that is reactive to active DNA polymerase enzyme from step (c);
• (e) contacting the signaling nanostructure with active DNA polymerase enzyme from step (c);
• (f) detecting signal development, wherein a change in the intensity of signal indicates the presence of target nucleic acid in the sample when using composition (b).