Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly

Abstract

Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise.

Figure 1

Scheme of CHA circuit. Complementary domain strands are indicated with an asterisk such that Domain 1 complements Domain 1*.

Figure 2

A) Fluorescence response of LT-CHA at 37 °C and 60 °C, with and without catalyst. B) Fluorescence response of HT-CHA at various temperatures, with and without catalyst. C) Initial rates of LT-CHA and HT-CHA at various temperatures, with and without catalyst, as calculated from the data in A & B. D) Initial rate vs. C_HT concentrations for HT-CHA at 60 °C (r² = 0.999). The sequences and conditions used in these experiments are found in Tables S3 and S4 respectively.

Figure 3

A) Scheme for CHA as a real-time detector of linear RCA. B) Concentration dependence of Circular T_HTRCA of RCA-CHA at 60 °C in the ThermoPol reaction, using EHT-CHA and Bst large fragment polymerase. C) Concentration dependence of Circular T_LTRCA of RCA-CHA at 37 °C in the Phi29 reaction, using EHT-CHA and Phi29 polymerase. The sequences and conditions used in these experiments are found in Tables S3 and S4 respectively.

Figure 4

High-temperature CHA transduction is sensitive and specific. A) 1% agarose gel of the product generated from a 2 hour RCA reaction with and without Circular T_HTRCA. B) The same RCA products from Figure A were diluted 4-fold with EHT-CHA, followed by a CHA reaction in ThermoPol reaction conditions at 60 °C. The concentrations listed are those before dilution. C) 1% agarose gel of the product generated from an overnight RCA reaction with and without Circular T_HTRCA. D) The same RCA products from Figure C were diluted 4-fold with HT-CHA, followed by a CHA reaction in ThermoPol reaction conditions at 60 °C. The concentrations listed are those before dilution.

Figure 5

A) Scheme for CHA as a real-time detector of SDA. B) Concentration dependence of Circular T_HTSDA of SDA-CHA at 60 °C in the NEBuffer 2 reaction using EHT-CHA, Bst large fragment polymerase, and Nb. BsrDI. C) Concentration dependence of Circular T_LTSDA of SDA-CHA at 37 °C in the NEBuffer 2 reaction using LT-CHA, Klenow (3’–5’exo-) polymerase, and Nb.BbvCI. The sequences and conditions used in these experiments are found in Tables S3 and S4 respectively.