Chem Commun. (2016) Oct 27;52(88):13043-13046. doi: 10.1039/c6cc07153h.

Spatial organization based reciprocal switching of enzyme-free nucleic acid circuits

Yidan Tang,ab Zhentong Zhu,ab Baiyang Lua and Bingling Lia

a State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun, Jilin, 130022, P. R. China.

b University of Chinese Academy of Sciences, Beijing, 100039, China.

 

Abstract

We report a new nucleic acid sensing strategy through an intelligent design of spatial organization based reciprocal switching of catalytic hairpin assembly (CHA). The so-called SORS-CHA not only turns a well-designed CHA circuit into a relatively universal detector for any targeting sequence, but also guarantees a much enhanced signal resolution and a believability to minimize the misreading induced by unexpected signal drifts. With more trustworthy results, but a simpler sequence design, nucleic acid circuits could become competitive in real-world applications.

PMID: 27757452

 

Supplement

CHA has been widely applied in the detection of various species since it can amplify ten-to-a-hundred fold with a remarkable signal-to-background ratio. However, while the CHA circuit has provided interesting opportunities in molecular diagnostics, its further use in more practical applications still suffers from several challenges. For example, in order to detect different targets the circuit sequences should be rebuilt which is laborious and usually takes great risk of encountering design failure [1]. Besides, even though the circuits are compatible with multiple analytical readouts, such as electrochemistry and fluorescence, many of them have a high noise level that can frequently produce target non-relevant signal drifts. These hard-to-avoid signal drifts can result in misreading and seriously affect the veracity of analysis [2]. With the purpose to provide a solution for the above shortcomings, we reported an enhanced nucleic acid sensing strategy through an intelligent design of spatial organization based reciprocal switching of CHA (SORS-CHA). This strategy could provide universal and intelligent transiting strategy to transduce different input oligonucleotide targets into well-designed output probes which makes nucleic acid circuits become competitive in real-world applications.

The concept of “spatial organization” derives  from our proceeding work, which means that two segments (TH and BM) can be brought close enough via an organizer sequence to trigger an efficient CHA reaction like a single-stranded catalyst (C1) [3]. This SORS-CHA strategy turns a well-designed CHA into a universal detector for different input targets. Meanwhile, this strategy allows a targeting sequence turning off (inhibiting) a CHA circuit while simultaneously turning on (activating) another one. The deviation signal (On signal- Off signal) can also illustrated as a reciprocal switch, which guarantee a much enhanced signal resolution and believability to minimize the misreading induced by unexpected signal drifts.

We have engineered two “identically behaved” CHA circuits to guarantee a high efficiency of SORS-CHA. This strategy can be examined in two tubes pattern using a fluorescein amidite (FAM) as a fluorescent probe to monitor both CHA reaction processes. By replacing the fluorescent probe of CHA2 from FAM with rhodamine (ROX), our design could be more conveniently integrated into only one tube which made the design flexible enough to meet different detection requirements.

We further employ this strategy to detect real world sequences Zaire Ebolavirus gene (ZEBOV). Though designing target sequence as one of the loop products of the ZEBOV LAMP reaction, we could directly use the SORS-CHA to monitor LAMP products amplified from a trance amount of the ZEBOV gene. As our proceeding work demonstration [4,5], a nucleic acid circuit could provide amplified and sequence-specific detection against false-positive signals generating from non-specific LAMP amplications. Here, more importantly, a reciprocal switching signal could provide a double confirmed confidence against unexpected false fluorescence drifts.

 

 

References

[1] Li B, Ellington AD, Chen X. Rational, modular adaptation of enzyme-free DNA circuits to multiple detection methods. Nucleic Acids Res. 2011; 39: e110.
[2] Zhang Y, Lei J, Ling P, Ju H. Catalytic hairpin assembly-programmed porphyrin–DNA complex as photoelectrochemical initiator for DNA biosensing. Anal Chem. 2015; 87: 5430–5436.
[3] Li B, Jiang Y, Chen X, Ellington AD. Probing spatial organization of DNA strands using enzyme-free hairpin assembly circuits. J Am Chem Soc. 2012; 134: 13918–13921.
[4] Du Y, Hughes RA, Bhadra S, Jiang Y, Ellington AD, Li B. A sweet spot for molecular diagnostics: coupling isothermal amplification and strand exchange circuits to glucometers. Sci Rep. 2015; 5: 11039.
[5] Li B, Chen X, Ellington AD. Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions. Anal Chem. 2012; 84: 8371-8377.