Strand-Exchange Nucleic Acid Circuitry with Enhanced Thermo-and Structure- Buffering Abilities Turns Gene Diagnostics Ultra-Reliable and Environmental Compatible

Abstract

Catalytic hairpin assembly (CHA) is one of the most promising nucleic acid amplification circuits based on toehold-mediated strand exchange reactions. But its performance is usually ruined by fluctuated environmental temperatures or unexpected self-structures existing in most real-world targets. Here we present an amide-assistant mechanism that successfully reduces the prevalence of these problems for CHA and maximizes its thermo- and structure- buffering abilities. Such an organic amide-promoted CHA (shortened as OHT-CHA) can unprecedentedly amplify through 4 °C to 60 °C without rebuilding sequences or concerning target complexity. We are then for the first time able to employ it as a direct and universal signal booster for loop mediated isothermal reaction (LAMP). LAMP is one of the most promising point-of-care (POC) gene amplifiers, but has been hard to detect precisely due to structured products and haunted off-target amplicons. OHT-CHA guarantees a significant and reliable signal for LAMP reaction amplified from as little as 10−19 M virus gene. And one single set of OHT-CHA is qualified to any detection requirement, either in real-time at LAMP running temperature (~60 °C), or at end-point on a POC photon counter only holding environmental temperatures fluctuating between 4 °C to 42 °C.

Establishment of a universal and rational gene detection strategy through three-way junction-based remote transduction

“… 17 , 20 , 26 , 27 , 30 , 31 Through these methods, sequence-specific detection of these targets could be realized over a wide temperature range from 4 °C to 60 °C. 31 , 32 In order to realize a universal well-designed output OSD or CHA to detect different input targets, we make an important modification on the trigger. As shown in Fig.…”

Effective design principles for leakless strand displacement systems

“…Despite these challenges, there is a body of work demonstrating strand displacement cascades with natural DNA and RNA inputs, including logic computation both in vitro (9) and in vivo (25), a molecular multigene classifier (21), regulation of cellular gene expression (26,27), and in situ fluorescence imaging of mRNA expression (55). Destabilizing organic solvents (56) and the hybridization probe method (21) can be generally adopted to overcome secondary structure in strand displacement. We envision that, with these techniques, leakless architectures could be used to interface with biological sequences.…”

One-tube smart genetic testing via coupling isothermal amplification and three-way nucleic acid circuit to glucometers

“…The whole reaction components are high temperature-resistant. Until now CHA has been the only type of enzyme-free circuitry that is well engineered to execute at very large temperature window, through 4 C to as high as 60 C [34,35]. Also we have previously engineered a thermo-stable invertase that can keep efficient at even 95 C [21].…”

“…1, the basic principle says that only the domain b in TP, and domain a in CHA H1 were relevant to the T1. Therefore, except domain b* of TP, and a* of H1, we directly borrow all other CHA component sequences from a well-performed CHA circuit previously designed for other purpose [31,35]. Classic fluorescence resonance energy transfer (FRET) experiments have been firstly performed to test the efficiency of 3W-CHA reaction (scheme shown in Fig.…”