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.
Highlights
Gene segments are containing highly self-folding structures that can block toehold binding and strand exchange steps involved in Catalytic hairpin assembly (CHA) pathway, inhibiting or ruining the whole reaction (Fig. 1B, column 2, Figure S1)
One single set of organic amide promoted HT-CHA system (OHT-CHA) was 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
The reaction followed a classic four-step pathway[9] shown in Fig. 1A and general rules summarized in our report of HT-CHA15
Summary
Gene segments are containing highly self-folding structures that can block toehold binding and strand exchange steps involved in CHA pathway, inhibiting or ruining the whole reaction (Fig. 1B, column 2, Figure S1) The prevalence of such inhibition induced by unexpected “temperature fluctuation” or “self-folding” has perniciously impeded the broader applications of CHA and other nucleic acid circuits, especially in achieving point-of-care (POC) gene or RNA diagnostics. In the following several decades, these properties of formamide have been successively employed for increasing reaction rate or accuracy of many novel nucleic acid interactions[20,21,22,23,24,25,26,27,28,29], including those milestones such as polymerase chain reaction[20], sequencing[21] and nanostructure assembly[26,27,29] Inspired by these early findings, here we deeply explored the amide (especially formamide)-assistant mechanism in the CHA reaction and showed dramatic enhancement in the buffering ability of a CHA against unexpected self-folding or temperature-cooling. Gene detection based on LAMP turns ultra-reliable and environmental compatible, being ready in POC applications
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