Self-priming phosphorothioated hairpin-mediated isothermal amplification

Highlights

• A novel and simple isothermal amplification method capable of detecting target nucleic acid without the requirement for complex multiple primer sets and/or enzymes is developed.

• Despite the ultra-simplified mechanism requiring only a single DNA probe and a single enzyme, the SP-HAMP reaction identified the target DNA down to a single copy.

• The practical diagnostic capability was also verified by reliably identifying practically long target DNA and target RNA molecules.

Abstract

We herein describe a novel technology, termed self-priming phosphorothioated hairpin-mediated isothermal amplification (SP-HAMP), enabling target nucleic acid detection. Isothermal amplification strategies are a simple process that efficiently raises the amount of nucleic acid at a constant temperature, but still has lots of problems such as the requirement of multiple exogenous primers and enzymes, which trigger non-specific background signal and increase the complexity of procedures. The key component for overcoming the above-mentioned limitations is the designed hairpin probe (HP) consisting of self-priming region along the 3′ stem and the 3′ overhang and phosphorothioate modifications at the 5′ overhang and the specific loop part. The HP was designed to open through binding to target nucleic acid. Upon opening of HP, its self-priming (SP) region is rearranged to form a smaller hairpin whose 3′ end could serve as a primer. The following extension produces the extended HP and displaces the bound target nucleic acid, which is then recycled to open another HP. Due to the reduced stability caused by the specific two phosphorothioate (PS) modifications, the 3′ end of EP1 is readily rearranged to form the foldback hairpin structure, which would promote the foldback extension to produce once more extended HP. Since the two PS modifications are always located at the same positions along the 5’ stem within the further extended HPs, the foldback reaction followed by the extension would be continuously repeated, consequently producing a large number of the long hairpin concatamers. Based on this unique design principle, we successfully detected even a single copy of target DNA with outstanding discrimination capability under an isothermal condition by employing only a single HP without the requirement for the complicated multiple primers. In conclusion, the sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling short target nucleic acid detection such as microRNAs or any target which is less than 200 mer.

Introduction

During the past few decades, the isothermal amplification strategies have shown great promise as an alternative technique to the conventional PCR method due to their intrinsic advantageous features, which include low hardware dependence eliminating the requirement for thermocycling machine, rapid amplification without separated denaturation step, and a simplified reaction procedure (Bae et al., 2020; Chen et al., 2017; Wang et al., 2017). Since these features are very critical for realizing point-of-care (POC) or on-site molecular diagnostics under resource-limited settings (Erlich et al., 1991; Gill and Ghaemi 2008; Li and Macdonald 2015; Saiki et al., 1988), various types of isothermal amplification methods such as strand displacement amplification (SDA) (Spargo et al., 1996; Walker et al., 1992), nucleic acid sequence-based amplification (NASBA) (Compton 1991; Guatelli et al., 1990), rolling-circle amplification (RCA) (Fire and Xu 1995; Lizardi et al., 1998; Park et al., 2018), helicase-dependent amplification (HDA) (An et al., 2005; Vincent et al., 2004), recombinase polymerase amplification (RPA) (Crannell et al., 2014; Piepenburg et al., 2006), exponential amplification reaction (EXPAR) (Park et al., 2016; Van Ness et al., 2003), isothermal chain amplification (ICA) (Jung et al. 2010, 2011), loop-mediated amplification (LAMP) (Mori et al., 2006; Notomi et al., 2000), and so on (Jang et al., 2019; Kim et al., 2020; Lee et al., 2020; Song et al., 2020) have been extensively developed.

Of these, the LAMP method has been considered as one of the most attractive isothermal amplification methods due to its powerful amplification efficiency by employing only a single enzyme and has proven its utility for molecular diagnostics in numerous clinical applications (Notomi et al., 2000; Parida et al., 2008; Golinelli and Hughes 2001; Tattersall and Ward 1976; Zhang et al., 2014). Notably, several diagnostic kits to identify SARS-COV-2 virus have been recently developed and already commercialized by relying on the LAMP method in the current ongoing pandemic of COVID-19 (Augustine et al., 2020; Zhu et al., 2020; Park et al., 2020; Yu et al., 2020; Zhang et al., 2020).

In the LAMP, the use of foldback structured primers and their subsequent extension simplifies the amplification step by eliminating the requirement for the complicated combination of enzymes or proteins utilized for other isothermal amplification methods such as NASBA, E-SDA, HRCA, HDA, and RPA (Zhao et al., 2015). However, the requirement for multiple exogenous primers to accomplish the amplification under an isothermal condition may give rise to the complexity of the primer design and cumbersome procedures. Moreover, the relatively high operating temperature of the LAMP (60–65 °C), when compared with several other methods working at lower temperatures around 40 °C (An et al., 2005; Fire and Xu 1995; Guatelli et al., 1990; Walker et al., 1992), also potentially increases device complexity and power consumption. Therefore, there has been a great incentive existing for the development of a more advanced method to isothermally amplify target nucleic acid by eliminating the above-mentioned limitations while preserving the high amplification efficiency of the LAMP.

Upon this background, we herein describe an ultra-simplified advanced version of loop-mediated isothermal amplification (LAMP) actuated by a single self-priming phosphorothioated hairpin probe, termed self-priming phosphorothioated hairpin-mediated isothermal amplification (SP-HAMP). We particularly focused on the recent finding that the phosphorothioate (PS)-modified DNA (PS-DNA) destabilizes base stacking interaction within the double-helical structure and consequently reduces the melting temperature (T_m) of the PS-DNA/DNA duplex (Boczkowska et al., 2002; LaPlanche et al., 1986). By taking advantage of this phenomenon, we designed hairpin probe (HP) to contain PS-DNA modification site, target recognition site, and self-priming (SP) region, and successfully verified that the SP-HAMP reaction based on the HP could isothermally amplify and reliably identify target nucleic acid in an ultra-simplified manner employing only a single HP and a single enzyme.