The stepwise endonuclease activity of a thermophilic Argonaute protein

Abstract

Thermophilic Argonaute proteins (Agos) can function as endonucleases via specific guide-target base-pairing cleavage for host defense. The ability to cleave target DNA sequences at any arbitrary sites endows them with reprogramed DNA capacity. Here, we identify that an Ago from the hyperthermophilic archaeon Pyrococcus furiosus (PfAgo) shows a stepwise endonuclease activity, which is demonstrated by the double strand DNA cleavage directed by a single guide DNA rather than canonical one pair of guide DNAs. We reveal that the cleavage products with 5'-phosphorylated ends can used as the renewed guide which is capable to induce next round of cleavage to complementary sequences of target DNA. By combining the PfAgo stepwise endonuclease activity followed by target DNA amplification, we establish a rapid and specific platform for the unambiguously multiplex gene detection, termed RADAR (Renewed-gDNA Assisted DNA-cleavage by Argonaute). In the end, RADAR was applied to distinguish of human papillomavirus of serotypes in patient samples in a single reaction, suggesting that our technique would be adopted for diagnosing application.

Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations

“…Here, we suggest the importance of these hot spots positions for gDNA design, which may contribute to conformational changes between the guide to WT or SNV target thus lead to the discrimination effect. In consistent with our previous results, the adjacent mismatches at 10–11 position of gDNA significantly affect cleavage activity of Pf Ago in a sequence-independent manner ( 39 ). Therefore, we establish the rule for gDNA design: a 16 nt gDNA with introduced single mismatch located at position adjunct with corresponding SNV site, to gain the continuous mismatches at 10–11 position for SNV target, and refer to single mismatch at position either 10 or 11 for WT target, to achieve the best discrimination.…”

“…This result suggests that a single-nucleotide difference in the gDNA for the WT and SNV alleles was not sufficient to precisely differentiate the undesired allele for DNA cleavage under the evaluated conditions. The single nucleotide mismatch between the gDNA to target has been systematically evaluated in our previous study, and the result showed some positions of mismatch affected the cleavage activity but in dependence of target sequences ( 39 ).…”

“…To explore the clinical utility of A-Star, we investigated the LOD of A-Star using a set of commercial cfDNA standards containing genomic DNA at three available VAFs of 0.1%, 1% and 5% for KRAS G12D, PIK3CA E545K and the EGFR indel. Because Pf Ago non-specifically binds DNA template in PCR, we found that a low amount of cfDNA mock samples with 33 ng input was not directly detectable using the A-Star method ( Supplementary Figure S19 ) ( 31 , 39 ). We surmise that Pf Ago bound the DNA sample to some extent and thus decreased the available amount of template for PCR amplification when the initial template concentration was low ( Supplementary Figure S20 ), Thus, we added a pre-amplification step to increase the amount of cfDNA for both the WT and variant alleles before conducting the A-Star process.…”

A Hyperthermophilic Argonaute From Ferroglobus placidus With Specificity on Guide Binding Pattern

“…An artificial restriction enzyme platform based on Pyrococcus furiosus Argonaute ( Pf Ago) was developed to recognize and cleave DNA sequences at virtually any arbitrary site ( Enghiad and Zhao, 2017 ). Furthermore, thermophilic pAgos have been recently used as programmable tools for specific target detection and rare single nucleotide variant enrichment in molecular diagnosis applications ( He et al, 2019 ; Xun et al, 2019 ; Liu et al, 2021a ; Song et al, 2020 ).…”

A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis

“…This device consists of 3D printed casing and internal structure with precise temperature control and fluorescence detection, whereas this assay combines RT-LAMP with an Argonaute protein from hyperthermophilic archaeon Pyrococcus furiosus (PfAgo) capable of precise recognition and cleavage of a target DNA at 95°C as directed by small 5′-phosphorylated single strand DNA (ssDNA) as guide DNA (gDNA) 10,11 . Due to the multi-turnover activity of PfAgo, its secondary cleavage mechanism can be harnessed for specific, sensitive, and multiplex nucleic acid detection 12,13 . For COVID-19 samples, although nasopharyngeal swab and nasal swab samples were recommended for detection of SARS-CoV-2, saliva samples are a more attractive alternative due to the ease, safety, and non-invasive nature of its collection 14,15 , and its relatively high viral load during the first week of infection 16 .…”