Characterizing the Impact of Primer-Template Mismatches on Recombinase Polymerase Amplification

Abstract

Recombinase polymerase amplification (RPA) is an isothermal amplification assay that has been ubiquitously utilized in the detection of infectious agents. Like any nucleic acid amplification technology, primer-template complementarity is critical to RPA reaction success. Mismatches arising in the primer-template complex are known to impact reaction kinetics, invalidate downstream analysis, such as nucleic acid quantification, and result in false negatives if used in a diagnostic capacity. Although the impact of specific primer-template mismatches has been well characterized for techniques such as PCR, characterization remains limited for RPA. Through our study, we systematically characterize the impact of mismatches on the RPA reaction, when located in the 3′-anchor region of the primer-template complex. Our investigation identified that the nucleotides involved, as well as position of each mismatch, influence the size of the impact, with terminal cytosine-thymine and guanine-adenine mismatches being the most detrimental. The presence of some mismatch combinations, such as a penultimate cytosine-cytosine and a terminal cytosine-adenine mismatch pairing, led to complete RPA reaction inhibition. Through the successful characterization of 315 mismatch combinations, researchers can optimize their RPA assay accordingly and seek to implement RPA technology for rapid, in-field genotyping. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that has been ubiquitously utilized in the detection of infectious agents. Like any nucleic acid amplification technology, primer-template complementarity is critical to RPA reaction success. Mismatches arising in the primer-template complex are known to impact reaction kinetics, invalidate downstream analysis, such as nucleic acid quantification, and result in false negatives if used in a diagnostic capacity. Although the impact of specific primer-template mismatches has been well characterized for techniques such as PCR, characterization remains limited for RPA. Through our study, we systematically characterize the impact of mismatches on the RPA reaction, when located in the 3′-anchor region of the primer-template complex. Our investigation identified that the nucleotides involved, as well as position of each mismatch, influence the size of the impact, with terminal cytosine-thymine and guanine-adenine mismatches being the most detrimental. The presence of some mismatch combinations, such as a penultimate cytosine-cytosine and a terminal cytosine-adenine mismatch pairing, led to complete RPA reaction inhibition. Through the successful characterization of 315 mismatch combinations, researchers can optimize their RPA assay accordingly and seek to implement RPA technology for rapid, in-field genotyping. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique (NAAT) that has been ubiquitously implemented in the detection of human and plant pathogens.1El Wahed A.A. Patel P. Maier M. Pietsch C. Rüster D. Böhlken-Fascher S. Kissenkötter J. Behrmann O. Frimpong M. Diagne M.M. Faye M. Dia N. Shalaby M.A. Amer H. Elgamal M. Zaki A. Ismail G. Kaiser M. Corman V.M. Niedrig M. Landt O. Faye O. Sall A.A. Hufert F.T. Truyen U. Liebert U.G. Weidmann M. Suitcase lab for rapid detection of SARS-CoV-2 based on recombinase polymerase amplification assay.Anal Chem. 2021; 93: 2627-2634Crossref PubMed Scopus (63) Google Scholar,2Babu B. Ochoa-Corona F.M. Paret M.L. Recombinase polymerase amplification applied to plant virus detection and potential implications.Anal Biochem. 2018; 546: 72-77Crossref PubMed Scopus (49) Google Scholar The RPA system relies on three T4 phage proteins, UvsX, UvsY, and Gp32. UvsX and UvsY facilitate priming of the DNA target through the assembly of a nucleoprotein filament, and Gp32 stabilizes the displaced single-stranded DNA during D-loop formation.3Piepenburg O. Williams C.H. Stemple D.L. Armes N.A. DNA detection using recombination proteins.PLoS Biol. 2006; 4: e204Crossref PubMed Scopus (1466) Google Scholar Together, this equates to the denaturation and primer annealing steps of a typical PCR cycle. RPA's performance at 37°C to 42°C makes it ideal for use in low-resource field settings, as demonstrated during the 2015 Ebola outbreak.4Yang M. Ke Y. Wang X. Ren H. Liu W. Lu H. Zhang W. Liu S. Chang G. Tian S. Wang L. Huang L. Liu C. Yang R. Chen Z. 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A polymorphic DNA marker genetically linked to Huntington’s disease.Nature. 1983; 306: 234-238Crossref PubMed Scopus (1725) Google Scholar under certain conditions, RPA can be performed in the absence of a heat block,5Crannell Z.A. Rohrman B. Richards-Kortum R. Equipment-free incubation of recombinase polymerase amplification reactions using body heat.PLoS One. 2014; 9: e112146Crossref PubMed Scopus (198) Google Scholar highlighting its potential as the basis for future diagnostics. RPA reaction success depends on robust primer design, like any NAAT.8Higgins M. Ravenhall M. Ward D. Phelan J. Ibrahim A. Forrest M.S. Clark T.G. Campino S. PrimedRPA: primer design for recombinase polymerase amplification assays.Bioinformatics. 2019; 35: 682-684Crossref PubMed Scopus (48) Google Scholar This process balances several factors, which include the following: ensuring primer specificity by maximizing Watson-Crick nucleotide base pairing,9Watson J.D. Crick F.H. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid.Nature. 1953; 171: 737-738Crossref PubMed Scopus (8860) Google Scholar minimizing the potential of off-site binding to nontarget DNA, and minimizing primer-derived secondary structures, which can impede the reaction with varying degrees of severity.10Lobato I.M. O’Sullivan C.K. Recombinase polymerase amplification: basics, applications and recent advances.Trends Analyt Chem. 2018; 98: 19-35Crossref PubMed Scopus (471) Google Scholar Technique-specific primer design software has been developed, such as PrimedRPA, Primer3, and PrimerExplorer, to assist this task and overcome limitations associated with manual primer design.8Higgins M. Ravenhall M. Ward D. Phelan J. Ibrahim A. Forrest M.S. Clark T.G. Campino S. PrimedRPA: primer design for recombinase polymerase amplification assays.Bioinformatics. 2019; 35: 682-684Crossref PubMed Scopus (48) Google Scholar,11Untergasser A. Nijveen H. Rao X. Bisseling T. Geurts R. Leunissen J.A.M. Primer3Plus, an enhanced web interface to Primer3.Nucleic Acids Res. 2007; 35: W71-W74Crossref PubMed Scopus (2003) Google Scholar However, the presence of unknown polymorphic sites in the primer binding region can compromise an NAAT reaction, causing nucleotide mismatches and reducing the stability of the primer-template complex.12Ziegler K. Steininger P. Ziegler R. Steinmann J. Korn K. Ensser A. SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene.Euro Surveill. 2020; 25: 2001650Crossref PubMed Scopus (100) Google Scholar,13Allawi H.T. SantaLucia Jr., J. Thermodynamics of internal C.T mismatches in DNA.Nucleic Acids Res. 1998; 26: 2694-2701Crossref PubMed Scopus (200) Google Scholar This reduction is particularly an issue for organisms with high genetic diversity, limited genomic characterization, or error-prone replication systems.14Cuevas J.M. Geller R. Garijo R. López-Aldeguer J. Sanjuán R. Extremely high mutation rate of HIV-1 in vivo.PLoS Biol. 2015; 13: e1002251Crossref PubMed Scopus (229) Google Scholar Previous work has shown how mismatches can acutely disrupt NAAT amplification.15Stadhouders R. Pas S.D. Anber J. Voermans J. Mes T.H.M. Schutten M. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5′ nuclease assay.J Mol Diagn. 2010; 12: 109-117Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar,16Kwok S. Kellogg D.E. McKinney N. Spasic D. Goda L. Levenson C. Sninsky J.J. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.Nucleic Acids Res. 1990; 18: 999-1005Crossref PubMed Scopus (853) Google Scholar Initial reports highlighted that RPA has a high tolerance to polymorphisms in the primer/probe binding sites,17Boyle D.S. Lehman D.A. Lillis L. Peterson D. Singhal M. Armes N. Parker M. Piepenburg O. Overbaugh J. Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.MBio. 2013; 4: e00135-13Crossref PubMed Scopus (203) Google Scholar but subsequent work has identified that primer-template mismatches can impact RPA reaction efficiency and success, although they were unable to predict the impact based on the presence of a single mismatch.18Daher R.K. Stewart G. Boissinot M. Boudreau D.K. Bergeron M.G. Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology.Mol Cell Probes. 2015; 29: 116-121Crossref PubMed Scopus (129) Google Scholar Single or multiple mismatches located toward the 3′ terminus of the primer result in the most severe disruption for PCR, significantly reducing amplification efficiency and, in certain cases, preventing amplification altogether.15Stadhouders R. Pas S.D. Anber J. Voermans J. Mes T.H.M. Schutten M. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5′ nuclease assay.J Mol Diagn. 2010; 12: 109-117Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar However, this phenomenon can be utilized to facilitate single-nucleotide polymorphism (SNP) genotyping, as demonstrated by amplification-refractory mutation system–PCR, kompetitive allele-specific PCR, and other techniques.19Little S. Amplification-refractory mutation system (ARMS) analysis of point mutations.Curr Protoc Hum Genet. 2001; (Chapter 9:Unit 9.8)Crossref PubMed Google Scholar,20Semagn K. Babu R. Hearne S. Olsen M. Single nucleotide polymorphism genotyping using kompetitive allele specific PCR (KASP): overview of the technology and its application in crop improvement.Mol Breed. 2014; 33: 1-14Crossref Scopus (648) Google Scholar The genotyping of informative SNPs can personalize treatment choices and inform related fields, such as pharmacogenomics, where these biomarkers can be integrated into treatment decision pathways.21Roden D.M. McLeod H.L. Relling M.V. Williams M.S. Mensah G.A. Peterson J.F. Van Driest S.L. Pharmacogenomics.Lancet. 2019; 394: 521-532Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar For example, several SNPs have been identified in metabolic genes that confer increased sensitivity or tolerance to the widely used warfarin anticoagulant drug,22Pirmohamed M. Warfarin: almost 60 years old and still causing problems.Br J Clin Pharmacol. 2006; 62: 509-511Crossref PubMed Scopus (248) Google Scholar and whose detection can inform the correct dosing in individuals with a high risk of thromboembolism, lowering the risk of adverse drug events due to underlying xenobiotic metabolism heterogeneity.23Budnitz D.S. Lovegrove M.C. Shehab N. Richards C.L. Emergency hospitalizations for adverse drug events in older Americans.N Engl J Med. 2011; 365: 2002-2012Crossref PubMed Scopus (1406) Google Scholar, 24Johnson J.A. Cavallari L.H. Warfarin pharmacogenetics.Trends Cardiovasc Med. 2015; 25: 33-41Crossref PubMed Scopus (110) Google Scholar, 25Perera M.A. Cavallari L.H. Limdi N.A. Gamazon E.R. Konkashbaev A. Daneshjou R. et al.Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study.Lancet. 2013; 382: 790-796Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar This study aims to build on previous work and systematically characterize the impact of mismatches on the RPA reaction across four human genetic loci with clinical relevance to warfarin treatment. Two loci are situated within the cyp2c9 gene, linked to warfarin clearance, whereas the other two loci are found within genes associated with altered warfarin dosage levels (vkorc1 and hbb). The study centers on mismatches located in the primer anchor region, defined as the pre-ante-penultimate to 3′-terminal position, and attempts to build a mismatch classification system that can predict the impact of a given mismatch on RPA reaction success and kinetics. For this DNA were utilized the used they as and as such influence reaction kinetics. the utilized for has which the of the mismatch loci under the impact on reaction is in the presence of a given mismatch compromise which on the such as nucleic acid Z.A. Rohrman B. Richards-Kortum R. Development of a recombinase polymerase amplification assay with an internal 2015; Google Scholar this characterization in the of RPA for in-field rapid genotyping. RPA reactions were performed using the were and SNPs linked to warfarin metabolic were identified The DNA each were and to the in with 1 were the four loci For each the located in a primer binding region, with primer variants through the of or two nucleotides the pre-ante-penultimate to the 3′ terminal position the impact of a single mismatches on amplification be for their of type a of the of which is a in the xenobiotic metabolism of This mutation has been shown to reducing of warfarin and, as M.A. Cavallari L.H. Limdi N.A. Gamazon E.R. Konkashbaev A. Daneshjou R. et al.Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study.Lancet. 2013; 382: 790-796Abstract Full Text Full Text PDF PubMed Scopus (217) Google mutation a the rate of warfarin is and individuals have increased M.A. Cavallari L.H. Limdi N.A. Gamazon E.R. Konkashbaev A. Daneshjou R. et al.Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study.Lancet. 2013; 382: 790-796Abstract Full Text Full Text PDF PubMed Scopus (217) Google the of complex by the The mutation highlighted and, as a dose of warfarin is for A.Y. Effects of and polymorphisms on warfarin sensitivity and during the of J. 2019; PubMed Scopus Google of the the underlying risk of warfarin treatment has been shown to the risk of adverse linked to S. U. R.K. M. 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Pas S.D. Anber J. Voermans J. Mes T.H.M. Schutten M. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5′ nuclease assay.J Mol Diagn. 2010; 12: 109-117Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar the stability of the primer-template complex anchor region the This result that the position and nucleotides in a mismatch are informative the stability of the primer-template complex. an with our of polymerase and the of which the nucleotides in a mismatch the impact on the polymerase due to in and in DNA Biochem. PubMed Scopus Google Scholar is to the by mismatch and, if this can be used to predict the impact of a given mismatch on RPA reaction kinetics. Our investigation the of in primer binding sites for RPA diagnostic as a single mismatch has the potential to the of reaction success to sensitivity and the impact of mismatch on reaction success is a potential issue for robust The of specific mismatches to the reaction, classification to the presence or absence of a the of our the of certain mismatch combinations, such as and the RPA reaction, whereas the single penultimate and the RPA reaction kinetics. the RPA reaction be to reaction success in the presence of in reaction kinetics, which be used for The reaction such as and be used to the and of a to the in mismatch impact on reaction success, a of be to sensitive and to mismatch we have systematically the impact of terminal mismatches on the RPA reaction across several Through a of we have the impact of 315 mismatch on the RPA reaction, highlighting to RPA the of primer design and a on which to build for genotyping. that our on RPA mismatch tolerance the of RPA primer design using such as PrimedRPA, in the design of robust in with high genetic The of genotyping and for and in high has the potential to inform clinical and decision to treatment of with and for to the with with with with

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