title: Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction.
abstract: The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community.
authors.affiliation: Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia.|School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Ellie
authors.initials: E
authors.lastname: Mok
authors.affiliation: Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia.
authors.forename: Eugene
authors.initials: E
authors.lastname: Wee
authors.affiliation: Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia.|School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Matt
authors.initials: M
authors.lastname: Trau
mesh_terms: D001622:Betaine / Q000737:chemistry; D015374:Biosensing Techniques; D004268:DNA-Binding Proteins / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D019855:Ethylene Glycol / Q000737:chemistry; D035683:MicroRNAs / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques; D019946:Propylene Glycol / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012045:Regulatory Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014199:Trehalose / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; D035683:MicroRNAs; D000644:Quaternary Ammonium Compounds; D012710:Serum Albumin, Bovine; D001622:Betaine; D019946:Propylene Glycol; D014199:Trehalose; D019855:Ethylene Glycol; C027917:tetramethylammonium; D004121:Dimethyl Sulfoxide