Inhibition of nonspecific polymerase activity using Poly(Aspartic) acid as a model anionic polyelectrolyte

DNA polymerases with strand-displacement activity allow to amplify nucleic acids under isothermal conditions but often lead to undesirable by-products. Here, we report the increase of specificity of isothermal amplification in the presence of poly (aspartic) acids (pAsp). We hypothesized that side reactions occur due to the binding of the phosphate backbone of synthesized DNA strands with surface amino groups of the polymerase, and weakly acidic polyelectrolytes could shield polymerase molecules from DNA and thereby inhibit nonspecific amplification. Suppression of nonspecific polymerase activity by pAsp was studied on multimerization as a model side reaction. It was found that a low concentration of pAsp (0.01%) provides successful amplification of specific DNA targets. The inhibitory effect of pAsp is due to its polymeric structure since aspartic acid did affect neither specific nor nonspecific amplification. Strongly acidic polyelectrolyte heparin does not possess the same selectivity since it suppresses any DNA synthesis. The applicability of pAsp to prevent nonspecific reactions and reliable detection of the specific target has been demonstrated on the genetic material of SARS-CoV-2 coronavirus using Loopmediated isothermal amplification.

Automated summary

This study found that the addition of poly(aspartic) acid (pAsp) can increase specificity and inhibit nonspecific activity of DNA polymerases during isothermal amplification. The inhibitory effect of pAsp is attributed to its polymeric structure, which prevents the formation of unwanted by-products and allows for reliable detection of specific DNA targets.