NEAREST-NEIGHBOUR PARAMETERS APPLIED TO LNA-MODIFIED LAMP PRIMERS

Locked nucleic acid (LNA) is a DNA chemical modification characterized by a methylene bridge that connects the 2’-oxygen of ribose with the 4’-carbon. It makes the nucleotide more stable, increasing the organization of the phosphate backbone and inducing stable duplexes with DNA and RNA. This stabilizing behavior is of interest to improve different molecular techniques such as PCR and LAMP. The latter is an isothermal amplification method which uses from four to six primers to identify different regions in the target. Achieving an ideal hybridisation between primer and target is crucial to the success of the process, e.g. the detection of the pathogen. A growing strategy which has been used is the incorporation of LNA-modified bases to LAMP primers looking forward to a more stabilization of the primer-target duplex to promote the amplification. However, the design of LAMP primers is complex and not so accurate as well as thermodynamic parameters for LNA-modified pairs. An improvement of these parameters could contribute to design more specific oligonucleotides. Although a systematic analysis of LNA melting temperatures has not been done yet, a recent work from our group developed LNA-NN parameters which showed an improvement in the parameters compared to previous sets. Here, we applied these thermodynamic parameters for LNA/DNA base pairs to obtain melting temperature of 24 LAMP primers designed to detect SARS-CoV-2 coronavirus. We compared their temperature to a control primer set and found the presence of LNA modification increased the temperatures for all primers. Especially, two or more LNA adenosine showed the largest increase. Our findings show a stabilization of LNA bases which could contribute to an improvement in the viral detection. Also our LNA-NN parameters set shows a potential as a new tool to the design of both PCR and LAMP primers as well as other oligonucleotide design.
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (Capes/Ação Emergencial, Brazil, Finance Code 001), CNPq and Fapemig.