Complete Mesoscopic Parameterization of Single LNA Modifications in DNA Applied to Oncogene Probe Design

Abstract

The use of mesoscopic models to describe the thermodynamic properties of locked nucleic acid (LNA)-modified nucleotides can provide useful insights into their properties, such as hydrogen-bonding and stacking interactions. In addition, the mesoscopic parameters can be used to optimize LNA insertion in probes, to achieve accurate melting temperature predictions, and to obtain duplex opening profiles at the base-pair level. Here, we applied this type of model to parameterize a large set of melting temperatures for LNA-modified sequences, from published sources, covering all possible nearest-neighbor configurations. We have found a very large increase in Morse potentials, which indicates very strong hydrogen bonding as the main cause of improved LNA thermodynamic stability. LNA-modified adenine−thymine (AT) was found to have similar hydrogen bonding to unmodified cytosine−guanine (CG) base pairs, while for LNA CG, we found exceptionally large hydrogen bonding. In contrast, stacking interactions, which were thought to be behind the stability of LNA, were similar to unmodified DNA in most cases. We applied the new LNA parameters to the design of BRAF, KRAS, and EGFR oncogene variants by testing all possible LNA modifications. Selected sequences were then synthesized and had their hybridization temperatures measured, achieving a prediction accuracy within 1 °C. We performed a detailed base-pair opening analysis to discuss specific aspects of these probe hybridizations that may be relevant for probe design.

Mutations in microRNA-128-2-3p identified with amplification-free hybridization assay

“…A potential pitfall and a scientific challenge of any miRNA detection method is the design of the specific detection probes. Recently, we applied a unique Peyrard-Bishop mesoscopic model to design oncogene-specific probes, which accounts for both hydrogen bonding and pistacking interactions between each individual nucleotide pair [29][30][31]. The model included locked nucleic acid (LNA) enriched probes that were designed specifically to target the desired DNA and RNA.…”

“…The model included locked nucleic acid (LNA) enriched probes that were designed specifically to target the desired DNA and RNA. LNA is a nucleic acid analogue which improves the probes' affinity and specificity especially for DNA:RNA recognition [24,29,[32][33][34]. Using this model, we optimized oligonucleotide reagents so that the target SNP in coding RNA was discriminated with high specificity [31].…”