Research Article: The ability of locked nucleic acid oligonucleotides to pre-structure the double helix: A molecular simulation and binding study

Date Published: February 12, 2019

Publisher: Public Library of Science

Author(s): You Xu, Olof Gissberg, Y. Vladimir Pabon-Martinez, Jesper Wengel, Karin E. Lundin, C. I. Edvard Smith, Rula Zain, Lennart Nilsson, Alessandra Villa, Freddie Salsbury.


Locked nucleic acid (LNA) oligonucleotides bind DNA target sequences forming Watson-Crick and Hoogsteen base pairs, and are therefore of interest for medical applications. To be biologically active, such an oligonucleotide has to efficiently bind the target sequence. Here we used molecular dynamics simulations and electrophoresis mobility shift assays to elucidate the relation between helical structure and affinity for LNA-containing oligonucleotides. In particular, we have studied how LNA substitutions in the polypyrimidine strand of a duplex (thus forming a hetero duplex, i.e. a duplex with a DNA polypurine strand and an LNA/DNA polypyrimidine strand) enhance triplex formation. Based on seven polypyrimidine single strand oligonucleotides, having LNAs in different positions and quantities, we show that alternating LNA with one or more non-modified DNA nucleotides pre-organizes the hetero duplex toward a triple-helical-like conformation. This in turn promotes triplex formation, while consecutive LNAs distort the duplex structure disfavoring triplex formation. The results support the hypothesis that a pre-organization in the hetero duplex structure enhances the binding of triplex forming oligonucleotides. Our findings may serve as a criterion in the design of new tools for efficient oligonucleotide hybridization.

Partial Text

Nucleic acid hybridization plays a key role in biotechnological and medical applications [1,2]. An effective approach is to design oligonucleotides (ONs) that bind a DNA target sequence with high affinity and specificity, using both Watson-Crick (WC) and Hoogsteen (HG) base pairing. To competitively bind an ON to a target, the binding affinity needs to be higher than in the original (DNA) duplex. This can be achieved by using synthetically modified nucleotides, for instance having a modified sugar moiety that restricts backbone and ribose conformational flexibility as in locked nucleic acid (LNA) [3–5].

We have used molecular dynamics simulation and the electrophoretic mobility shift assay to systematically study the propensity of hetero duplexes to form triple-helix structures while having the same sequence but with different LNA/DNA contents in the polypyrimidine strand. All hetero duplexes form stable helical structure but with different conformations. The LNA/DNA mixmer oligonucleotides modulate duplex conformation. Duplexes conformationally similar to triple-helix-type conformation show high propensity to bind a TFO. Thus, the combination of simulation and experimental binding assays elucidated that the best strategy to design a duplex with a high triplex propensity is to have a ratio of LNA:DNA of 1:1 or 1:2 and avoiding two or more consecutive LNA nucleotides in the sequence.