Stability and Mismatch Discrimination of Locked Nucleic Acid–DNA Duplexes

Abstract

Locked nucleic acids (LNA; symbols of bases, +A, +C, +G, and +T) are introduced into chemically synthesized oligonucleotides to increase duplex stability and specificity. To understand these effects, we have determined thermodynamic parameters of consecutive LNA nucleotides. We present guidelines for the design of LNA oligonucleotides and introduce free online software that predicts the stability of any LNA duplex oligomer. Thermodynamic analysis shows that the single strand–duplex transition is characterized by a favorable enthalpic change and by an unfavorable loss of entropy. A single LNA modification confines the local conformation of nucleotides, causing a smaller, less unfavorable entropic loss when the single strand is restricted to the rigid duplex structure. Additional LNAs adjacent to the initial modification appear to enhance stacking and H-bonding interactions because they increase the enthalpic contributions to duplex stabilization. New nearest-neighbor parameters correctly forecast the positive and negative effects of LNAs on mismatch discrimination. Specificity is enhanced in a majority of sequences and is dependent on mismatch type and adjacent base pairs; the largest discriminatory boost occurs for the central +C·C mismatch within the +T+C+C sequence and the +A·G mismatch within the +T+A+G sequence. LNAs do not affect specificity in some sequences and even impair it for many +G·T and +C·A mismatches. The level of mismatch discrimination decreases the most for the central +G·T mismatch within the +G+G+C sequence and the +C·A mismatch within the +G+C+G sequence. We hypothesize that these discrimination changes are not unique features of LNAs but originate from the shift of the duplex conformation from B-form to A-form.

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

“…We used different types of nucleic acids targeting H. pylori: LNA, 2′OMe, unlocked nucleic acid (UNA), PNA, and DNA (Table 1). Different designs of oligo-nucleotide probes were tested to find the best discrimination possible based on earlier published criteria to improve mismatch discrimination You et al 2006;Langkjaer et al 2009;Wengel 2010, 2011;Owczarzy et al 2011). As DNA has a restricted flexibility of oligonucleotide design, only one probe was designed (Table 1).…”

“…In contrast, LNA, 2′OMe, and UNA bases can be positioned anywhere within an oligonucleotide sequence, which means that the design is much more flexible and that probe fine-tuning is possible (You et al 2006). As a general rule, these probes were designed with a triplet of LNA-modified nucleotides positioned at the center of the mismatch site to improve the discrimination (You et al 2006;Owczarzy et al 2011). The use of a higher density of LNA residues in each probe is an-other important parameter to improve mismatch discrimination (You et al 2006).…”

“…More recently, other types of synthetic nucleic acids such as peptide nucleic acid (PNA) and locked nucleic acids (LNA) have been used in FISH with very promising results (Guimaraes et al 2007;Mook et al 2007;Tavares et al 2008;Campbell and Wengel 2011;Almeida et al 2013;Cerqueira et al 2013). To understand the specificity of these novel molecules towards DNA and RNA, several thermal dissociation studies have been conducted to analyze the effects of mismatches on duplex stability (Owczarzy et al 2011;Yan et al 2012;Matsumoto et al 2013). However, this type of analysis has always been performed with naked DNA in standard chemical solutions.…”

Dendrimers as Drug Nanocarriers: The Future of Gene Therapy and Targeted Therapies in Cancer

“…LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever preferred and hybridize with DNA or RNA according to Watson-Crick base-pairing rules. Due to the high stability of LNA-RNA it started to be used in a biotechnology field in a pharmaceutical business [73]. The multi-valent folate (FA)-conjugated 3WJ RNP constructed to harbor anti-miR-21 LNA sequences (FA-3WJ-LNA-miR21).…”

microRNA in the control of stem-like phenotype of cancer cells

“…In these oligonucleotide variants, the ribose ring is modified with an additional bond between the 2'-oxygen and 4'-carbon. This modification significantly increases melting temperature and stabilization of the RNA duplexes, specificity for target miRNAs, and resistance to endonucleases [120]. The mentioned methods showed promising results in in vitro studies, however, the biggest drawbacks of this approach are to specifically deliver antagomiRs or miRNA mimics to cancer cells, and to obtain permanent therapeutic effect.…”