Thermodynamic control of mismatch discrimination for extensive splicing regulation of PKM pre-mRNA.

In this article, we present an approach to maximizing the splicing regulatory properties of splice-switching oligonucleotide (SSO) designed to regulate alternative splicing of PKM pre-mRNA. The studied SSO interacts with the regulatory element in exon 10 of PKM pre-mRNA and contributes to a significant reduction of PKM2 level with a simultaneous increase of the PKM1 isoform. This SSO forms a duplex not only with the regulatory fragment of exon 10 but also with a similar RNA fragment of intron 9.

Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of Gene.

Oligonucleotide tools, as modulators of alternative splicing, have been extensively studied, giving a rise to new therapeutic approaches. In this article, we report detailed research on the optimization of bifunctional antisense oligonucleotides (BASOs), which are targeted towards interactions with hnRNP A1 protein. We performed a binding screening assay, Kd determination, and UV melting experiments to select sequences that can be used as a high potency binding platform for hnRNP A1.

Evaluation of Gene Expression Knock-Down by Chemically and Structurally Modified Gapmer Antisense Oligonucleotides.

We analyzed the effect of modified nucleotides within gapmer antisense oligonucleotides on RNase H mediated gene silencing. Additionally, short hairpins were introduced into antisense oligonucleotides as structural motifs, and their influence on biological and physicochemical properties of pre-structured gapmers was investigated for the first time. The results indicate that two LNA residues in specified positions of the gap flanking regions are sufficient and favorable for efficient knock-down of the β-actin gene.

Gapmer Antisense Oligonucleotides Containing 2',3'-Dideoxy-2'-fluoro-3'-C-hydroxymethyl-β-d-lyxofuranosyl Nucleotides Display Site-Specific RNase H Cleavage and Induce Gene Silencing.

Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNase H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-β-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs.

The regulation properties of RNA secondary structure in alternative splicing.

The RNA secondary structure is important for many functional processes in the cell. The secondary and tertiary structures of cellular RNAs are essential for the activity of these molecules in processes such as transcription, splicing, translation, and localization. New high-throughput analytical methods, including next generation sequencing, have allowed for the in-depth characterization of the 'RNA structurome': a new term describing how the RNA structure controls the activity of RNA by itself and how it regulates the expression of genes.

A Disease-Causing Intronic Point Mutation C19G Alters Tau Exon 10 Splicing via RNA Secondary Structure Rearrangement.

Alternative splicing of MAPT cassette exon 10 produces tau isoforms with four microtubule-binding repeat domains (4R) upon exon inclusion or three repeats (3R) upon exon skipping. In human neurons, deviations from the ∼1:1 physiological 4R:3R ratio lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Certain FTDP-17-associated mutations affect a regulatory hairpin that sequesters the exon 10 5' splice site (5'ss, located at the exon 10-intron 10 junction).

A systematic study on the influence of thermodynamic asymmetry of 5'-ends of siRNA duplexes in relation to their silencing potency.

siRNA molecules possess high potential as molecular tools and can be used as effective therapeutics in humans. One of the key steps in the action of these molecules is the choice of antisense strand by the RNA-induced silencing complex (RISC). To explain this process, we verified the theory which states that antisense strand selection is based on the thermodynamically less stable 5' end of siRNA.

Contribution of 3'T and 3'TT overhangs to the thermodynamic stability of model siRNA duplexes.

Herein, we report comprehensive thermodynamic studies on 36 RNA/DNA duplexes designed as siRNA mimics to determine the energetic contribution of 3'T and 3'TT dangling ends. The thermodynamic effect induced by the presence of 3'T overhangs on the stability of RNA duplexes ranges from -0.28 to -0.

Thermodynamic, Anticoagulant, and Antiproliferative Properties of Thrombin Binding Aptamer Containing Novel UNA Derivative.

Thrombin is a serine protease that plays a crucial role in hemostasis, fibrinolysis, cell proliferation, and migration. Thrombin binding aptamer (TBA) is able to inhibit the activity of thrombin molecule via binding to its exosite I. This 15-nt DNA oligonucleotide forms an intramolecular, antiparallel G-quadruplex structure with a chair-like conformation.

Influence of mismatched and bulged nucleotides on SNP-preferential RNase H cleavage of RNA-antisense gapmer heteroduplexes.

This study focused on determining design rules for gapmer-type antisense oligonucleotides (ASOs), that can differentiate cleavability of two SNP variants of RNA in the presence of ribonuclease H based on the mismatch type and position in the heteroduplex. We describe the influence of structural motifs formed by several arrangements of multiple mismatches (various types of mismatches and their position within the ASO/target RNA duplex) on RNase H cleavage selectivity of five different SNP types. The targets were mRNA fragments of APP, SCA3, SNCA and SOD1 genes, carrying C-to-G, G-to-C, G-to-A, A-to-G and C-to-U substitutions.

Thermodynamic, structural and fluorescent characteristics of DNA hairpins containing functionalized pyrrolo-2'-deoxycytidines.

Herein, we present comprehensive physicochemical and structural analysis of various DNA hairpins modified with pyrrolo-2'-deoxycytidine (Py-dC) derivatives. The introduction of modified Py-dC in most cases causes minor decrease of hairpin thermodynamic stability. The energetically unfavorable effect is more pronounced when modified residue is present within hairpin loop.

Correction: A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides.

[This corrects the article DOI: 10.1371/journal.pone.

A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides.

Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed.