Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM 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. Newly designed BASOs were applied to regulate the mutually exclusive alternative splicing of the PKM gene. Our studies demonstrate that at least three repetitions of regulatory sequence are necessary to increase expression of the PKM1 isoform. On the other hand, PKM2 expression can be inhibited by a lower number of regulatory sequences. Importantly, a novel branched type of BASOs was developed, which significantly increased the efficiency of splicing modulation. Herein, we provide new insights into BASOs design and show, for the first time, the possibility to regulate mutually exclusive alternative splicing via BASOs.

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. Based on the studies presented herein, we observed that for the tested siRNA duplexes, the difference in the thermodynamic stability of the terminal, penultimate and pre-penultimate pairs in the duplex siRNA is not the dominant factor in antisense strand selection. We found that both strands in each tested siRNA molecule are used as an antisense strand. The introduction of modified nucleotides, whose impact on the thermodynamic stability of siRNA duplexes was studied, results in changes in antisense strand selection by the RISC complex. The presence of a modified residue often caused predominant selection of only one antisense strand which is at variance with the theory of siRNA strand bias.

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. In this review, we present many examples of the influence of structural motifs of RNA, long range interactions and global RNA structure on the alternative splicing processes. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.