In mammalian cells, small non-coding RNAs (sncRNAs) negatively regulate gene expression in a pathway known as RNA interference (RNAi). RNAi can be categorized into post-transcriptional gene silencing (PTGS), which involves the cleavage of target messenger RNA (mRNA) or inhibition of translation in the cytoplasm, and transcriptional gene silencing (TGS), which is mediated by the establishment of repressive epigenetic marks at target loci. Transfer RNAs (tRNAs), which are essential for translation, can be processed into small ncRNAs, termed tRNA-derived small RNAs (tsRNAs). The biogenesis of tsRNAs and their role in gene expression regulation has not yet been fully understood. Here, we show that Dicer dependent tsRNAs promote gene silencing through a mechanism distinct from PTGS and TGS. tsRNAs can lead to downregulation of target genes by targeting introns via nascent RNA silencing (NRS) in nuclei. Furthermore, we show that Ago2 slicer activity is required for this mechanism. Synthetic tsRNAs can significantly reduce expression of a target gene at both RNA and protein levels. Target genes regulated by NRS are associated with various diseases, which further underpins its biological significance. Finally, we show that NRS is evolutionarily conserved and has the potential to be explored as a novel synthetic sRNA based therapeutic.

Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs) and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs) or restoring the anomalous levels of non-coding RNAs (ncRNAs) that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs), carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

Abstract.The discovery and examination of the post-transcriptional gene regulatory mechanism known as RNA interference (RNAi), contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA-based drugs are currently under clinical testing. In this work we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off-target effects such as saturation of the RNAi machinery, passenger strand-mediated silencing, immunostimulation and miRNA-like off-target effects as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps towards the development of reliable siRNA molecules for therapeutic use.

Cellular uptake studies of G-quadruplex constructs having the Tetrahymena telomeric repeat sequence d(TGGGGT) modified with amino and guanidinium residues at the 3′-termini and an antisense oligonucleotide at 5′-termini were studied.