RNA Interference in Mammalia Cells by RNA-3’-PNA Chimeras

The discovery of siRNAs as the mediators of RNA interference has led to an increasing interest in their therapeutic applications. Chemical modifications are introduced into siRNAs to optimize the potency, the stability and the pharmacokinetic properties in vivo. Here, we synthesize and test the effects of RNA-3’-PNA chimeras on siRNA functioning and stability. We demonstrate that the chemical modifications are compatible with the siRNA machinery, because all the PNA-modified siRNAs can efficiently mediate specific gene silencing in mammalian cells. Furthermore, we find that the modification on the sense strand of siRNA results in an increased persistence of the activity, whereas modification on both strands results in enhanced nuclease resistance in serum

Investigation of the Stereochemical-Dependent DNA and RNA Binding of Arginine-Based Nucleopeptides

Nucleopeptides represent an intriguing class of nucleic acid analogues, in which nucleobases are placed in a peptide structure. The incorporation of D- and/or L-amino acids in nucleopeptide molecules allows the investigation of the role of backbone stereochemistry in determining the formation of DNA and RNA hybrids. Circular Dichroism (CD) spectroscopic studies indicated the nucleopeptide as having fully l-backbone configuration-formed stable hybrid complexes with RNA molecules. Molecular Dynamics (MD) simulations suggested a potential structure of the complex resulting from the interaction between the l-nucleopeptide and RNA strand. From this study, both the backbone (ionics and H-bonds) and nucleobases (pairing and pi-stacking) of the chiral nucleopeptide appeared to be involved in the hybrid complex formation, highlighting the key role of the backbone stereochemistry in the formation of the nucleopeptide/RNA complexes.This research was supported by Scientific Independence of Young Researchers (SIR) 2014 (RBSI142AMA) and University of Campania Luigi Vanvitelli (Valere) to S.D.M

Synthesis, biophysical characterization and anti-HIV activity of d(TG3AG) Quadruplexes bearing hydrophobic tails at the 5'-end

Novel conjugated G-quadruplex-forming d(TG3AG) oligonucleotides, linked to hydrophobic groups through phosphodiester bonds at 50-end, have been synthesized as potential anti-HIV aptamers, via a fully automated, online phosphoramidite-based solid-phase strategy. Conjugated quadruplexes showed pronounced anti-HIV activity with some preference for HIV-1, with inhibitory activity invariably in the low micromolar range. The CD and DSC monitored thermal denaturation studies on the resulting quadruplexes, indicated the insertion of lipophilic residue at the 50-end, conferring always improved stability to the quadruplex complex (20 < DTm < 40 C). The data suggest no direct functional relationship between the thermal stability and anti-HIV activity of the folded conjugated G-quartets. It would appear that the nature of the residue at 50 end of the d(TG3AG) quadruplexes plays an important role in the thermodynamic stabilization but a minor influence on the anti-HIV activity. Moreover, a detailed CD and DSC analyses indicate a monophasic behaviour for sequences I and V, while for ODNs (II–IV) clearly show that these quadruplex structures deviate from simple two-state melting, supporting the hypothesis that intermediate states along the dissociation pathway may exis

Tailoring the Structure of Cell Penetrating DNA and RNA Binding Nucleopeptides

Synthetic nucleic acid interactors represent an exciting research field due to their biotechnological and potential therapeutic applications. The translation of these molecules into drugs is a long and difficult process that justifies the continuous research of new chemotypes endowed with favorable binding, pharmacokinetic and pharmacodynamic properties. In this scenario, we describe the synthesis of two sets of homo-thymine nucleopeptides, in which nucleobases are inserted in a peptide structure, to investigate the role of the underivatized amino acid residue and the distance of the nucleobase from the peptide backbone on the nucleic acid recognition process. It is worth noting that the CD spectroscopy investigation showed that two of the reported nucleopeptides, consisting of alternation of thymine functionalized L-Orn and L-Dab and L-Arg as underivatized amino acids, were able to efficiently bind DNA and RNA targets and cross both cell and nuclear membranes

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies

SYNTHESIS OF 4-SUBSTITUTED PYRIMIDINE 2',3'-DIDEOXYNUCLEOSIDES

Reaction of 5'-O-(4,4'-dimethoxytriphenylmethyl)-3'-deoxythymidine with triphenylphosphine/carbon tetrachloride, followed by deprotection of the 5'-hydroxyl group, afforded the 4-chloro derivative 3 from which some 4-substituted pyrimidin-2(1H)one-2',3'-dideoxyribosides were obtained by nucleophilic substitution under very mild conditions. © 1991, Taylor & Francis Group, LLC. All rights reserved

Current Methods in Synthesis of Cyclic Oligonucleotides and Analogues

Cyclic oligonucleotides and analogues (cONs) are highly specific ligands for DNA, RNA and proteins. cONs are described in several diagnostic and therapeutic applications, as well as in elucidating enzyme mechanisms and as lead structures for development of new drugs because of their unusual chemical and biological properties in comparison with linear counterparts. In numerous structural studies it has been shown that conformational constraints imposed by circularity cause unique topologies, which in some cases can result into peculiar ON nanostructures. The wide field of applications of cONs has called for the definition of easy and efficient strategies to produce such compounds. In this review, we will describe the enzymatic and chemical approaches reported for the synthesis of cONs with a particular focus on the chemical ones. We proceed following a chronological order from the earliest to the latest methods and improvements, describing at first the natural cyclic ONs and then modified linkages and cyclic analogues. © 2012 Bentham Science Publishers

SYNTHESIS OF CYCLIC BRANCHED OLIGODEOXYRIBONUCLEOTIDES

The cyclic branched oligodeoxyribonucleotides 7 and 8 have been prepared via the intermediate 4 synthesized using a phosphotriester approach

PNA-DNA chimeras forming quadruplex structures

H-NMR, CD, and UV spectroscopy have been used to investigate the structure of PNA/DNA chimeras forming quadruplex structures. In particular, we synthesized 5′TGGG3′-t (1) and 5′TGG3′-gt (2), where lower and upper case letters indicate PNA and DNA residues, respectively. CD spectrum and all NMR data of (1) are typical of quadruplexes involving four parallel strands. UV melting profile of (1) indicates that its thermal stability is quite similar to that observed for the reference structure [d(TGGGT)]4. 1H-NMR spectrum for 5′TGG3′_gt (2) shows that this oligonucleotide is not able to fold into a single, well-defined species