siRNA-Mediated Timp1 Silencing Inhibited the Inflammatory Phenotype during Acute Lung Injury.

Acute lung injury is a complex cascade process that develops in response to various damaging factors, which can lead to acute respiratory distress syndrome. Within this study, based on bioinformatics reanalysis of available full-transcriptome data of acute lung injury induced in mice and humans by various factors, we selected a set of genes that could serve as good targets for suppressing inflammation in the lung tissue, evaluated their expression in the cells of different origins during LPS-induced inflammation, and chose the tissue inhibitor of metalloproteinase Timp1 as a promising target for suppressing inflammation. We designed an effective chemically modified anti-TIMP1 siRNA and showed that Timp1 silencing correlates with a decrease in the pro-inflammatory cytokine IL6 secretion in cultured macrophage cells and reduces the severity of LPS-induced acute lung injury in a mouse model.

"Bind, cleave and leave": multiple turnover catalysis of RNA cleavage by bulge-loop inducing supramolecular conjugates.

Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.

Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences.

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5-1 h. Artificial RNases based on the catalytic peptide [(ArgLeu)2Gly]2 were able to take progress a step further by demonstrating an ability to cleave miRNA-21 in tumour cells and provide a significant reduction of tumour growth in mice.

Strict conformational demands of RNA cleavage in bulge-loops created by peptidyl-oligonucleotide conjugates.

Potent knockdown of pathogenic RNA in vivo is an urgent health need unmet by both small-molecule and biologic drugs. 'Smart' supramolecular assembly of catalysts offers precise recognition and potent destruction of targeted RNA, hitherto not found in nature. Peptidyl-oligonucleotide ribonucleases are here chemically engineered to create and attack bulge-loop regions upon hybridization to target RNA. Catalytic peptide was incorporated either via a centrally modified nucleotide (Type 1) or through an abasic sugar residue (Type 2) within the RNA-recognition motif to reveal striking differences in biological performance and strict structural demands of ribonuclease activity. None of the Type 1 conjugates were catalytically active, whereas all Type 2 conjugates cleaved RNA target in a sequence-specific manner, with up to 90% cleavage from 5-nt bulge-loops (BC5-α and BC5L-ß anomers) through multiple cuts, including in folds nearby. Molecular dynamics simulations provided structural explanation of accessibility of the RNA cleavage sites to the peptide with adoption of an 'in-line' attack conformation for catalysis. Hybridization assays and enzymatic probing with RNases illuminated how RNA binding specificity and dissociation after cleavage can be balanced to permit turnover of the catalytic reaction. This is an essential requirement for inactivation of multiple copies of disease-associated RNA and therapeutic efficacy.

Non-enzymatic recombination of RNA: Ligation in loops.

BACKGROUND: While the RNA world hypothesis is widely accepted, it is still far from complete: the existence of self-replicating ribozyme, consisting of potentially hundreds of nucleotides, is a core assumption for the majority of RNA world models. The appearance of such long RNA molecules under prebiotic conditions is not self-evident. Recombination seems to be a plausible way of creating RNA diversity, resulting in the appearance of functional RNAs, capable of self-replicating. METHODS: We report here on the study of recombination process modelled with two 96 nts RNA fragments. Detection of recombination products was performed with RT-PCR followed by TA-cloning and Sanger sequencing. RESULTS: A wide range of recombinant products was detected. We found that (i) the most efficient ligation was observed for RNA species forming bulges or internal loops, with ligation partners located within the loop; (ii) a strong preference was observed for formation of a few types of major products with a large variety of minor products; (iii) ligation could occur with participation of either 2',3'-cyclophosphate or 5'-ppp; (iv) the presence of key reaction components, i.e. 5'ppp-RNAs, enabled the formation of additional types of product; (v) molecular dynamics simulations of one of the most abundant products suggests that the ligation results in a preferable formation of 2'-5'- rather than 3'-5'-linkages. CONCLUSIONS: The study demonstrates regularities of new RNA molecules formation with non-enzymatic recombination process. GENERAL SIGNIFICANCE: Our findings provide new data supporting the RNA World hypothesis and show the way of new RNA sequences emergence under prebiotic conditions.

'Dual' peptidyl-oligonucleotide conjugates: Role of conformational flexibility in catalytic cleavage of RNA.

Traditional therapeutic interventions against abnormal gene expression in disease states at the level of expressed proteins are becoming increasingly difficult due to poor selectivity, off-target effects and associated toxicity. Upstream catalytic targeting of specific RNA sequences offers an alternative platform for drug discovery to achieve more potent and selective treatment through antisense interference with disease-relevant RNAs. We report a novel class of catalytic biomaterials, comprising amphipathic RNA-cleaving peptides placed between two RNA recognition motifs, here demonstrated to target the TΨC loop and 3'- acceptor stem of tRNAPhe. These unique peptidyl-oligonucleotide 'dual' conjugates (DCs) were created by phosphoramidate or thiol-maleimide conjugation chemistry of a TΨC-targeting oligonucleotide to the N-terminus of the amphipathic peptide sequence, followed by amide coupling of a 3'-acceptor stem-targeting oligonucleotide to the free C-terminal carboxylic acid functionality of the same peptide. Hybridization of the DCs bearing two spatially-separated recognition motifs with the target tRNAPhe placed the peptide adjacent to a single-stranded RNA region and promoted cleavage within the 'action radius' of the catalytic peptide. Up to 100% cleavage of the target tRNAPhe was achieved by the best candidate (i.e. DC6) within 4 h, when conformational flexibility was introduced into the linker regions between the peptide and oligonucleotide components. This study provides the strong position for future development of highly selective RNA-targeting agents that can potentially be used for disease-selective treatment at the level of messenger, micro, and genomic viral RNA.

Peptidyl-oligonucleotide conjugates demonstrate efficient cleavage of RNA in a sequence-specific manner.

Described here is a new class of peptidyl-oligonucleotide conjugates (POCs) which show efficient cleavage of a target RNA in a sequence-specific manner. Through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to amphiphilic peptide sequences containing leucine, arginine, and glycine, zero-linker conjugates are created which exhibit targeted phosphodiester cleavage under physiological conditions. tRNA(Phe) from brewer's yeast was used as a model target sequence in order to probe different structural variants of POCs in terms of selective TΨC-arm directed cleavage. Almost quantitative (97-100%) sequence-specific tRNA cleavage is observed for several POCs over a 24 h period with a reaction half-life of less than 1 h. Nontargeted cleavage of tRNA(Phe) or HIV-1 RNA is absent. Structure-activity relationships reveal that removal of the peptide's central glycine residue significantly decreases tRNA cleavage activity; however, this can be entirely restored through replacement of the peptide's C-terminal carboxylic acid group with the carboxamide functionality. Truncation of the catalytic peptide also has a detrimental effect on POC activity. Based on the encouraging results presented, POCs could be further developed with the aim of creating useful tools for molecular biology or novel therapeutics targeting specific messenger, miRNA, and genomic viral RNA sequences.