Chemically Synthesized,Self-Assembling Small Interfering RNA-Prohead RNA Molecules Trigger Dicer-Independent Gene Silencing

RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery in vivo. An innovative delivery strategy involves fusing siRNAs to a three-way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA-3WJ molecules are presumed to enter the RNAi pathway through Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA-3WJs are substrates for Dicer in vitro. However, our results reveal that siRNA-3WJs transfected into Dicer-deficient cell lines trigger potent gene silencing. Interestingly, siRNA-3WJs transfected into an Argonaute 2-deficient cell line also retain some gene silencing activity. siRNA-3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5′ of the 3WJ (5′-siRNA-3WJ) relative to 3′ of the 3WJ (3′-siRNA-3WJ). This work sheds light on the functional properties of siRNA-3WJs and offers a design rule for maximizing their potency in the human RNAi pathway.     

多吡啶钌(Ⅱ)配合物的合成及其与RNA相互作用的光谱学研究

RNA是生物的重要组成物质 ,它参与蛋白质的生物合成 ,在基因的调控中起着重要的作用 ,并与一些疾病 ,如艾滋病等的诊断与治疗相关联 .与 DNA相比 ,RNA为单链 ,其戊糖为核糖 ,在碱基组成上没有胸腺嘧啶 ,取而代之的是结构十分相近的尿嘧啶 .RNA能自身回折 ,碱基在局部区域内象DN     

Oligo(rU)_(15)对SDS/DEAB混合胶束向囊泡转化的诱导作用

囊泡是由表面活性剂分子分散在水中形成的具有密闭双分子层结构的球形或者椭球形的分子有序组合体,在生物学、材料学、化学和医药学等领域具有极为重要和广泛的应用~([1-4]).     

Modular and Versatile Trans-Encoded Genetic Switches

Current bacterial RNA switches suffer from lack of versatile inputs and are difficult to engineer. We present versatile and modular RNA switches that are trans-encoded and based on tRNA-mimicking structures (TMSs). These switches provide a high degree of freedom for reengineering and can thus be designed to accept a wide range of inputs, including RNA, small molecules, and proteins. This powerful approach enables control of the translation of protein expression from plasmid and genome DNA.     

Histidine affinity chromatography-based methodology for the simultaneous isolation of Escherichia coli small and ribosomal RNA

Research on RNA has led to many important biological discoveries and the improvement of therapeutic technologies. In particular, there is a great focus on small RNA and ribosomal RNA owing to their key functions in the cell, which make them excellent therapeutic targets. Although the study of these RNA classes is progressing, some limitations have been found regarding the use of suitable techniques that are able to produce and isolate biologically competent and chemically stable RNA. To address this, we have developed a novel histidine affinity chromatography-based isolation methodology for small and ribosomal RNA molecules. The new procedure involves three main steps: (1) cell lysis with guanidinium buffer, (2) RNA primary isolation with ammonium sulfate precipitation and (3) histidine affinity chromatography to specifically purify small RNA and ribosomal RNA from other Escherichia coli impurities (genomic DNA and proteins). The RNA quality assessment revealed that both RNA species were obtained with a high recovery, integrity and purity. The potential of this method to achieve a reproducible RNA isolation with appropriate quality has been demonstrated and it should have broad application in the structural, biophysical and biomedical investigation of systems involving RNA components.     

Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA was observed at high temperatures (75 °C) in contrast to ssDNA. It is proposed that the presence of duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity, through contributions of the nucleic sequence and the degree of secondary structure present. In addition, the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg²⁺ during chromatography. This novel chromatographic procedure permits analysis of the temperature dependent formation of dimeric RNA species.     

Cleavage of RNA bulge loops by artificial RNases

Sensitivity of phosphodiester bonds in RNA bulge loops to cleavage by short cationic peptides and compounds based on 1,4-diazabicyclo[2.2.2]octane and its conjugates with imidazole was studied. Bulge loops containing from one to seven nucleotides were formed in RNA upon its hybridization with partially complementary oligodeoxyribonucleotides. The efficiency of RNA cleavage depends on the length of a bulge loop, the position of the cleaved phosphodiester bond in the loop, and the nature of the RNA-binding fragment of chemical ribonuclease (1,4-diazabicyclo[2.2.2]octane or a cationic peptide). In the absence of Mg²⁺ ions, the phosphodiester bond in the CA motif located in the apical position in 4-, 6-, or 7-membered loops is cleaved with the highest efficiency. In the presence of magnesium ions, the selectivity of RNA cleavage within bulge loops is substantially enhanced. In the case of 1,4-diazabicyclo[2.2.2]octane-based compounds, RNA is subjected to cleavage predominantly at the bonds in 4-, 6-, and 7-membered loops, whereas cleavage of other bonds is greatly suppressed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1236–1246, July, 2006.     

Base pairing in RNA structures: A computational analysis of structural aspects and interaction energies

The base pairing patterns in RNA structures are more versatile and completely different as compared to DNA. We present here results of ab-initio studies of structures and interaction energies of eight selected RNA base pairs reported in literature. Interaction energies, including BSSE correction, of hydrogen added crystal geometries of base pairs have been calculated at the HF/6-31G** level. The structures and interaction energies of the base pairs in the crystal geometry are compared with those obtained after optimization of the base pairs. We find that the base pairs become more planar on full optimization. No change in the hydrogen bonding pattern is seen. It is expected that the inclusion of appropriate considerations of many of these aspects of RNA base pairing would significantly improve the accuracy of RNA secondary structure prediction.     

Steady‐state electrophoresis of RNA against a gradient of cationic charges in a polyacrylamide matrix

A novel method for separation of RNA fragments is reported here, based on migrating the polyanionic RNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations are typically performed in a 0–10 mM, pK 10.3 Immobiline gradient under denaturing conditions (6 M urea). In the 100–1000 bp length, it is shown that separations of RNA are optimal and very sharp bands can be obtained, in comparison with conventional electrophoresis, due to the “focusing” effect originated by the charge balancing between the positively charged gel matrix and the negatively charged RNA species. Excellent separations are also obtained from micro‐RNAs, single‐stranded RNA molecules of 21–23 nucleotides in length, which appear to regulate gene expression in animal and plant tissues. As a third example, 2‐D runs in control and polycationic gels are shown. Under native conditions, RNAs are not aligned in a diagonal, suggesting that molecular shape has a strong influence on the interaction between RNA and the charged gel matrix. Thus, 2‐D runs in cationic matrices might be exploited for structural studies of RNA molecules.     

Chemical synthesis of an artificially branched hairpin ribozyme variant with RNA cleavage activity

Due to the development in the field of RNA synthesis over the past decade of years, preparation of RNA oligonucleotides longer than 50 nucleotides is possible today. In this report, we describe the chemical preparation of a branched RNA molecule with RNA cleavage activity consisting of 81 nucleotides. It is derived from the hairpin ribozyme, a small catalytic RNA occurring in nature. The hairpin ribozyme consists of two separately folded domains (loop A and loop B domain), which can be joined in a number of different ways without loss of activity. In the construct presented here, 2′-deoxy-N4-(6-hydroxyhexyl)-5-methylcytidine was introduced to connect the loop B domain with the loop A domain via an artificial branch. The synthesized branched RNA is able to catalyze the cleavage of a number of suitable substrates. Compared with the corresponding non-branched reverse-joined ribozyme it cleaves its substrates only 5-fold slower. Surprisingly, no ligation activity could be detected.     

Environment-Responsive Peptide Dimers Bind and Stabilize Double-Stranded RNA

Double-stranded RNAs (dsRNA) possess immense potential for biomedical applications. However, their therapeutic utility is limited by low stability and poor cellular uptake. Different strategies have been explored to enhance the stability of dsRNA, including the incorporation of modified nucleotides, and the use of diverse carrier systems. Nevertheless, these have not resulted in a broadly applicable approach thereby preventing the wide-spread application of dsRNA for therapeutic purposes. Herein, we report the design of dimeric stapled peptides based on the RNA-binding protein TAV2b. These dimers are obtained via disulfide formation and mimic the natural TAV2b assembly. They bind and stabilize dsRNA in the presence of serum, protecting it from degradation. In addition, peptide binding also promotes cellular uptake of dsRNA. Importantly, peptide dimers monomerize under reducing conditions which results in a loss of RNA binding. These findings highlight the potential of peptide-based RNA binders for the stabilization and protection of dsRNA, representing an appealing strategy towards the environment-triggered release of RNA. This can broaden the applicability of dsRNA, such as short interfering RNAs (siRNA), for therapeutic applications.