Antisense Strand Of siRNA Research Articles

Small Interfering RNAs Containing Dioxane- and Morpholino-Derived Nucleotide Analogues Show Improved Off-Target Profiles and Chirality-Dependent In Vivo Knock-Down.

To expand the applicability of recently developed dioxane- and morpholino-based nucleotide analogues, their seed region destabilizing properties in small interfering RNAs (siRNAs) were investigated in order to improve potential off-target profiles. For this purpose, the corresponding adenosine analogues were synthesized in two diastereomeric series as building blocks for the automated oligonucleotide synthesis. The obtained nucleotide precursors were integrated at position 7 of an siRNA antisense strand, targeting transthyretin messenger RNA. Evaluation of the melting temperatures revealed significant differences in the obtained duplex stabilities between the two diastereomeric series, while the influence of the central scaffold was small. All siRNAs containing these novel nucleotide structures showed improved off-target profiles in vitro compared to their parent sequence with the common 2'-OMe-modified adenosine at the same position. In contrast, in vivo potencies were highly dependent on the chirality within the six-membered nucleotide scaffolds and showed high mRNA downregulations for the (2R,6R)-configured diastereomers.

Elimination of Off-Target Effect by Chemical Modification of 5'-End of siRNA.

In this study, the efficiency of RNA interference of small interfering RNAs (siRNAs) bearing 5'-O-methyl-2'-deoxythymidine (X) and 5'-amino-2', 5'-dideoxythymidine (Z) at the 5'-end of the sense strand and the antisense strand of siRNA was investigated in HeLa cells stably expressing enhanced green fluorescent protein. The results indicated that when one strand of siRNA was modified with X or Z and the other was unmodified, the X or Z modification was predominant in the process of strand selection and the unmodified strand was selected as a guide strand. When both strands are modified with X or Z, the modified antisense strand with X or Z will be selected as a guide strand with a certain probability. The resulting mature RNA-induced silencing complex exerted reduced, but still moderate silencing activity remained. These results suggest that the modification of the sense strand with X or Z eliminates the off-target effects caused by the sense strand without affecting the silencing efficiency of the siRNA.

Optimization in Chemical Modification of Single-Stranded siRNA Encapsulated by Neutral Cytidinyl/Cationic Lipids.

Single-stranded siRNA (ss-siRNA) refers to the antisense strand of siRNA, which plays the role of gene silencing. Since single-stranded RNA is unstable, the present study employed a homemade neutral cytidinyl/cationic lipid delivery system and chemical modifications to improve its stability. The results showed that with the aid of mixed lipids, ss-siRNA could knock down 40% of target mRNA at 25 nM. With 2ʹ-F/2ʹ-OMe, phosphorothioate and 5ʹ-terminal phosphorylation, the optimized ss-siRNA could knock down 80% of target mRNA at 25 nM. Further knocking down AGO2, the ss-siRNAs could not effectively silence target mRNAs. Analysis of the biodistribution in vivo showed that ss-siRNA was less durable than siRNA, but spread more quickly to tissues. This study provides a safe and efficient ss-siRNA delivery and modification strategy, which lays the foundation for future works.

Feasibility of cRGD conjugation at 5′-antisense strand of siRNA by phosphodiester linkage extension

Small interfering RNAs (siRNAs) are widely studied for their highly specific gene silencing activity. However, obstacles remain to the clinical application of siRNAs. Attaching conjugates to siRNAs can improve their stability and broaden their application, and most functional conjugates of siRNAs locate at the 3′-terminus of the sense or antisense strand. In this work, we found that conjugating a group at the 5′-terminus of the antisense strand via phosphodiester was practicable, especially when the group was a flexible moiety such as an alkyl linker. When conjugating a bulky ligand, such as cRGD, the length of the 5′-phosphodiester linker between the ligand and the 5′-terminus of the antisense strand was the key in terms of RNA interference (RNAi). With a relative longer linker, the conjugates showed potency similar to siRNA. A highly efficient transfection system composed of a neutral cytidinyl lipid (DNCA) and a gemini-like cationic lipid (CLD) was employed to deliver siRNAs or their conjugates. The cRGD conjugates showed superior targeting delivery and antitumor efficacy in vivo and also selective cellular uptake in vitro. This unity of encapsulation and conjugation strategy may provide potential strategies for siRNA-based gene therapy.

The 2′-caged-tethered-siRNA shows light-dependent temporal controlled RNAi activity for GFP gene into HEK293T cells

Small interfering RNA (siRNA) exhibits gene-specific RNAi activity by the formation of RISC complex with mRNA of gene. The structural modification of siRNA at appropriate positions affects the structure of RISC complex and then RNAi activity. The modified siRNA are mostly prepared from the incorporation of sugar ring modified, and nucleobase modified RNA nucleotides. It is learned that the introduction of the sterically hindered nucleoside at the specific position of siRNA, severely affects siRNA-RISC complex formation. This report describes the syntheses of bulkier siRNA from 2′-caged-tethered-siRNAs, containing bulkier photolabile protecting group (o-nitrobenzyl) at 2′-position of ribose nucleoside. Importantly, these 2′-caged-siRNAs exhibit the light-dependent RNA interference (RNAi) activity into HEK293T cells for the GFP gene expression. The 2′-caged-siRNAs are synthesized by caging the sense and antisense strand of siRNA. The biochemical evaluations of these caged-siRNAs show that antisense-strand caged-siRNAs decrease RNAi activity temporarily in dark while enhancing RNAi activity, almost like control, after exposure withUV- light. However, 2′-caged sense strand siRNA increase RNAi activity temporarily while decreasing RNAi activity after exposure with light. These caged-siRNAs are also stable in the serum (fetal bovine serum) as like native siRNA. Hence these results strongly support that 2′-caged-tethered-siRNAs are promising analogues to control RNAi activity by UV-light.

Synergistic Targeting HER2 and EGFR with Bivalent Aptamer-siRNA Chimera Efficiently Inhibits HER2-Positive Tumor Growth.

HER2 overexpression is identified on 20–30% breast cancer and other cancers at different levels. Although HER2 targeted monoclonal antibody combined with chemical drugs has shown improved outcomes in HER2 expressing patients, drug resistance and toxicity have limited their efficacy. To overcome drug resistance, cotargeting multiple HER receptors was proven to be effective. EGFR/HER2 dimerization can active PI3K/AKT pathway, and resistance to HER2-targeted drugs is associated with upregulation of EGFR. Here, we developed a novel HER2/EGFR targeted nucleic acid therapeutic to address current drug limits. The new therapeutic is constructed by fusing HER2 aptamer-EGFR siRNA sense strand with HER2 aptamer-EGFR siRNA antisense strand into one molecule: a bivalent HER2 aptamer-EGFR siRNA aptamer chimera (HEH). In breast cancer cell lines, HEH can be selectively taken up into HER2 expressing cells and successfully silence EGFR gene and down regulate HER2 expression. In breast cancer xenograft models, HEH is capable of triggering cell apoptosis, decreasing HER2 and EGFR expression, and suppressing tumor growth. The therapeutic efficacy of HEH is superior to HER2 aptamer only, which suggests that HEH has synergistic effect by targeting HER2 and EGFR. This study demonstrated that HEH has great potential as a new HER2 targeted drug to address toxicity and resistance of current drugs and may provide a cure for many HER2 positive cancers.

Prediction of VIGS efficiency by the Sfold program and its reliability analysis in Gossypium hirsutum

Genetic transformation in some plant species, including cotton (Gossypium hirsutum), is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing (VIGS) by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different cDNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA (siRNA) in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of siRNA in target sequence: the disruption energy of the target (ΔGdisruption), the differential stability of siRNA duplex ends (DSSE), and the internal stability at positions 9–14 of the siRNA antisense strand (AIS), which are calculated by Sfold program (http://sfold.wadsworth.org). We find that the siRNAs with low ΔGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, GhPDS, GhCLA1, GhAOS1, and GhCXE1 via choosing different target sequences for each gene. Therefore, the Sfold program can be used to analyze target sequences when carrying out VIGS design to increase gene-silencing effects in plants.

Practical and Reliable Synthesis of 1,2-Dideoxy-d-ribofuranose and its Application in RNAi Studies

ABSTRACTWe developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-d-ribofuranose (dRH) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dRH by the standard phosphoramidite solid-phase method, dRH was converted to the corresponding phosphoramidite derivative and linked to a solid support (controlled pore glass resin). Chemically modified small interfering RNAs (siRNAs) possessing dRH at their 3′-overhang regions were synthesized. Introducing dRH to the 3′-end of the antisense strand of siRNA reduced its knockdown effect.

Gene Knockdown by EpCAM Aptamer-siRNA Chimeras Suppresses Epithelial Breast Cancers and Their Tumor-Initiating Cells.

Effective therapeutic strategies for in vivo siRNA delivery to knockdown genes in cells outside the liver are needed to harness RNA interference for treating cancer. EpCAM is a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells (TIC, also known as cancer stem cells). Here, we show that aptamer-siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sense strand and annealed to the siRNA antisense strand) are selectively taken up and knock down gene expression in EpCAM(+) cancer cells in vitro and in human cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere formation (in vitro TIC assays) and tumor initiation by EpCAM(+) luminal and basal-A triple-negative breast cancer (TNBC) cell lines, but not EpCAM(-) mesenchymal basal-B TNBCs, in nude mice. Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM(+) Her2(+) and TNBC tumors and suppress their growth. Thus, EpCAM-AsiCs provide an attractive approach for treating epithelial cancer.

Abstract LB-299: Gene knockdown by EpCAM aptamer-siRNA chimeras suppresses epithelial breast cancers and their tumor-initiating cells

Abstract Effective therapeutic strategies for in vivo siRNA delivery to knockdown genes in cells outside the liver are needed to harness RNA interference for treating cancer. EpCAM is a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells (T-IC, also known as cancer stem cells). Here we show that aptamer-siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sense strand and annealed to the siRNA antisense strand) are selectively taken up and knockdown gene expression in EpCAM+ cancer cells in vitro and in human cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere formation (in vitro T-IC assays) and tumor initiation by EpCAM+ luminal and basal-A triple negative breast cancer (TNBC) cell lines, but not EpCAM- mesenchymal basal-B TNBCs, in nude mice. Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM+ Her2+ and TNBC tumors and suppress their growth. Thus EpCAM-AsiCs provide an attractive approach for treating epithelial cancer. Note: This abstract was not presented at the meeting. Citation Format: Adi Gilboa-Geffen, Peter Hamar, Lee Adam Wheeler, Radiana Trifonova, Fabio Petrocca, Anders Wittrup, Judy Lieberman. Gene knockdown by EpCAM aptamer-siRNA chimeras suppresses epithelial breast cancers and their tumor-initiating cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-299. doi:10.1158/1538-7445.AM2015-LB-299

TNF-α Gene Silencing Using Polymerized siRNA/Thiolated Glycol Chitosan Nanoparticles for Rheumatoid Arthritis

Among various proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), tumor necrosis factor (TNF)-α plays a pivotal role in the release of other cytokines and induction of chronic inflammation. Even though siRNA has the therapeutic potential, they have a challenge to be delivered into the target cells because of their poor stability in physiological fluids. Herein, we design a nanocomplex of polymerized siRNA (poly-siRNA) targeting TNF-α with thiolated glycol chitosan (tGC) polymers for the treatment of RA. Poly-siRNA is prepared through self-polymerization of thiol groups at the 5' end of sense and antisense strand of siRNA and encapsulated into tGC polymers, resulting in poly-siRNA-tGC nanoparticles (psi-tGC-NPs) with an average diameter of 370 nm. In the macrophage culture system, psi-tGC-NPs exhibit rapid cellular uptake and excellent in vitro TNF-α gene silencing efficacy. Importantly, psi-tGC-NPs show the high accumulation at the arthritic joint sites in collagen-induced arthritis (CIA) mice. Treatment monitoring data obtained by the matrix metalloproteinase 3-specific nanoprobe and microcomputed tomography show that intravenous injection of psi-tGC-NPs significantly inhibits inflammation and bone erosion in CIA mice, comparable to methotrexate (5 mg/kg). Therefore, the availability of psi-tGC-NP therapy that target specific cytokines may herald new era in the treatment of RA.

In-silico analysis for RNA-interference mechanism of α-synuclein to treat Parkinson's disease

Parkinson's Disease (PD) causing mutations in α-synuclein gene are ALA30PRO, GLU46LYS and ALA53THR. The conformational changes in proteins with respect to all the three mutations were analysed. These were used to predict the structures of Short Interfering RNA (siRNA) antisense strand and siRNA region. The siRNA binds with the argonaute protein forming RNA Induced Silencing Complex (RISC). Then, siRNA antisense-strand was attached to RISC. The structure of dicer (RNase-III-enzyme) cleaves double-stranded RNA (dsRNA) into two siRNA-strands. Incorporation of single siRNA-strand into RISC guides to pair with the complementary α-synuclein target-messenger RNA (mRNA) thereby enabling it to cleave the target.

Analysis of energetically biased transcripts of viruses and transposable elements

RNA interference (RNAi) is a natural endogenous process by which double-stranded RNA molecules trigger potent and specific gene silencing in eukaryotic cells and is characterized by target RNA cleavage. In mammals, small interfering RNAs (siRNAs) are the trigger molecules of choice and constitute a new class of RNA-based antiviral agents. In an efficient RNAi response, the antisense strand of siRNAs must enter the RNA-induced silencing complex (RISC) in a process mediated by thermodynamic features. In this report, we hypothesize that silent mutations capable of inverting thermodynamic properties can promote resistance to siRNAs. Extensive computational analyses were used to assess whether continuous selective pressure that promotes such mutations could lead to the emergence of viral strains completely resistant to RNAi (i.e., prone to transfer only the sense strands to RISC). Based on our findings, we propose that, although synonymous mutations may produce functional resistance, this strategy cannot be systematically adopted by viruses since the longest RNAi-refractory sequence is only 10 nt long. This finding also suggests that all mRNAs display fluctuating thermodynamic landscapes and that, in terms of thermodynamic features, RNAi is a very efficient antiviral system since there will always be sites susceptible to siRNAs.

Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-target Silencing

Off-target gene silencing is a major concern when using RNA interference. Imperfect pairing of the antisense strand with unintended mRNA targets is one of the main causes of small interfering RNA (siRNA) off-target silencing. To overcome this, we have developed "bulge-siRNA," a modified siRNA backbone structure with a single nucleotide (nt) bulge placed in the antisense strand. We found that siRNAs with a bulge at position 2 of the antisense strand were able to discriminate better between perfectly matched and mismatched targets, with no loss in silencing of the intended target. Genome-wide analysis also revealed that the bulge-siRNAs significantly reduced off-target silencing of transcripts with complementarity to the seed region of the siRNA antisense strand. When compared to 2'-methoxy ribosyl (2'-OMe) modified siRNAs previously developed to alleviate antisense off-target silencing; the bulge modification could better discriminate between on- versus off-targets. Our results suggest that the bulge-siRNA structure is a simple, yet superior alternative to chemical modifications for minimizing off-target silencing triggered by conventional siRNA structures.

Modeling RNA interference in mammalian cells

BackgroundRNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers.ResultsWe tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects.ConclusionsOur model has a simple mathematical form, amenable to analytical investigations and a small set of parameters with an intuitive physical meaning, that makes it a unique and reliable mathematical tool. The findings here presented will be a useful instrument for better understanding RNAi biology and as modelling tool in Systems and Synthetic Biology.

Computational models with thermodynamic and composition features improve siRNA design

BackgroundSmall interfering RNAs (siRNAs) have become an important tool in cell and molecular biology. Reliable design of siRNA molecules is essential for the needs of large functional genomics projects.ResultsTo improve the design of efficient siRNA molecules, we performed a comparative, thermodynamic and correlation analysis on a heterogeneous set of 653 siRNAs collected from the literature. We used this training set to select siRNA features and optimize computational models. We identified 18 parameters that correlate significantly with silencing efficiency. Some of these parameters characterize only the siRNA sequence, while others involve the whole mRNA. Most importantly, we derived an siRNA position-dependent consensus, and optimized the free-energy difference of the 5' and 3' terminal dinucleotides of the siRNA antisense strand. The position-dependent consensus is based on correlation and t-test analyses of the training set, and accounts for both significantly preferred and avoided nucleotides in all sequence positions. On the training set, the two parameters' correlation with silencing efficiency was 0.5 and 0.36, respectively. Among other features, a dinucleotide content index and the frequency of potential targets for siRNA in the mRNA added predictive power to our model (R = 0.55). We showed that our model is effective for predicting the efficiency of siRNAs at different concentrations.We optimized a neural network model on our training set using three parameters characterizing the siRNA sequence, and predicted efficiencies for the test siRNA dataset recently published by Novartis. On this validation set, the correlation coefficient between predicted and observed efficiency was 0.75. Using the same model, we performed a transcriptome-wide analysis of optimal siRNA targets for 22,600 human mRNAs.ConclusionWe demonstrated that the properties of the siRNAs themselves are essential for efficient RNA interference. The 5' ends of antisense strands of efficient siRNAs are U-rich and possess a content similarity to the pyrimidine-rich oligonucleotides interacting with the polypurine RNA tracks that are recognized by RNase H. The advantage of our method over similar methods is the small number of parameters. As a result, our method requires a much smaller training set to produce consistent results. Other mRNA features, though expensive to compute, can slightly improve our model.

A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites

The specificity of small interfering RNA (siRNA)-mediated gene silencing is a critical consideration for the application of RNA interference (RNAi). While the discovery of potential off-target effects by siRNAs is of concern, no systematic analysis has been conducted to explore the specificity of RNAi. Here, we present a study where a functionally validated siRNA (siCD46) was examined for silencing specificity on all possible 57 permutated target sites, each carrying a single-nucleotide mutation that would generate a mismatch when paired with siRNA antisense strand. We found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing. Surprisingly, mismatches formed between adenine (A) and cytosine (C), in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46. Northern blots showed that the silencing of fusion genes harboring the mutated target sites involved target mRNA degradation. This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is instrumental in the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.

The Activity of siRNA in Mammalian Cells is Related to the Kinetics of siRNA-target Recognition In Vitro: Mechanistic Implications

The specificity of siRNA-mediated suppression of gene expression involves base-base recognition between siRNA and its single-stranded RNA target. We investigated the kinetics of this process in vitro by using full-length ICAM-1 target RNA and biologically active and inactive ICAM-1-directed siRNA, respectively. To mimic the situation in living cells we used the well-characterised facilitator of RNA-RNA annealing and strand exchange cetyltrimethylammonium bromide, which increases strongly the kinetics of RNA-RNA interactions at conditions that do not affect RNA structure nor the presumed structure-function relationship. For the biologically active siRNA si2B, we find faster binding, i.e. recognition of the target and a slower backward reaction when compared with the biologically inactive siRNA si1. This is reflected by an approximately 400-fold more favorable equilibrium constant of si2B. Kinetic evidence favors an associative mechanism of recognition of the target strand by the double-stranded siRNA. The minimal model for siRNA-target recognition described here is consistent with the high biological activity of si2B only if one assumes a step subsequent to target recognition, which might be degradation of the target RNA when complexed with the antisense strand of siRNA or when considering rapid destruction of the released sense strand of siRNA.