Guide Strand Research Articles

Amide Modifications in the Seed Region of the Guide Strand Improve the On-Target Specificity of Short Interfering RNA.

RNA interference (RNAi) is a well-established research tool and is also maturing as a novel therapeutic approach. For the latter, microRNA-like off-target activity of short interfering RNAs (siRNAs) remains as one of the main problems limiting RNAi drug development. In this communication, we report that replacement of a single internucleoside phosphodiester in the seed region (nucleotides 2 to 7) of the guide strand with an amide linkage suppressed the undesired microRNA-like off-target activity by at least an order of magnitude. For the specific siRNA targeting the PIK3CB gene, an amide modification between the third and fourth nucleotides of the guide strand showed the strongest enhancement of specificity (completely eliminated off-target silencing) while maintaining high on-target activity. These results are important because off-target activity is one of the main remaining roadblocks for RNA based drug development.

Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position.

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.

2′-O-Methyl at 20-mer Guide Strand 3′ Termini May Negatively Affect Target Silencing Activity of Fully Chemically Modified siRNA

Small interfering RNAs (siRNAs) have the potential to treat a broad range of diseases. siRNAs need to be extensively chemically modified to improve their bioavailability, safety, and stability in vivo. However, chemical modifications variably impact target silencing for different siRNA sequences, making the activity of chemically modified siRNA difficult to predict. Here, we systematically evaluated the impact of 3′ terminal modifications (2′-O-methyl versus 2′-fluoro) on guide strands of different length and showed that 3′ terminal 2′-O-methyl modification negatively impacts activity for >60% of siRNA sequences tested but only in the context of 20- and not 19- or 21-nt-long guide strands. These results indicate that sequence, modification pattern, and structure may cooperatively affect target silencing. Interestingly, the introduction of an extra 2′-fluoro modification in the seed region at guide strand position 5, but not 7, may partially compensate for the negative impact of 3′ terminal 2′-O-methyl modification. Molecular modeling analysis suggests that 2′-O-methyl modification may impair guide strand interactions within the PAZ domain of argonaute-2, which may affect target recognition and cleavage, specifically when guide strands are 20-nt long. Our findings emphasize the complex nature of modified RNA-protein interactions and contribute to design principles for chemically modified siRNAs.

How to Computationally Stack the Deck for Hit-to-Lead Generation: In Silico Molecular Interaction Energy Profiling for de Novo siRNA Guide Strand Surrogate Selection.

The Argonaute-2 protein is part of the RNA-induced silencing complex (RISC) and anchors the guide strand of the small interfering RNA (siRNA). The 3'-end of the RNA contains two unpaired nucleotides (3'-overhang) that interact with the PAZ (PIWI/Argonaute/Zwille) domain of the protein. Theoretical and experimental evidence points toward a direct connection between the PAZ/3'-overhang binding affinity and siRNA's potency and specificity. Among the challenges to overcome when deploying siRNA molecules as therapeutics are their ready degradation under physiological conditions and off-target effects. It has been demonstrated that nuclease resistance can be improved via replacement of the dinucleotide overhang by small molecules which retain the interactions of the RNA guide strand with the PAZ domain. Most commonly, nucleotide analogues are used to substitute the siRNA overhang. However, in this study we adopt a de novo approach to its modification. The X-ray structure of human Argonaute-2 PAZ domain served to perform virtual screening and molecular interaction energy profiling (i.e., decomposition of the force field calculated protein-ligand interaction energies) of tailored-to-purpose fragment libraries. The binding of fragments to the PAZ domain was validated experimentally by NMR spectroscopy. The in silico guided protocol led to the efficient discovery of a number of PAZ domain ligands with affinities comparable to that of a reference dinucleotide (UpU, Kd = 33 μM). Originally starting from a generic fragment library, hits progress from 930 μM down to 14 μM within three iterations for the fragments selected via in silico molecular interaction energy profiling from a bespoke library. These dinucleotide siRNA guide strand surrogates represent potential new siRNA-based therapeutics (when attached to siRNA to form bioconjugates) featuring improved efficacy, specificity, stability, and cellular uptake. This project yielded a portfolio of seven patent applications, four of which have been granted to date.

A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs.

Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2. Eliminating off-target activity of the passenger strand will be important for improving therapeutic potential of RNAi.

Non-Specific Blocking of miR-17-5p Guide Strand in Triple Negative Breast Cancer Cells by Amplifying Passenger Strand Activity.

Conventional wisdom holds that only one of the two strands in a micro ribonucleic acid (miRNA) precursor duplex is selected as the active miRNA guide strand. The complementary miRNA passenger strand, however, is thought to be inactive. High levels of the oncogenic miRNA (oncomiR) guide strand called miR-17-5p is overexpressed in triple negative breast cancer (TNBC) and can inhibit ribosomal translation of tumor suppressor gene mRNAs, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, antisense knockdown of oncomiR miR-17-5p guide strand reduced PDCD4 and PTEN proteins by 1.8±0.3 fold in human TNBC cells instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3’UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knock down miR-17-5p reduced PDCD4 and PTEN protein expression instead of raising them. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand.

Dynamic Anchoring of the 3′-End of the Guide Strand Controls the Target Dissociation of Argonaute–Guide Complex

Argonaute (Ago) is the catalytic core of small RNA-based gene regulation. Despite plenty of mechanistic studies on Ago, the dynamical aspects and the mechanistic determinants of target mRNA binding and dissociation of Ago-guide strand remain unclear. Here, by using single-molecule fluorescence resonance energy transfer (FRET) assays and Thermus thermophilus Ago (TtAgo), we reveal that the 3'-end of the guide strand dynamically anchors at and releases from the PAZ domain of Ago, and that the 3'-end anchoring of the guide strand greatly accelerates the target dissociation by destabilizing the guide-target duplex. Our results indicate that the target binding/dissociation of Ago-guide is executed through the dynamic interplays among Ago, guide, and target.

Sa1015 Long Duration of Effect From RNAI Therapeutic to Treat Chronic Hepatitis B Virus Infection Correlates With Persistence of the Phosphorylated Guide Strand

Chronic hepatitis B virus (HBV) infection is a major disease for which there remains an unmet medical need. Current therapies for chronic hepatitis B include reverse transcriptase inhibitors and interferon. These therapies either require life-long administration or have significant side effects and limited efficacy. We have taken a novel approach toward the treatment of chronic hepatitis B by developing an siRNA-based therapeutic. In contrast to current therapies, our approach has the promise of significantly decreasing viral protein load which is primarily responsible for disease progression. In our formulation, liver-tropic cholesterol-conjugated siRNAs against HBV (chol-siHBVs) are co-injected intravenously with a reversibly masked, hepatocyte-targeted melittin-like peptide (NAG-MLP). Co-injection of chol-siHBVs and NAG-MLP results in multi-log repression of viral RNA, proteins and viral DNA with long duration of effect in transient and transgenic mouse models of chronic HBV infection, without toxicity. Using a hybridization/HPLC-based method of detection, we are able to correlate the degree of repression of the virus with the amount of the active form of the siRNA guide strand in the liver. In addition, this form of the guide strand can be detected up to one month after a single administration, correlating with the duration of effect. High efficacy, a long duration of effect and establishment of a robust pharmacokinetic/ pharmacodynamic relationship in the liver suggest co-injection of NAG-MLP and cholsiHBVs holds great promise as a novel therapeutic for chronically HBV infected patients.

Promiscuous 8-Alkoxyadenosines in the Guide Strand of an SiRNA: Modulation of Silencing Efficacy and Off-Pathway Protein Binding

8-Alkoxyadenosines have the potential to exist in anti or syn conformations around the glycosidic bond when paired opposite to U or G in the complementary strands, thereby placing the sterically demanding 8-alkoxy groups in the major or minor groove, respectively, of duplex RNA. These modified bases were used as "base switches" in the guide strands of an siRNA to prevent off-pathway protein binding during delivery via placement of the alkoxy group in the minor groove, while maintaining significant RNAi efficacy by orienting the alkoxy group in the major groove. 8-Alkoxyadenosine phosphoramidites were synthesized and incorporated into the guide strand of caspase 2 siRNA at four different positions: two in the seed region, one at the cleavage junction, and another nearer to the 3'-end of the guide strand. Thermal stabilities of the corresponding siRNA duplexes showed that U is preferred over G as the base-pairing partner in the complementary strand. When compared to the unmodified positive control siRNAs, singly modified siRNAs knocked down the target mRNA efficiently and with little or no loss of efficacy. Doubly modified siRNAs were found to be less effective and lose their efficacy at low nanomolar concentrations. SiRNAs singly modified at positions 6 and 10 of the guide strand were found to be effective in blocking binding to the RNA-dependent protein kinase PKR, a cytoplasmic dsRNA-binding protein implicated in sequence-independent off-target effects.

Nucleoside Optimization for RNAi: A High-Throughput Platform

The RNA induced silencing complex (RISC) contains at its core the endonuclease Argonaute (Ago) that allows for guide strand (GS)-mediated sequence-specific cleavage of the target mRNA. Functionalization of the sugar/phosphodiester backbone of the GS, which is in direct contact with Ago, presents a logical opportunity to affect RISC's activity. A systematic evaluation of modified nucleosides requires the synthesis of phosphoramidites corresponding to all four canonical bases (A, U, C, and G) and their sequential evaluation at each position along the 21-nucleotide-long GS. With the use of a platform approach, the sequential replacement of canonical bases with inosine greatly simplifies the problem and defines a new activity baseline toward which the corresponding sugar-modified inosines are compared. This approach was validated using 2'-O-benzyl modification, which demonstrated that positions 5, 8, 15, and 19 can accommodate this large group. Application of this high-throughput methodology now allows for hypothesis-driven rational design of highly potent, immunologically silent and stable siRNAs suitable for therapeutic applications.

SiRNA Repositioning for Guide Strand Selection by Human Dicer Complexes

The human ribonuclease Dicer and its double-stranded RNA (dsRNA)-binding protein (dsRBP) partners TRBP and PACT play important roles in the biogenesis of regulatory RNAs. Following dicing, one dsRNA product strand is preferentially assembled into an RNA-induced silencing complex (RISC). The mechanism of strand selection in humans and the possible role of Dicer in this process remain unclear. Here we demonstrate that dsRNAs undergo significant repositioning within Dicer complexes following dicing. This repositioning enables directional binding of RNA duplexes, thereby biasing their orientation for guide strand selection according to the thermodynamic properties of the helix. Our findings indicate that Dicer is itself capable of sensing siRNA thermodynamic asymmetry regardless of the dsRBP to which it is bound. These results support a model in which Dicer employs two distinct RNA-binding sites-one for dsRNA processing and the other for sensing of siRNA thermodynamic asymmetry-during RISC loading in humans.

Enhanced specificity of HPV16 E6E7 siRNA by RNA–DNA chimera modification

Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecific cytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1-8 and its complementary part of the passenger strand plus the 3' overhang, which is designated as a double-strand RNA-DNA chimera (dsRDC). In this study, we found that the specificity of potent siRNAs targeting human papillomavirus 16 (HPV16) E6 and E7 oncogenes, which we previously reported, could be enhanced by short dsRDC modification (first six nucleotides from the 5' end of the GS and its complementary nucleotides of the passenger strand). Such dsRDC modification reduced nonspecific cytotoxicity in two of three siRNAs (497 and 752), although not in the other (573), which correlated with their off-target effects. In addition, silencing activity was marginally impaired in two dsRDCs (497 and 573) and moderately in one (752). Finally, dsRDC-497 induced E6E7-specific growth suppression of cervical cancer cells as well as E6E7-immortalized human keratinocytes. Our results show that dsRDC modification enhances the specificity of E6E7 siRNA, which is required for use in in vivo settings.

Parallel assessment of silencing potency of conventional siRNAs vs. Dicer substrate interfering RNAs

RNA interference (RNAi) is a sequence specific gene silencing mechanism that can decrease the expression of target proteins. RNAi's sequence specificity has made it a powerful tool for cell biologists and is being developed for therapeutics. RNAi is triggered by short interfering RNA (siRNA), which are ~21bp RNA duplexes designed such that one of the strands (the guide strand (GS)) is complementary to the mRNA of the target protein. Biasing strand selection in favor of the guide strand, rather than the non‐guide strand (the passenger strand (PS)) will improve gene silencing potency. Our lab has developed longer, asymmetric, and chemically modified siRNA variants, termed Dicer substrate interfering RNA (dsiRNA). DsiRNA are engineered such that the GS is preferentially selected. Here we present a parallel screening of gene silencing ability of conventional siRNA vs. dsiRNA. Using a psiCHECK assay and calculating the 50% inhibitory concentration (IC50), we found that the PS of conventional siRNA exhibited > 8‐fold lower IC50 value than the GS (IC50(PS) = 9.75 pM vs. IC50(GS) = 71.55 pM). Meanwhile, dsiRNA reduced the silencing potency of the PS (IC50(PS) = 24.40 pM) while simultaneously increasing the silencing potency of the GS (IC50(GS) = 29.90 pM). Thus, relative to conventional siRNA, the biased strand selection of the dsiRNA improves the silencing ability of the GS and reduces the silencing ability of the PS.

The Mechanism Selecting the Guide Strand from Small RNA Duplexes is Different Among Argonaute Proteins

Double-stranded RNA induces RNA silencing and is cleaved into 21-24 nt small RNA duplexes by Dicer enzyme. A strand of Dicer-generated small RNA duplex (called the guide strand) is then selected by a thermodynamic mechanism to associate with Argonaute (AGO) protein. This AGO-small RNA complex functions to cleave mRNA, repress translation or modify chromatin structure in a sequence-specific manner. Although a model plant, Arabidopsis thaliana, contains 10 AGO genes, their roles and molecular mechanisms remain obscure. In this study, we analyzed the roles of Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine (85.7%) and that with AGO5 was mainly cytosine (83.5%). Small RNAs that were abundantly cloned from the AGO2 immunoprecipitation fraction (miR163-LL, which is derived from the Lower Left of mature miR163 in pre-miR163, and miR390) and from the AGO5 immunoprecipitation fraction (miR163-UL, which is derived from the Upper Left of mature miR163 in pre-miR163, and miR390(*)) are derived from the single small RNA duplexes, miR163-LL/miR163-UL and miR390/miR390(*). Each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner rather than in a thermodynamic stability-dependent manner. Furthermore, we showed that both AGO2 and AGO5 have the ability to bind cucumber mosaic virus-derived small RNAs. These results clearly indicate that the mechanism selecting the guide strand is different among AGO proteins and that multiple AGO genes are involved in anti-virus defense in plants.

997. Loop Composition as Well as Orientation of the Guide Strand in shRNA May Dramatically Modulate Activity of the shRNA: Effect on the Development of RNAi-Based Inhibitors

RNA interference (RNAi) is a new promising technology for therapeutic drug development. It can be initiated either by chemically synthesized double-stranded RNA delivered to a cell, or by transcription of a short hairpin RNA in the cell nucleus from a DNA template. It has been realized that many RNA sequences although |[ldquo]|active|[rdquo]| as siRNAs, are often inactive as shRNAs. The composition of the loop sequence in shRNA has been suggested to play a role in determining its activity by affecting shRNA transport from the cell nucleus to the cytoplasm (Kawasaki and Taira, Nucleic Acids Res. 2003, 31(2):700-7). In the present study we identified several siRNA sequences that efficiently inhibit HCV replication in the replicon system. In subsequent attempts to generate active shRNAs spanning these validated sequences, we tested the effect of two different loop sequences, as well as the guide strand orientation on the shRNA activity. Functional tests were performed in the HCV replicon systems, as well as in a model luciferase-HCV fusion systems. We demonstrate that the context of the loop within the shRNA has a dramatic effect on shRNA activity, and a loop that is |[ldquo]|inactive|[rdquo]| in one shRNA can be fully active in the context of another shRNA. We also demonstrate that the orientation of the guide strand in the shRNAs (sense vs antisense) may dramatically influence the shRNA activity. Changes in the extent and precision of the shRNA processing by Dicer will be presented. The effect of these findings on the development of ddRNAi-based inhibitors will be discussed.

An analysis of mismatched duplex DNA unzipping through a bacterial nanopore.

A 50-base Guide strand was synthesized that consisted of a central 10-base probe sequence flanked by two tracts of 20 adenine residues. Target sequences of 10 bases containing up to three mismatches were prepared and hybridized to the Guide strand in 1 M KCl. The transport of these constructs through single alpha-hemolysin pores was analysed by measuring the current blockade as a function of time. Complementary dsDNA takes significantly longer (840 +/- 60 micro s) to pass through the pore than a sequence of the same length containing a single (590 +/- 45 micro s) and a double (270 +/- 50 micro s) mismatch. Constructs involving three mismatches were indistinguishable from Guide ssDNA transport (120 +/- 30 micro s). The results suggest that dsDNA must unzip as it is transported through the nanopore. Duplexes containing mismatches unzip more quickly and can be distinguished from those with perfect complementarity.