ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy

Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado–Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.     

RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2’-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC₅₀): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC₅₀: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.     

Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis

RNA interference can be considered as an antisense mechanism of action that utilizes a double-stranded RNase to promote hydrolysis of the target RNA. We have performed a comparative study of optimized antisense oligonucleotides designed to work by an RNA interference mechanism to oligonucleotides designed to work by an RNase H-dependent mechanism in human cells. The potency, maximal effectiveness, duration of action, and sequence specificity of optimized RNase H-dependent oligonucleotides and small interfering RNA (siRNA) oligonucleotide duplexes were evaluated and found to be comparable. Effects of base mismatches on activity were determined to be position-dependent for both siRNA oligonucleotides and RNase H-dependent oligonucleotides. In addition, we determined that the activity of both siRNA oligonucleotides and RNase H-dependent oligonucleotides is affected by the secondary structure of the target mRNA. To determine whether positions on target RNA identified as being susceptible for RNase H-mediated degradation would be coincident with siRNA target sites, we evaluated the effectiveness of siRNAs designed to bind the same position on the target mRNA as RNase H-dependent oligonucleotides. Examination of 80 siRNA oligonucleotide duplexes designed to bind to RNA from four distinct human genes revealed that, in general, activity correlated with the activity to RNase H-dependent oligonucleotides designed to the same site, although some exceptions were noted. The one major difference between the two strategies is that RNase H-dependent oligonucleotides were determined to be active when directed against targets in the pre-mRNA, whereas siRNAs were not. These results demonstrate that siRNA oligonucleotide- and RNase H-dependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays.     

Single-Stranded RNAs Use RNAi to Potently and Allele-Selectively Inhibit Mutant Huntingtin Expression

Mutant huntingtin (HTT) protein causes Huntington disease (HD), an incurable neurological disorder. Silencing mutant HTT using nucleic acids would eliminate the root cause of HD. Developing nucleic acid drugs is challenging, and an ideal clinical approach to gene silencing would combine the simplicity of single-stranded antisense oligonucleotides with the efficiency of RNAi. Here, we describe RNAi by single-stranded siRNAs (ss-siRNAs). ss-siRNAs are potent (>100-fold more than unmodified RNA) and allele-selective (>30-fold) inhibitors of mutant HTT expression in cells derived from HD patients. Strategic placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles. ss-siRNAs require Argonaute protein and function through the RNAi pathway. Intraventricular infusion of ss-siRNA produced selective silencing of the mutant HTT allele throughout the brain in a mouse HD model. These data demonstrate that chemically modified ss-siRNAs function through the RNAi pathway and provide allele-selective compounds for clinical development.     

Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA ‘gapmer’ oligonucleotide comprising a phosphorothioate central sequence flanked by 5′ and 3′ HNA sequences to conventional phosphorothioate oligonucleotides and to a 2′-O-methoxyethyl (2′-O-ME) phosphorothioate ‘gapmer’. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2′-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate ‘gap’) was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

Short-term and long-term modulation of gene expression by antisense therapeutics

To achieve effective modulation of gene expression by antisense oligonucleotides, novel oligonucleotide chemistries that do not promote RNase H degradation of target RNA are needed. In addition to short-term oligonucleotide effects, long-term gene regulation can be accomplished by intracellularly expressed antisense RNAs delivered by viral vectors.     

Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells

RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5’ untranslated region (5’UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5’UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2’-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5’UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.     

2'-fluoro-4'-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity

The synthesis of oligonucleotides containing 2′-deoxy-2′-fluoro-4′-thioarabinonucleotides is described. 2′-Deoxy-2′-fluoro-5-methyl-4′-thioarabinouridine (4′S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4′S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4′S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4′-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2′-Deoxy-2′-fluoro-4′-thioarabinouridine (4′S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2′F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.     

Thermodynamic criteria for high hit rate antisense oligonucleotide design

Antisense oligonucleotides are used for therapeutic applications and in functional genomic studies. In practice, however, many of the oligonucleotides complementary to an mRNA have little or no antisense activity. Theoretical strategies to improve the ‘hit rate’ in antisense screens will reduce the cost of discovery and may lead to identification of antisense oligonucleotides with increased potency. Statistical analysis performed on data collected from more than 1000 experiments with phosphorothioate-modified oligonucleotides revealed that the oligo-probes, which form stable duplexes with RNA (ΔG^o₃₇ ≤ –30 kcal/mol) and have small self-interaction potential, are more frequently efficient than molecules that form less stable oligonucleotide–RNA hybrids or more stable self-structures. To achieve optimal statistical preference, the values for self-interaction should be (ΔG^o₃₇) ≥ –8 kcal/mol for inter-oligonucleotide pairing and (ΔG^o₃₇) ≥ –1.1 kcal/mol for intra-molecular pairing. Selection of oligonucleotides with these thermodynamic values in the analyzed experiments would have increased the ‘hit rate’ by as much as 6-fold.     

Anti-Influenza Virus Activities of Nicked and Circular Dumbbell RNA/DNA Chimeric Oligonucleotides

Abstract

We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of nicked and circular dumbbell DNMA chimeric oligonucleotides.     

Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides

Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase H degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC(50) values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS-[2'meRNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially unaffected. Indeed, a PS-FANA oligomer containing a single deoxyribonucleotide residue core retained significant antisense activity. These findings correlated exactly with the ability of the various chimeric antisense molecules to elicit RNase H degradation of the target RNA in vitro, and suggest that this mode of inhibition is likely the most important determinant for potent antisense activity.     

Oligonucleotide N3'-->P5' phosphoramidates as potential therapeutic agents

Uniformly modified nucleic acids analogues, oligonucleotide N3'-->P5' phosphoramidates, containing 3'-amino instead of 3'-hydroxyl nucleosides, were synthesized and studied. These compounds form very stable duplexes with complementary native phosphodiester DNA and exceptionally stable duplexes with RNA strands. Increases in duplex melting temperature, deltaTm, relatively to their phosphodiester counterparts, reaches 2.9-3.5 degrees C per modified nucleoside. Moreover, the phosphoramidate compounds form extremely stable triple stranded complexes with single or double stranded DNA oligomers under near physiological salt and pH conditions. Melting temperatures of these triplexes usually exceed that of the isosequential phosphodiester counterparts by up to 35 degrees C. For 11-15-mers 2'-deoxyphosphoramidates are structurally and functionally similar to the native RNA molecules and thus can be used as RNA decoys. They are resistant to enzymatic digestion by nucleases both in vitro and in vivo. Oligonucleotide phosphoramidates apparently are cell permeable, and they have a good bioavailability and biodistribution, while being non-toxic in mice at therapeutically relevant doses. Duplexes of the several studied phosphoramidates with complementary RNA strands apparently are not substrates for RNase H in vitro. Despite that, these compounds exerted high sequence-specific antisense activity in various cell lines and in SCID mice. The observed in vitro lack of RNase H recognition of the RNA:phosphoramidate duplexes may result in better specificity in biological activity of these compounds relative to RNase H inducing oligonucleotides. Experimental results also indicate that oligonucleotide phosphoramidates can be used as efficient and specific modulators of gene expression by an antigene mechanism of action. Finally, the oligo-2'-deoxyphosphoramidate double stranded complexes can structurally mimic native RNA complexes, which could be efficiently and specifically recognized by the RNA binding proteins, such as HIV-1 Rev and Tat.     

Cleavage of RNA in hybrid duplexes by ribonuclease H ofE. coli: I. Substrate properties of complexes formed by RNA and a tandem of short oligodeoxyribonucleotides

We studied theE. coli RNase H cleavage of a 5′-labeled RNA fragment within two hybrid duplexes with identical sequences, one of which is formed by RNA and a 20-mer oligodeoxyribonucleotide (RNA/p20) whereas the second, by RNA and a tandem of short oligodeoxyribonucleotides (octanucleotide : tetranucleotide : octanucleotide) (RNA/tandem). It was shown that RNA in the RNA/p20 complex is hydrolyzed from the 3′-end to yield consecutively the 17-, 14-, 11-, 8-, and 5-mer 5′-labeled fragments. On hydrolysis of RNA in complex RNA/tandem, the same products were registered but their accumulation rates in this case differed. Thus, the initial rates of accumulation of the 17- and 8-mer were close. Moreover, the accumulation of the final 5-mer differed considerably: in the RNA/tandem complex it appeared within first minutes of the reaction but only after a considerable lag period in complex RNA/p20. These data testify that the tandem is involved not only in the consecutive accumulation of the shortened products (which is characteristic of complexes including extended oligonucleotides) but also in the parallel accumulation. This results from hydrolysis of each duplex segment formed by RNA and the short oligonucleotide of the tandem. Although the order of recognition and cleavage of RNA target by ribonuclease H at certain bonds depends on the type of the hybrid duplex, the destruction of RNA target within complex RNA/tandem and in complex with the full-size oligonucleotide occurs with a close effectiveness.     

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.     

Properties of isonucleotide-incorporated oligodeoxynucleotides and inhibition of the expression of spike protein of SARS-CoV

Antisense oligonucleotides are recognized to be very efficient tools for the inhibition of gene expression in a sequence specific way. For the discovery of a novel efficient way to modify oligonucleotides, a series of single isonucleotide-incorporated antisense oligodeoxynucleotides have been synthesized, in which an isonucleotide was introduced at different positions of the sequences. The binding behaviors of modified oligodeoxynucleotides to the complementary sequence were studied by UV, CD, and molecular dynamics simulation. The results showed that although the incorporated isonucleotides at certain positions of the sequence interfere with the binding ability to a different extent, B-form duplexes were maintained and the binding abilities of the 3'-end-modified duplexes were better than the corresponding mismatched duplexes. The digestion of modified oligodeoxynucleotides by snake venom phosphodiesterase showed that an isonucleotide strongly antagonizes hydrolysis. The DNA/RNA hybrid formed by a modified oligodeoxynucleotide and its target RNA could activate RNase H. The 3'-end-modified antisense oligodeoxynucelotides inhibited S-glycoprotein expression of SARS-CoV at the mRNA levels in insect Sf9 cells. This study indicated the possibility of designing a novel and effective antisense oligodeoxynucleotide by incorporating an isonucleotide at the 3'-end of the sequence.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA–protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176–183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 (‘slicer’ enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson–Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (T_M) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers.