siRNA function in RNAi: a chemical modification analysis

Abstract

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.

FIGURE 1.

Structures of EGFP siRNA and chemical modifications. (A) Graphical representation of dsRNAs used for targeting EGFP mRNA. EGFP was encoded by the pEGFP-C1 reporter plasmid. siRNAs were synthesized with 2-nt deoxythymidine overhangs at the 3′-end. The position of the first nucleotide of the mRNA target site is indicated relative to the start codon of EGFP mRNA. The sequence of the antisense strand of siRNA is exactly complementary to the mRNA target site. (B) Structure and nomenclature of chemical modifications.

FIGURE 2.

siRNA 2′-OH is not required to guide mRNA cleavage. (A) Sequence and structure of siRNA duplexes with modification at the 2′-position of the sugar unit. Nucleotides with 2′-hydroxyl groups (-OH) are black. Nucleotides with 2′-deoxy groups (-H) are cyan. Nucleotides with 2′-fluoro groups (-F) are red. The cleavage site on the target mRNA is also shown (red arrow). (B) Ratios of normalized GFP to RFP fluorescence intensity in lysates from modified siRNA-treated HeLa cells. The fluorescence intensity ratio of target (GFP) to control (RFP) fluorophores was determined in the presence of EGFP siRNA duplexes with modifications at the 2′-position of the sugar unit. Normalized ratios at <1.0 indicate specific RNA interference effects. For comparison, results from unmodified duplex siRNA-treated cells are included.

FIGURE 3.

Extending the half-life of siRNA duplexes prolongs the persistence of RNA interference in vivo. (A) Comparing the stability of unmodified siRNAs with siRNAs containing 2′-fluoro-uridine and 2′-fluoro-cytidine (2′-FU, 2′-FC) modifications (a) and thioate linkage (P–S) modifications (b). Unmodified or modified EGFP antisense strand siRNAs (AS) were 5′-labeled with [γ-³²P]ATP by T4 polynucleotide kinases. Duplex siRNAs were formed by annealing equal molar ratios of sense-strand (SS) siRNAs with the 5′-³²P-labeled antisense strand. To analyze siRNA stability in HeLa cell extract, 50 pmole of siRNA was incubated with 500 μg of HeLa cell extract in 50 μL of reaction mixture containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10 mM NaCl, 2 mM MgCl₂, and 10% glycerol. At various time points, siRNAs were extracted and analyzed on 20% polyacrylamide gels containing 7 M urea followed by phosphorimage analysis (Fugi). (B) Kinetics of RNAi effects of duplex siRNA with 2′-fluoro-uridine and 2′-fluoro-cytidine modification in HeLa cells over a 144-h time course. The fluorescence intensity ratio of target (GFP) to control (RFP) protein was determined in the presence of unmodified dsRNA (blue bars) and duplex siRNA with 2′-fluoro-uridine and -cytidine modifications (DS-2′-FU, 2′-FC, cyan bar) and normalized to the ratio observed in the presence of mock-treated cells (red bars). Normalized ratios at <1.0 indicated specific RNA interference.

FIGURE 4.

RNA interference mediated by siRNAs harboring modifications that stabilize base-pairing interactions. 5-Bromo-uridine (U[5Br]) or 5-iodo-uridine (U[5I]) replaced uridine or 2,6-diaminopurine (DAP) replaced adenine in siRNAs to stabilize base-pairing interactions. The activity of siRNAs with base modifications was quantified by the dual fluorescence assay. For comparison, results from unmodified duplex siRNA (DS, lanes 2–6)-treated cells are included.

FIGURE 5.

Structural integrity of the 5′ half of siRNA duplexes, as defined by the antisense strand, was functionally more important than at the 3′ half. (A) Graphical description of asymmetric requirement of duplex siRNA structure. The structure of unmodified (WT DS) siRNA duplexes, siRNAs with 2′-fluoro-uridine and 2′-fluoro-cytidine, 2′-deoxy modification at the 3′ half (SS/AS-2′-FU, 2′-FC + [9–19] dA, dG) or 5′ half (SS/AS-2′-FU, 2′-FC + [1–13] dA, dG) of the antisense strand, and siRNA duplexes with mismatches within the antisense 3′ half (SS/AS-[18,19] mm) or 5′ half (SS/AS-[1,2] mm) of the siRNA duplex are shown here. (B) Results from cells treated with duplex siRNAs with asymmetrically modified siRNA duplexes. For comparison, results from unmodified duplex siRNAs (DS, lanes 2–6)-treated cells are included.

FIGURE 6.

For analysis of siRNA-mediated target mRNA cleavage in vitro, 10 nM cap-labeled target EGFP mRNA was incubated with 100 nM siRNA and HeLa cytoplasmic extracts, as described in Materials and Methods. Reaction products were resolved on an 8% polyacrylamide-7 M urea gel. Arrows indicate the capped target EGFP mRNA and the 5′ cleavage product, which were expected to be 124 nt and 55 nt, respectively. The identity of the cleavage product was assigned according to RNase T1 partial digestion and a molecular weight marker of RNA (data not shown). The 3′ fragment is unlabeled, and therefore, invisible.

FIGURE 7.

Model for RNAi in human cells highlighting the requirement of the A-form helix and major groove for mRNA cleavage with the steps not requiring the RNA 2′-OH of the guide antisense siRNA. See text for details.