RNA interference using boranophosphate siRNAs: structure-activity relationships

Abstract

In RNA interference (RNAi), short double-stranded RNA (known as siRNA) inhibits expression from homologous genes. Clinical or pre-clinical use of siRNAs is likely to require stabilizing modifications because of the prevalence of intracellular and extracellular nucleases. In order to examine the effect of modification on siRNA efficacy and stability, we developed a new method for synthesizing stereoregular boranophosphate siRNAs. This work demonstrates that boranophosphate siRNAs are consistently more effective than siRNAs with the widely used phosphorothioate modification. Furthermore, boranophosphate siRNAs are frequently more active than native siRNA if the center of the antisense strand is not modified. Boranophosphate modification also increases siRNA potency. The finding that boranophosphate siRNAs are at least ten times more nuclease resistant than unmodified siRNAs may explain some of the positive effects of boranophosphate modification. The biochemical properties of boranophosphate siRNAs make them promising candidates for an RNAi-based therapeutic.

Figure 1

Synthesis of modified siRNAs through T7 in vitro transcription.

Figure 2

(A) Gel analysis of enzymatically synthesized single-stranded RNA samples used for mass spectrometry experiments. The sequence of the RNA is 5′-pppGpUpUpCpApCpCpUpUpGpApUpGpCpCpGpUpUpCpUpU-3′. The samples were analyzed with 20% PAGE/7 M urea. The gel was stained with SYBR® Green II and visualized with a UV transilluminator. Lane Std is a chemically synthesized 21-nt RNA. Lane M is 10-bp DNA marker. Lane PO is enzymatically synthesized normal RNA. Lane BP is enzymatically synthesized boranophosphate RNA. (B) Mass spectrometry spectra of enzymatically synthesized RNA samples: PO for normal; PS for phosphorothioate; BP: boranophosphate.

Figure 3

(A) Native and modified siRNA species. Modified nucleotides are shown in red. For control siRNAs, the inverted sequence is underlined. A—antisense strand, S—sense strand, b—boranophosphate, t—phosphorothioate, n—native, a—adenosine, c—cytidine, u—uridine, 3—adenosine, cytidine and uridine. (B) Structure of native (left), phosphorothioate (Rp isomer, center) and boranophosphate (Sp isomer, right) ribonucleic acid backbone linkages.

Figure 4

Position-specific effects of phosphorothioate and boranophosphate modifications on silencing activity. (A) Percent inhibition of GFP fluorescence in cells treated with native (grey bar), phosphorothioate (black bars) and boranophosphate (white bars) EGFP1 siRNA at 25 nM. To account for variations in transfection efficiency, the results of each individual experiment were normalized to results for native siRNA-treated cells in that experiment. Error bars represent the standard error. (B) Percent inhibition of GFP fluorescence in cells treated with boranophosphate modified or unmodified EGFP2 siRNA at 25 nM. Error bars represent the standard error. A—antisense strand, S—sense strand, b—boranophosphate, t—phosphorothioate, n—native, a—adenosine, c—cytidine, u—uridine, cont—inverted control sequence.

Figure 5

Effect of increased boranophosphate modification on siRNA activity. Percent inhibition of GFP fluorescence in cells treated with native (grey bar), or boranophosphate siRNAs with the EGFP1 sequence at 25 nM. siRNAs with <3 central modifications are represented by black bars. Those with ≥3 central modifications are represented by white bars. Error bars represent the standard error. The numbers below the graph correspond to the percent of residues in the duplex region that are modified. A—antisense strand, S—sense strand, b—boranophosphate, n—native, a—adenosine, c—cytidine, u—uridine, 3—adenosine, cytidine and uridine.

Figure 6

Effect of boranophosphate modification on siRNA potency. SiRNA dose–response as measured by the decrease in GFP fluorescence. The effect of each siRNA (EGFP1 sequence) at 25 nM is normalized to an arbitrary value of 100 to reveal changes in efficacy. (A) Effect of A^b₃ S^b₃ (open triangles), A^b_c S^b₃ (closed triangles), A^b_c S^b_c (open circles), and native siRNA (closed circles). A—antisense strand, S—sense strand, b—boranophosphate, n—native, a—adenosine, c—cytidine, u—uridine, 3—adenosine, cytidine and uridine. (B) Effect of boranophosphate-modified cytidines on the antisense (open circle) or sense (closed circle) strand. (C) Effect of boranophosphate-modified cytidines and adenosines on the antisense (open circle) or sense (closed circle) strand.

Figure 7

Effect of boranophosphate-modified EGFP1 siRNA on mRNA and protein levels. Percent reduction in mRNA (white triangle) and protein (black circle) levels as determined by northern and FACS analysis when transfected with 12.5 nM siRNA. Samples of cells from the same population were used for both analyses. EGFP mRNA levels were normalized to β-actin expression. A—antisense strand, S—sense strand, b—boranophosphate, c—cytidine, 3—adenosine, cytidine and uridine, cont—native control EGFP1 sequence.

Figure 8

Stability of native and boranophosphate siRNAs. siRNAs were incubated with bovine pancreatic RNases for the times indicated, and then assessed for degradation by agarose gel electrophoresis. Above: Plot of siRNA degradation by RNase over time. Half-lives were calculated based on regression using the data shown. Error bars represent standard error. Below: photograph of siRNA samples after RNase incubation and electrophoresis. A—antisense strand, S—sense strand, b—boranophosphate, n—native, a—adenosine, c—cytidine, u—uridine, 3—adenosine, cytidine and uridine.