Limits...
Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi.

Watanabe C, Cuellar TL, Haley B - RNA Biol (2016)

Bottom Line: Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences.Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds.Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.

View Article: PubMed Central - PubMed

Affiliation: a Departments of Bioinformatics and Computational Biology , South San Francisco , CA 94080.

ABSTRACT
Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.

No MeSH data available.


Potential liabilities identified for miR-3G and miR-E-based RNAi. (A) Total guide RNA values were determined by adding all reads with the designated guide RNA 5′ nt, as well as those +/− 1 nt 5′ shifted from the appropriate start site. Displayed are the ratios, for each targeting sequence and hairpin context, of guides with the appropriate 5′ nt relative to the total guide RNA read count. (B) Total guide RNA read counts (computed as in A) and passenger strand reads, which included the dominant passenger strand read and all reads with a +/− 1 nt 5′ shift are compared for miR-3G relative to miR-E.
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f0005: Potential liabilities identified for miR-3G and miR-E-based RNAi. (A) Total guide RNA values were determined by adding all reads with the designated guide RNA 5′ nt, as well as those +/− 1 nt 5′ shifted from the appropriate start site. Displayed are the ratios, for each targeting sequence and hairpin context, of guides with the appropriate 5′ nt relative to the total guide RNA read count. (B) Total guide RNA read counts (computed as in A) and passenger strand reads, which included the dominant passenger strand read and all reads with a +/− 1 nt 5′ shift are compared for miR-3G relative to miR-E.

Mentions: Detailed assessment of the guide and passenger strand reads from miR-3G and miR-E suggests strengths and liabilities, with respect to targeting specificity, for each system that may be improved through refined target sequence prediction schemes and alterations of the stem loop sequence/structure. In particular, we noted that while >80% of the sequences for each miR-E derived guide RNA initiated from the designated 5′ nt, 2/6 miR-3G derived guides did not reach this threshold (Fig. 5A). This is consistent with the finding that the 5′ terminal nt of miRNAs derived from the 5p arm of their stem loops, like miR-3G derived guides, can be less precisely defined than the 5′ ends of 3p arm-derived small RNAs, such as those from miR-E26. Contrary to those observations, however, and not unlike observations made in the Drosophila system, 4/6 miR-3G-derived guide RNAs were processed with equal precision to those from miR-E, suggesting that siRNA-intrinsic sequences may impact small RNA processing fidelity, independent of the miR-16-2 scaffold, and definition of these sequence features could be exploited to improve the design and expression of miR-3G adapted siRNAs.39 Separately, a comparison of guide vs. passenger strand accumulation patterns (inclusive of all small RNAs containing +/− 1 5´nt of the predicted guide RNA and +/− 1 5 nt of the dominant passenger sequence) revealed that miR-3G generates small RNAs with a ∼2-fold average greater ratio of asymmetry, relative to those derived from miR-E (Fig. 5B). The observed skewing of guide strand accumulation for miR-3G compared to miR-E occurs despite similar trends in sequence-by-sequence asymmetry for the 2 contexts. This could reflect the intrinsically higher asymmetric processing of endogenous miR-16-2, with respect to miR-30a, and suggests that further manipulation of the miR-E stem loop may lead to more favorable guide versus passenger strand ratios and decreased off-target silencing potential.Figure 5.


Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi.

Watanabe C, Cuellar TL, Haley B - RNA Biol (2016)

Potential liabilities identified for miR-3G and miR-E-based RNAi. (A) Total guide RNA values were determined by adding all reads with the designated guide RNA 5′ nt, as well as those +/− 1 nt 5′ shifted from the appropriate start site. Displayed are the ratios, for each targeting sequence and hairpin context, of guides with the appropriate 5′ nt relative to the total guide RNA read count. (B) Total guide RNA read counts (computed as in A) and passenger strand reads, which included the dominant passenger strand read and all reads with a +/− 1 nt 5′ shift are compared for miR-3G relative to miR-E.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829305&req=5

f0005: Potential liabilities identified for miR-3G and miR-E-based RNAi. (A) Total guide RNA values were determined by adding all reads with the designated guide RNA 5′ nt, as well as those +/− 1 nt 5′ shifted from the appropriate start site. Displayed are the ratios, for each targeting sequence and hairpin context, of guides with the appropriate 5′ nt relative to the total guide RNA read count. (B) Total guide RNA read counts (computed as in A) and passenger strand reads, which included the dominant passenger strand read and all reads with a +/− 1 nt 5′ shift are compared for miR-3G relative to miR-E.
Mentions: Detailed assessment of the guide and passenger strand reads from miR-3G and miR-E suggests strengths and liabilities, with respect to targeting specificity, for each system that may be improved through refined target sequence prediction schemes and alterations of the stem loop sequence/structure. In particular, we noted that while >80% of the sequences for each miR-E derived guide RNA initiated from the designated 5′ nt, 2/6 miR-3G derived guides did not reach this threshold (Fig. 5A). This is consistent with the finding that the 5′ terminal nt of miRNAs derived from the 5p arm of their stem loops, like miR-3G derived guides, can be less precisely defined than the 5′ ends of 3p arm-derived small RNAs, such as those from miR-E26. Contrary to those observations, however, and not unlike observations made in the Drosophila system, 4/6 miR-3G-derived guide RNAs were processed with equal precision to those from miR-E, suggesting that siRNA-intrinsic sequences may impact small RNA processing fidelity, independent of the miR-16-2 scaffold, and definition of these sequence features could be exploited to improve the design and expression of miR-3G adapted siRNAs.39 Separately, a comparison of guide vs. passenger strand accumulation patterns (inclusive of all small RNAs containing +/− 1 5´nt of the predicted guide RNA and +/− 1 5 nt of the dominant passenger sequence) revealed that miR-3G generates small RNAs with a ∼2-fold average greater ratio of asymmetry, relative to those derived from miR-E (Fig. 5B). The observed skewing of guide strand accumulation for miR-3G compared to miR-E occurs despite similar trends in sequence-by-sequence asymmetry for the 2 contexts. This could reflect the intrinsically higher asymmetric processing of endogenous miR-16-2, with respect to miR-30a, and suggests that further manipulation of the miR-E stem loop may lead to more favorable guide versus passenger strand ratios and decreased off-target silencing potential.Figure 5.

Bottom Line: Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences.Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds.Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.

View Article: PubMed Central - PubMed

Affiliation: a Departments of Bioinformatics and Computational Biology , South San Francisco , CA 94080.

ABSTRACT
Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.

No MeSH data available.