Conditional Dicer substrate formation via shape and sequence transduction with small conditional RNAs

Abstract

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) enables knockdown of a gene of choice, executing the logical operation: silence gene Y. The fact that the siRNA is constitutively active is a significant limitation, making it difficult to confine knockdown to a specific locus and time. To achieve spatiotemporal control over silencing, we seek to engineer small conditional RNAs (scRNAs) that mediate 'conditional RNAi' corresponding to the logical operation: if gene X is transcribed, silence independent gene Y. By appropriately selecting gene X, knockdown of gene Y could then be restricted in a tissue- and time-specific manner. To implement the logic of conditional RNAi, our approach is to engineer scRNAs that, upon binding to mRNA 'detection target' X, perform shape and sequence transduction to form a Dicer substrate targeting independent mRNA 'silencing target' Y, with subsequent Dicer processing yielding an siRNA targeting mRNA Y for destruction. Toward this end, here we design and experimentally validate diverse scRNA mechanisms for conditional Dicer substrate formation. Test tube studies demonstrate strong OFF/ON conditional response, with at least an order of magnitude increase in Dicer substrate production in the presence of the cognate mRNA detection target. By appropriately dimensioning and/or chemically modifying the scRNAs, only the product of signal transduction, and not the reactants or intermediates, is efficiently processed by Dicer, yielding siRNAs. These mechanism studies explore diverse design principles for engineering scRNA signal transduction cascades including reactant stability vs metastability, catalytic vs noncatalytic transduction, pre- vs post-transcriptional transduction, reactant and product molecularity, and modes of molecular self-assembly and disassembly.

Figure 1

Molecular logic of conditional and conventional RNAi. Conditional RNAi (if gene X is transcribed, silence independent gene Y) provides a conceptual framework for exerting spatiotemporal control over gene knockdown. Toward this end, small conditional RNAs (scRNAs) interact and change conformation to transduce between binding of mRNA ‘detection target’ X and production of a Dicer substrate targeting independent mRNA ‘silencing target’ Y. Inset: Conventional RNAi (silence gene Y) employs constitutively active Dicer substrates, making it difficult to control the locus and time of gene knockdown. We consider conditional formation of Dicer substrates that are either DsiRNAs or shRNAs.

Figure 2

Conditional catalytic DsiRNA formation using metastable scRNAs. (a) Mechanism 1. scRNA A detects mRNA detection target X (containing subsequence ‘a-b-c-d’) to form catalyst X·A, which mediates production of DsiRNA Dicer substrate B·C targeting mRNA silencing target Y (containing independent subsequence ‘v-w-x-y-z’). scRNAs A, B, and C coexist metastably in the absence of X. Successive toehold-mediated 3-way branch migrations enable assembly of X with A (step 1), X·A with B (step 2), X·A·B with C (step 3a), and disassembly of DsiRNA Dicer substrate B·C from catalyst X·A (step 3b). Domain lengths: |a| = 10, |b| = 10, |c| = 5, |d| = 2, |e| = 2, |v| = 2, |w| = 5, |x| = 2, |y| = 6, |z| = 5. Chemical modifications (2′OMe-RNA): A and parts of B and C (dashed backbone). (b) Conditional catalytic Dicer substrate formation. OFF state: minimal production of Dicer substrate B·C in the absence of detection target X, the presence of mRNA silencing target Y, or the presence of mRNA off-target Z. ON state: strong production of B·C in the presence of substoichiometric or stoichiometric short RNA detection target X_s (‘a-b-c-d’) or the presence of full-length mRNA detection target X. (c) Quantification of the Dicer substrate band (B·C) in panel (b). (d) Conditional Dicer processing. OFF state: minimal processing of the reactants (lane 2). ON state: efficient processing of Dicer substrate B·C (lane 4), yielding canonical 21- and 23-nt siRNAs (boxed bands). The non-siRNA remainder of the cleaved substrate is labeled ‘waste’. See Section S2 for additional computational and experimental studies of Mechanism 1.

Figure 3

Conditional DsiRNA formation using stable scRNAs. (a) Mechanism 2. scRNA A·B detects mRNA detection target X (containing subsequence ‘a-b-c’), leading to production of DsiRNA Dicer substrate B·C targeting mRNA silencing target Y (containing independent subsequence ‘w-x-y-z’). scRNAs A·B and C are stable in the absence of X. A swaps B for X (step 1) via toehold-mediated 3-way branch migration and spontaneous dissociation. B assembles with C (step 2) via loop/toehold nucleation and 3-way branch migration to form DsiRNA Dicer substrate B·C. Domain lengths: |a| = 6, |b| = 4, |c| = 8, |s| = 5, |w| = 2, |x| = 12, |y| = 4, |z| = 3. Chemical modifications (2′OMe-RNA): A and part of C (dashed backbone). (b) Conditional Dicer substrate formation. OFF state: minimal production of Dicer substrate B·C in the absence of detection target X, the presence of mRNA silencing target Y, or the presence of mRNA off-target Z. ON state: strong production of B·C in the presence of short RNA detection target X_s (‘a-b-c’) or full-length mRNA detection target X. (c) Quantification of the Dicer substrate band (B·C) in panel (b). (d) Conditional Dicer processing. OFF state: minimal Dicer processing of the reactants (lane 2). ON state: efficient Dicer processing of DsiRNA Dicer substrate B·C (lane 4), yielding canonical 21–23-nt siRNAs (boxed bands). The non-siRNA remainder of the cleaved substrate is labeled ‘waste’. See Section S3 for additional computational and experimental studies of Mechanism 2.

Figure 4

Conditional shRNA formation using a single stable scRNA. (a) Mechanism 3. scRNA A·B detects mRNA detection target X (containing subsequence ‘a-b-c’), leading to production of shRNA Dicer substrate B targeting mRNA silencing target Y (containing independent subsequence ‘y-z’). scRNA A·B is stable in the absence of X. X partially displaces A from B via toehold-mediated 3-way branch migration, exposing a previously sequestered internal toehold, ‘c’, within B, mediating a further 3-way branch migration that disassembles B from X·A to yield shRNA Dicer substrate B. Domain lengths: |a| = 12, |b| = 14, |c| = 3, |y| = 2, |z| = 19. Chemical modifications (2′OMe-RNA): A (dashed backbone). (b) Conditional Dicer substrate formation. OFF state: minimal production of Dicer substrate B in the absence of detection target X, the presence of mRNA silencing target Y, or the presence of mRNA off-target Z. ON state: strong production of B in the presence of short RNA detection target X_s (‘a-b-c’) or full-length mRNA detection target X. (c) Quantification of the Dicer substrate band (B) in panel (b). (d) Conditional Dicer processing. OFF state: minimal processing of the reactants (lane 2). ON state: efficient processing of shRNA Dicer substrate B (lane 4), yielding canonical 21- and 22-nt siRNAs (boxed bands). See Section S4 for additional computational and experimental studies of Mechanism 3.

Figure 5

Conditional DsiRNA formation via template-mediated 4-way branch migration. (a) Mechanism 4. scRNAs A·B and C·D detect mRNA detection target X (containing subsequence ‘a-b-c-d-e’), leading to production of DsiRNA Dicer substrate B·C targeting mRNA silencing target Y (containing independent subsequence ‘x-y-z’). scRNAs A·B and C·D coexist metastably in the absence of X. X templates conucleation of A·B and C·D, mediating a short 3-way branch migration that enables toehold/toehold nucleation between B and C to create a 5-way junction (step 1a). Subsequent 4-way branch migration and spontaneous dissociation disassemble DsiRNA Dicer substrate B·C from X·A·D (step 1b). Domain lengths: |a| = 8, |b| = 6, |c| = 6, |d| = 7, |e| = 11, |x| = 2, |y| = 19, |z| = 2. Chemical modifications (2′OMe-RNA): A and D (dashed backbone). (b) Conditional Dicer substrate formation. OFF state: minimal production of Dicer substrate B·C in the absence of detection target X, the presence of mRNA silencing target Y, or the presence of mRNA off-target Z. ON state: strong production of B·C in the presence of short RNA detection target X_s (‘a-b-c-d-e’) or full-length mRNA detection target X. (c) Quantification of the Dicer substrate band (B·C) in panel (b). (d) Conditional Dicer processing. OFF state: minimal processing of the reactants (lane 2). ON state: efficient processing of DsiRNA Dicer substrate B·C (lane 4), yielding canonical 21–24-nt siRNAs (boxed bands). Additional Dicer products are produced by unexpected cleavage of the substrate within domains ‘y’ and ‘y*’. See Section S5 for additional computational and experimental studies of Mechanism 4.

Figure 6

Conditional shRNA transcription using scDNAs. (a) Mechanism 5. scDNA A detects mRNA detection target X (containing subsequence ‘a-b’) and assembles with B to form a transcription template (containing promoter, coding, and termination sequences), leading to transcription of the shRNA Dicer substrate C targeting mRNA silencing target Y (containing independent subsequence ‘z’). scDNAs A and B coexist metastably in the absence of X. X assembles with A via toehold-mediated 3-way branch migration (step 1). Subsequently, X·A assembles with B via toehold-mediated 4-way branch migration to produce a dsDNA transcription template (step 2), mediating transcription of shRNA Dicer substrate C with catalytic turnover. Domain lengths: |a| = 10, |b| = 8, |p| = 8, |q| = 9, |t| = 7, |y| = 6, |z| = 19. (b) Conditional transcription template formation. OFF state: minimal production of transcription template A·B in the absence of short DNA detection target X_s (‘a-b’), the presence of mRNA silencing target Y, or the presence of mRNA off-target Z. ON state: strong production of transcription template X_s·A·B in the presence of X_s. (c) Conditional Dicer substrate transcription and processing. OFF state: minimal transcription of Dicer substrate C in the absence of short DNA detection target X_s (lane 1). ON state: strong transcription of C in the presence of X_s (lane 3) and efficient Dicer processing of shRNA Dicer substrate C (lane 4). (d) Quantification of the Dicer substrate band (C) in lanes 1 and 3 of panel (c). See Section S6 for additional computational and experimental studies of Mechanism 5.