Sequence-specific cleavage of RNA by Type II restriction enzymes

Abstract

The ability of 223 Type II restriction endonucleases to hydrolyze RNA-DNA heteroduplex oligonucleotide substrates was assessed. Despite the significant topological and sequence asymmetry introduced when one strand of a DNA duplex is substituted by RNA we find that six restriction enzymes (AvaII, AvrII, BanI, HaeIII, HinfI and TaqI), exclusively of the Type IIP class that recognize palindromic or interrupted-palindromic DNA sequences, catalyze robust and specific cleavage of both RNA and DNA strands of such a substrate. Time-course analyses indicate that some endonucleases hydrolyze phosphodiester bonds in both strands simultaneously whereas others appear to catalyze sequential reactions in which either the DNA or RNA product accumulates more rapidly. Such strand-specific variation in cleavage susceptibility is both significant (up to orders of magnitude difference) and somewhat sequence dependent, notably in relation to the presence or absence of uracil residues in the RNA strand. Hybridization to DNA oligonucleotides that contain endonuclease recognition sites can be used to achieve targeted hydrolysis of extended RNA substrates produced by in vitro transcription. The ability to 'restrict' an RNA-DNA hybrid, albeit with a limited number of restriction endonucleases, provides a method whereby individual RNA molecules can be targeted for site-specific cleavage in vitro.

Figure 1.

Initial screening experiments using REases AvaII, AvrII, BanI, BstNI, HinfI and TaqI in assays using RNA-DNA heteroduplex substrates in which either both DNA and RNA strands or only the RNA strand contain a FAM fluorophore. Sequences of RNA-DNA heteroduplex substrates with boxes defining REase recognition sites and arrows indicating expected sites of phosphodiester bond cleavage. Electrophoretic analysis of cleavage products. Lanes 1, 3, 5 and 7: IM 16B RNA-IM 13B DNA heteroduplex substrate (both with 3′-FAM label). Lanes 2, 4, 6 and 8: IM 16B RNA-IM 13C DNA heteroduplex substrate (RNA only with 3′-FAM label). Lanes 9, 11, 13 and 15: IM 2B RNA-IM 4B DNA heteroduplex substrate (both with 5′-FAM label). Lanes 10, 12, 14 and 16: IM 2B RNA-IM 4C DNA heteroduplex substrate (RNA only with 5′-FAM label). Lanes 1, 2, 9 and 10 show the uncut heteroduplex substrates. Asterisks indicate fluorophore-labelled RNA (*R) and DNA (*D) strands. Expected sizes of RNA and DNA products are: AvaII (30, 14 nucleotides); HinfI (25, 19 nucleotides); TaqI (22, 21 nucleotides); AvrII (18, 20 nucleotides); BanI (22, 16 nucleotides); and BstNI (29, 12 nucleotides), respectively. Arrows indicate RNA product of HinfI cleavage. Note that BstNI only appears to cleave the DNA strand of the RNA-DNA heteroduplex.

Figure 2.

Cleavage of IM399R–IM398D RNA–DNA heteroduplex substrate by AvaII, AvrII, BanI and TaqI restriction enzymes. (A) Sequence of IM399R–IM398D heteroduplex substrate with boxes defining REase recognition sites and arrows indicating expected sites of phosphodiester bond cleavage. (B) Lanes 1 and 10: no enzyme. Lanes 2 and 3: AvaII 100/50 U. Lanes 4 and 5: AvrII 40/20 U. Lanes 6 and 7: BanI 20/10 U. Lanes 8 and 9: TaqI 40/20 U. (C) Additional assays using: different RNA:DNA ratio in substrate annealing reaction. Lanes 1–4: R:D ratio 1:1. Lanes 5–8, R:D ratio 0.5:1. Lanes 1 and 5: no enzyme. Lanes 2 and 6: AvaII 200 U. Lanes 3 and 7: AvrII 125 U. Lanes 4 and 8: BanI 200 U. Green bands, RNA; Red bands, DNA; Yellow bands, merged RNA and DNA. All assays were incubated for 180 min in NEBuffer 4 at 37°C with the exception of TaqI assay (65°C).

Figure 3.

Specificity of RNA–DNA heteroduplex cleavage. (A) Lanes 1, 4, 7 and 10: IM397D–IM398D DNA duplex (1 pmol) incubated with 10 U of AvaII, AvrII, Ban I and TaqI for 60 min. Lanes 2, 5, 8 and 11: IM399R–IM398D RNA–DNA heteroduplex (1 pmol) incubated with AvaII (200 U), AvrII (200 U), BanI (200 U) and TaqI (100 U) for 180 min. Lanes 3, 6, 9 and 12: synthetic RNA markers (Table 1) corresponding to products of cognate RNA cleavage by AvaII (IM403R), AvrII (IM402R), Ban I (IM401R) and TaqI (IM404R). Green bands, RNA; red bands, DNA. REase cleavage sites and products are equivalent to those shown in (A). (B) Capillary electrophoretic analysis of products from Panel A lanes 1–3 (AvaII data). Shaded black peaks: DNA products. Unfilled blue peaks: RNA products. Upper trace: DNA–DNA duplex products. Middle trace: DNA–RNA heteroduplex products. Lower trace: synthetic RNA marker. The different peak heights for RNA and DNA products in the middle trace are a consequence of the higher fluorescent yield of the FAM fluorophore (RNA) compared to TAMRA (DNA). Equivalent data for the other enzymes are presented in Supplementary Figure S1.

Figure 4.

DNA and RNA strands of an RNA–DNA heteroduplex are cleaved at different rates by different restriction enzymes. (A) Sequence of IM399R–IM398D heteroduplex substrate with boxes defining REase recognition sites and arrows indicating expected sites of phosphodiester bond cleavage. (B) Time course analyses of IM399R–IM398D heteroduplex (2 pmol) cleavage using 50 U AvaII (Lanes 1-6) and 10 U BanI (lanes 7–12). Time points are: lanes 1 and 7, 10 min. Lanes 2 and 8, 20 min. Lanes 3 and 9, 30 min. Lanes 4 and 10, 40 min. Lanes 5 and 11, 50 min. Lanes 6 and 12, 60 min. (C) Time course analysis of IM399R–IM398D heteroduplex cleavage (2 pmol) using 20 U HinfI. Time points are: 10 min, 20 min, 30 min, 40 min, 50 min and 60 min for lanes 1–6.

Figure 5.

Replacing rA with rU in the recognition site impairs hydrolysis of an RNA–DNA heteroduplex substrate by AvaII. Time course analysis of IM399R–IM398D heteroduplex (2 pmol, lanes 1–6) and IM432R–IM431D heteroduplex (2 pmol, lanes 7–12) cleavage by 50 U AvaII. Time points are: lanes 1 and 7, 10 min. Lanes 2 and 8, 20 min. Lanes 3 and 9, 30 min. Lanes 4 and 10, 40 min. Lanes 5 and 11, 50 min. Lanes 6 and 12, 60 min. Note that these heteroduplexes are identical except that the IM399R–IM398D substrate contains a central rA:dT base pair in the AvaII site that is replaced by rU:dA in the IM432R–IM431D substrate.

Figure 6.

Oligonucleotide-directed hydrolysis of in vitro transcribed RNA by restriction endonucleases. (A) Predicted sequence of FAM-tagged RNA created by ligation of oligoribonucleotide IM411R (lower case script) to IVT product that initiated at nucleotide G23 of the template DNA (upper case script). Unique HinfI, AvaII, BanI, HinfI and TaqI restriction sites are shown with arrows indicating expected sites of phosphodiester bond cleavage. (B) Specific cleavage of FAM-tagged RNA. Oligonucleotides IM437D, IM438D, IM439D, IM440D and IM442D were hybridized with the FAM-tagged RNA to produce five different heteroduplex substrates that include restriction sites for the HinfI, AvaI, BanI, AvrII and TaqI REases, respectively. All five substrates were incubated in the absence (−) and presence (+) of the cognate REase. Assays using HinfI (50 U), AvaII (500 U), BanI (160 U) and AvrII (10 U) were incubated for 60 min at 37°C. The TaqI assay (50 U) was incubated at 65°C for 60 min. Approximately 250 fmol of RNA were loaded in each gel lane. Size standards (M) were produced by restriction of a FAM-labeled DNA duplex with the same sequence as the RNA substrate and using the equivalent REases.