Use of cis- and trans-ribozymes to remove 5' and 3' heterogeneities from milligrams of in vitro transcribed RNA.

Abstract

In vitro transcription with phage T7 RNA polymerase is the method of choice for obtaining multi-milligram quantities of RNA for structural studies. However, run-off transcription with this enzyme results in molecules that are heterogeneous at their 3′-, and depending on template sequence, 5′-termini. For transcripts longer than ∼50 nucleotides (nt), these impurities cannot be removed by preparative purification techniques. Use of cis-delta, or trans-VS ribozymes allows preparation of homogeneous RNA with any 3′-terminal sequence. If present, 5′ heterogeneity can be overcome with a cis-hammerhead ribozyme. During the course of a structural investigation of Group II self-splicing introns, we sought to prepare a 70 nt RNA molecule comprising domains V and VI (d56) of the ai5γ intron (Fig. 1). Run-off transcription from a plasmid linearized with the restriction enzyme BsaI to generate a DNA terminus ...TAGCC-3′ on the template strand resulted in six to eight different molecules, the shortest of which (∼30% of the full-length transcript) is the desired product (Fig. 2, lane A). Although resolved on an analytical gel, these different molecules could not be separated on a preparative scale either by gel electrophoresis or chromatography, making the resulting RNA inadequate for biophysical studies. Addition of random nucleotides to the 3′-terminus of run-off transcripts by T7 RNA polymerase is well documented (1); we chose to use a ribozyme to cleave the 3′-end of our transcript to make it homogeneous. The hammerhead (HH) and hairpin ribozymes have been employed previously to cleave transcripts (2, and references therein). However, the well-characterized hammerhead has sequence requirements 5′ to the cleavage site that are incompatible with our desired product. This catalytic RNA needs the sequence UX (X G) to precede the cleavage site. The hairpin ribozyme, which needs (G/C/U)N instead, is prone to aberrant cleavage (2). Furthermore, when used to remove 3′-termini, both require sequence complementarity with nucleotides internal to the product, necessitating ribozymes of different sequence for cleaving different constructs. In contrast, the hepatitis delta virus ribozyme (δ) and the Neurospora Varkud satellite RNA ribozyme (VS) have minimal sequence requirements: δ will cut after any base other than G (3, and references therein), while VS will cleave efficiently after any base other than C (4). Thus, these two ribozymes used in concert should allow cleavage after any desired sequence. Figure 1. Schematic representation of the hammerhead-d56-VSsl construct. The sequences of domains V and VI are shown in upper case letters, in their conventional base-paired representation. The hammerhead ribozyme (HH) and VS ribozyme substrate stem–loop (VSsl) are in lower case. Bonds that are cleaved by the ribozymes are shown in broken lines. Note that the first 11 nt of domain V are complementary to a portion of HH. VSsl is longer than that of (4) because the template plasmid was linearized with BamHI rather than AvaI.