RESUMO
Affinity purification of RNA using the ARiBo tag technology currently provides an ideal approach to quickly prepare RNA with 3' homogeneity. Here, we explored strategies to also ensure 5' homogeneity of affinity-purified RNAs. First, we systematically investigated the effect of starting nucleotides on the 5' heterogeneity of a small SLI RNA substrate from the Neurospora VS ribozyme purified from an SLI-ARiBo precursor. A series of 32 SLI RNA sequences with variations in the +1 to +3 region was produced from two T7 promoters (class III consensus and class II 2.5) using either the wild-type T7 RNA polymerase or the P266L mutant. Although the P266L mutant helps decrease the levels of 5'-sequence heterogeneity in several cases, significant levels of 5' heterogeneity (≥1.5%) remain for transcripts starting with GGG, GAG, GCG, GGC, AGG, AGA, AAA, ACA, AUA, AAC, ACC, AUC, and AAU. To provide a more general approach to purifying RNA with 5' homogeneity, we tested the suitability of using a small CRISPR RNA stem-loop at the 5' end of the SLI-ARiBo RNA. Interestingly, we found that complete cleavage of the 5'-CRISPR tag with the Cse3 endoribonuclease can be achieved quickly from CRISPR-SLI-ARiBo transcripts. With this procedure, it is possible to generate SLI-ARiBo RNAs starting with any of the four standard nucleotides (G, C, A, or U) involved in either a single- or a double-stranded structure. Moreover, the 5'-CRISPR-based strategy can be combined with affinity purification using the 3'-ARiBo tag for quick purification of RNA with both 5' and 3' homogeneity.
Assuntos
Bacteriófago T7/genética , Cromatografia de Afinidade/métodos , RNA Polimerases Dirigidas por DNA/química , Neurospora/genética , RNA Líder para Processamento/isolamento & purificação , RNA Viral/isolamento & purificação , Proteínas Virais/química , Marcadores de Afinidade/química , Bacteriófago T7/química , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/genética , Heterogeneidade Genética , Sequências Repetidas Invertidas , Neurospora/química , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/genética , Regiões Promotoras Genéticas , Clivagem do RNA , Estabilidade de RNA , RNA Catalítico/química , RNA Catalítico/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Thermus thermophilus/química , Thermus thermophilus/genética , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
Common approaches for purification of RNAs synthesized in vitro by the T7 RNA polymerase often denature the RNA and produce RNAs with chemically heterogeneous 5'- and 3'-ends. Thus, native affinity purification strategies that incorporate 5' and 3' trimming technologies provide a solution to two main disadvantages that arise from standard approaches for RNA purification. This chapter describes procedures for nondenaturing affinity purification of in vitro transcribed RNA using a 3'-ARiBo tag, which yield RNAs with a homogeneous 3'-end. The applicability of the method to RNAs of different sequences, secondary structures, and sizes (29-614 nucleotides) is described, including suggestions for troubleshooting common problems. In addition, this chapter presents three complementary approaches to producing 5'-homogeneity of the affinity-purified RNA: (1) selection of the starting sequence; (2) Cse3 endoribonuclease cleavage of a 5'-CRISPR tag; or (3) self-cleavage of a 5'-hammerhead ribozyme tag. The additional steps to express and purify the Cse3 endonuclease are detailed. In light of recent results, the advantages and limitations of current approaches to achieve 5'-homogeneity of affinity-purified RNA are discussed, such that one can select a suitable strategy to purify the RNA of interest.