ArticleRNA (New York, N.Y.)2013
Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags.
Article in RNA (New York, N.Y.), 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 31 citations in OpenAlex.
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- An integrative NMR-SAXS approach for structural determination of large RNAs defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme.Nucleic acids research · 2021Article
- Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies.Molecules (Basel, Switzerland) · 2021Review
- Solid-Phase Chemical Synthesis of Stable Isotope-Labeled RNA to Aid Structure and Dynamics Studies by NMR Spectroscopy.Molecules (Basel, Switzerland) · 2019Review
- High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension.BMC biotechnology · 2018Article
- Article
- Harnessing "A Billion Years of Experimentation": The Ongoing Exploration and Exploitation of CRISPR-Cas Immune Systems.The CRISPR journal · 2018Review
- AnIranian journal of microbiology · 2017Article
- ARiBo pull-down for riboproteomic studies based on label-free quantitative mass spectrometry.RNA (New York, N.Y.) · 2016Article
- Rapid NMR screening of RNA secondary structure and binding.Journal of biomolecular NMR · 2015Article
- Progress and outlook in structural biology of large viral RNAs.Virus research · 2014Review
- Advances in methods for native expression and purification of RNA for structural studies.Current opinion in structural biology · 2014Review
- Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors.Nucleic acids research · 2014Article
- CRISPR-based technologies: prokaryotic defense weapons repurposed.Trends in genetics : TIG · 2014Review
- Syn5 RNA polymerase synthesizes precise run-off RNA products.Nucleic acids research · 2014Article
- A CRISPR method for genome engineering.F1000prime reports · 2014Review
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.