Efficient Detection of Structure and Dynamics in Unlabeled RNAs: The SELOPE Approach

Schlagnitweit, Judith; Steiner, Emilie; Karlsson, Hampus; Petzold, Katja

doi:10.1002/chem.201800992

Cited by 28 publications

(55 citation statements)

References 36 publications

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“…In summary,t he measurement of dynamics in RNA has come a long way.M any issues are addressedw ith tweaking pulse sequences from protein dynamics, [12,103,151,152] selectively labeling atoms [41] and carefully analyzing data. However,o ne needs to keep in mind that many more artifacts can occur in RNA than in proteins and much work is still to be done.…”

Section: Discussionmentioning

confidence: 99%

“…Labelings chemes for CPMG:N umerous schemese xist, as this is essential for successful CPMG characterization and many have been developed by the Kreutz group. [17,40,41,75,84,85] Examples are C2'/C4'-labeling of ribose, [88] 2'-O-13 CH 3 -uridine, 6-13 Curidine and 6-13 C-cytidine, [41] [8][9][10][11][12][13] Cp urines, [75,84] 5-13 C-uridine and a2 ,8-13 C 2 -adenine. [17] 3.3.…”

Section: Cpmgmentioning

confidence: 99%

“…bly used, therefore exchange parameters as well as chemical shifts and populations are obtained from fits by solving the Bloch-McConnell equations for at wo-state exchange process (e.g.,r ef. [12]). Ad etailed description of how to set up and analyze experiments can be found in ref.…”

Section: Cpmgmentioning

confidence: 99%

“…Isotopically labeled RNAs are produced either by T7 in vitro transcription or solid-phase synthesis with subsequent purification. [11] Some exceptionse xist to the typical 13 C, 15 Nl abeling,s uch as:1 )nol abel is required for 1 H R 11 , [12] off-resonance ROESY [2,13] or 1 H, 1 HE XSY,a nd 2) limitations of methods can requirea tom-specific labeling (e.g., 13 CCPMG) or deuteration (solid-state NMR spectroscopy). Methodological breakthroughs in the last 10 years have led to the discoveryo fm any RNA excited states [6,8,14] and attempts have been made to investigate correlation to their function.…”

Section: Introductionmentioning

confidence: 99%

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RNA Dynamics by NMR Spectroscopy

2019

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An ever‐increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so‐called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined—Lego like—than in proteins, hence the numerous revelations of RNA excited states.

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Section: Discussionmentioning

confidence: 99%

Section: Cpmgmentioning

confidence: 99%

Section: Cpmgmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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RNA Dynamics by NMR Spectroscopy

2019

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“…Regarding 3-D methods in structure determination, highresolution structure determination methods have several challenges with long RNA molecules and complexes with their interacting partners 14 . For example, while many excellent NMR studies of biomolecules have been performed [18][19][20][21][22] , this method typically has been limited to proteins smaller than 50 kDa 23 and RNAs smaller than 100-300 nucleotides 24 . In addition, crystallographic studies of RNA molecules are typically more challenging than DNA and proteins.…”

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confidence: 99%

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

et al. 2020

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Long non-coding RNAs (lncRNAs) constitute a significant fraction of the transcriptome, playing important roles in development and disease. However, our understanding of structure-function relationships for this emerging class of RNAs has been limited to secondary structures. Here, we report the 3-D atomistic structural study of epigenetic lncRNA, Braveheart (Bvht), and its complex with CNBP (Cellular Nucleic acid Binding Protein). Using small angle X-ray scattering (SAXS), we elucidate the ensemble of Bvht RNA conformations in solution, revealing that Bvht lncRNA has a well-defined, albeit flexible 3-D structure that is remodeled upon CNBP binding. Our study suggests that CNBP binding requires multiple domains of Bvht and the RHT/AGIL RNA motif. We show that RHT/AGIL, previously shown to interact with CNBP, contains a highly flexible loop surrounded by more ordered helices. As one of the largest RNA-only 3-D studies, the work lays the foundation for future structural studies of lncRNA-protein complexes.

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Production of Structured RNA Fragments by In Vitro Transcription and HPLC Purification

et al. 2021

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The understanding of the functional importance of RNA has increased enormously in the last decades. This has required research on the RNA molecules themselves, with the concomitant need for obtaining purified RNA samples, such as for structural studies by NMR or other methods. The main method to create labeled and unlabeled RNA, T7 in vitro transcription, suffers from sequence‐dependent yield and often low homogeneity for short constructs (<100 nt) and requires laborious purification. Additionally, the design of structured RNA fragments mimicking the structure of a larger biological RNA is often not straightforward. Secondary structure simulations can be used to make reliable predictions about the folding of a particular RNA fragment. In this article, we describe how to design an RNA construct of interest from a larger sequence, and we combine several previously published improvements of the in vitro transcription method, such as the use of 2′‐methoxy modifications and dimethyl sulfoxide or the use of tandem repeats, to increase yield and purity of in vitro–transcribed RNA. Together with a high‐performance liquid chromatography (HPLC) purification procedure using both reversed‐phase ion‐pairing and ion‐exchange HPLC, we provide a robust protocol to obtain highly pure RNA of short to intermediate length in large quantities. The protocol optimizes yield, especially for RNA starting with nucleotides other than G. At the same time, it is simplified, and the required time is reduced. The protocols described here constitute a versatile pipeline for the production of purified RNA samples and are suitable for users with little experience in liquid chromatography. © 2021 The Authors. Basic Protocol 1: RNA construct design Basic Protocol 2: DNA template production and in vitro transcription Alternate Protocol: Tandem transcription and RNase H cleavage Basic Protocol 3: Reversed‐phase ion‐pairing HPLC purification Basic Protocol 4: Ion‐exchange HPLC purification

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Efficient Detection of Structure and Dynamics in Unlabeled RNAs: The SELOPE Approach

Cited by 28 publications

References 36 publications

RNA Dynamics by NMR Spectroscopy

RNA Dynamics by NMR Spectroscopy

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

Production of Structured RNA Fragments by In Vitro Transcription and HPLC Purification

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