Short Oligonucleotides Facilitate Co-transcriptional Labeling of RNA at Specific Positions

Wang, Siyu; Chen, Dian; Gao, Lingzhi; Liu, Yu

doi:10.1021/jacs.2c00020

Cited by 5 publications

(3 citation statements)

References 42 publications

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“…Furthermore, under conditions of 0.5 mM Mg 2+ and 10.0 mM Gua + , the proportions of folded structures (E FRET ∼ 0.8) in EC-88 and EC-105 were approximately 36% and 14% lower, respectively, compared to those observed in the isolated post-transcriptional riboG-apt and riboG-term (Figures 6D, 6I, Figure 2–figure supplement 6G and Figure 3–figure supplement 4F). The T7 RNA polymerase (RNAP) sequestered about 8 nt of the nascent RNA, preventing the EC-88 construct from forming the P2 stem (Durniak et al, 2008; Huang & Sousa, 2000; Lubkowska et al, 2011; Tahirov et al, 2002; Wang et al, 2022; Yin & Steitz, 2002). Consequently, a pseudoknot structure potentially formed instead of the expected KL.…”

Section: Resultsmentioning

confidence: 99%

Ligand Response of Guanidine-IV riboswitch at Single-molecule Level

Gao,

Chen,

Liu

2023

Preprint

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Riboswitches are non-coding RNA elements that can specifically sense ligands and regulate gene expression. A recent report introduced a type of riboswitch known as the guanidine-IV riboswitch, which responds to guanidine levels and controls the transcription of downstream genes. However, there is currently a lack of clarity regarding the mechanism by which the riboswitch senses guanidine and undergoes conformational changes. This lack of understanding has impeded the application of this riboswitch. Therefore, our study aimed to address this gap by investigating the conformational changes that occur in a guanidine-IV riboswitch RNA. We examined the aptamer, terminator, and full-length riboswitch using single-molecule fluorescence resonance energy transfer (smFRET). Our findings indicated that the aptamer portion was more sensitive to guanidine compared to the terminator and full-length riboswitch. Additionally, we mimicked the structural changes in the guanidine-IV riboswitch that occur during transcription at a single-nucleotide and single-molecule level using Position-specific Labelling of RNA (PLOR) and smFRET. The results showed that guanidine caused a less pronounced change in the riboswitch RNA after transcribing 88 nucleotides. Based on our findings, we have proposed a folding model for the guanidine-IV riboswitch in the absence and presence of guanidine.Impact statementThe guanidine-IV riboswitch’s aptamer domain exhibits a greater sensitivity towards guanidine in comparison to both the terminator and full-length riboswitch. This results in the riboswitch showcasing a narrow transcriptional window that is responsive to ligand binding. And a comprehensive structure-function model for the guanidine-IV riboswitch under both guanidine-free and guanidine-present conditions.

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Section: Resultsmentioning

confidence: 99%

Ligand Response of Guanidine-IV riboswitch at Single-molecule Level

Gao,

Chen,

Liu

2023

Preprint

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“…Next to isotope-labeled nucleotides, certain modifications or even oligonucleotides could be incorporated by the same protocol, as their 5'-incorporation has been shown before [18,38,39]. Such modifications, next to isotopically nucleotides for NMR, open the possibility of sitespecific analysis of RNA with methods like fluorescence spectroscopy [40] or EPR spectroscopy [41]. The recently published SMRITI-method for introduction of several modifications during IVT [40] could also be modified to introduce a labeled segment via an engineered elongation complex [42].…”

Section: Discussionmentioning

confidence: 99%

“…Such modifications, next to isotopically nucleotides for NMR, open the possibility of sitespecific analysis of RNA with methods like fluorescence spectroscopy [40] or EPR spectroscopy [41]. The recently published SMRITI-method for introduction of several modifications during IVT [40] could also be modified to introduce a labeled segment via an engineered elongation complex [42].…”

Section: Discussionmentioning

confidence: 99%

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

et al. 2022

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Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5’ position, which allows 5’-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

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Observation of structural switch in nascent SAM-VI riboswitch during transcription at single-nucleotide and single-molecule resolution

Xue

Dian

et al. 2023

Nat Commun

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Growing RNAs fold differently as they are transcribed, which modulates their finally adopted structures. Riboswitches regulate gene expression by structural change, which are sensitive to co-transcriptionally structural biology. Here we develop a strategy to track the structural change of RNAs during transcription at single-nucleotide and single-molecule resolution and use it to monitor individual transcripts of the SAM-VI riboswitch (riboSAM) as transcription proceeds, observing co-existence of five states in riboSAM. We report a bifurcated helix in one newly identified state from NMR and single-molecule FRET (smFRET) results, and its presence directs the translation inhibition in our cellular translation experiments. A model is proposed to illustrate the distinct switch patterns and gene-regulatory outcome of riboSAM when SAM is present or absent. Our strategy enables the precise mapping of RNAs’ conformational landscape during transcription, and may combine with detection methods other than smFRET for structural studies of RNAs in general.

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Short Oligonucleotides Facilitate Co-transcriptional Labeling of RNA at Specific Positions

Cited by 5 publications

References 42 publications

Ligand Response of Guanidine-IV riboswitch at Single-molecule Level

Ligand Response of Guanidine-IV riboswitch at Single-molecule Level

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Observation of structural switch in nascent SAM-VI riboswitch during transcription at single-nucleotide and single-molecule resolution

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