Chemo-enzymatic synthesis of [2-13C, 7-15 N]-ATP for facile NMR analysis of RNA

Olenginski, Lukasz T.; Dayie, T. Kwaku

doi:10.1007/s00706-020-02667-6

Cited by 6 publications

(24 citation statements)

References 47 publications

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“…Our newly synthesized [2- 13 C, 7- 15 N]-ATP (Olenginski and Dayie 2020 ) removes unwanted 13 C- 13 C and 13 C- 15 N dipolar interactions and was therefore used along with commercially available [U- 13 C/ 15 N]-ATP to empirically test our simulations. To this end, we measured adenosine R 1,C2 rates for [U- 13 C/ 15 N]-ATP or [2- 13 C, 7- 15 N]-ATP labeled HBV ε (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…DNA template and [U- 13 C/ 15 N]-ATP were purchased from Integrated DNA Technologies and Cambridge Isotope Laboratories, respectively. The [2- 13 C, 7- 15 N]-ATP was synthesized as recently described (Olenginski and Dayie 2020 ). After transcription, samples were extracted with acid-phenol:chloroform, ethanol precipitated, purified by preparative denaturing gel electrophoresis, and electroeluted.…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, R 1 measurements at lower temperature (mimicking increased molecular weight) revealed exacerbated R 1,C2 discrepancies, which further corroborates our simulations and argues that selective 13 C2 labeled probes obviates the need to account for the significant contributions to meausured R 1,C2 that arise from neighboring 13 C atom(s) in RNAs with a > 20 ns. Our [2- 13 C, 7- 15 N]-ATP (Olenginski and Dayie 2020 ) also showed better spectroscopic properties than [U- 13 C/ 15 N]-ATP, providing and additional advantage to using selectively labeled samples to measure RNA dynamics.…”

Section: Introductionmentioning

confidence: 98%

“…To empirically test our simulations, we measured adenosine C2 relaxation in the 61 nucleotide (nt) human hepatitis B virus encapsidation signal ε (HBV ε) RNA (Flodell et al 2006 ; Lee 1997 ; Knaus and Nassal 1993 ; Hirsch et al 1990 ) labeled with [U- 13 C/ 15 N]-ATP or [2- 13 C]-ATP. To this end, we used our recently synthesized [2- 13 C, 7- 15 N]-ATP (Olenginski and Dayie 2020 ) as a selective adenosine 13 C2 labeled probe. We demonstrate that the removal of long-range 13 C- 13 C dipolar coupling partners reveals discrepancies in measured adenosine C2 R 1 values between uniformly and selectively labeled samples.…”

Section: Introductionmentioning

confidence: 99%

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Quantifying the effects of long-range 13C-13C dipolar coupling on measured relaxation rates in RNA

Olenginski

Dayie

2021

J Biomol NMR

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Selective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove 13C-13C dipolar couplings that complicate 13C relaxation analyses. While these phenomena are well documented for sites with adjacent 13C nuclei (e.g. ribose C1′), less is known about so-called isolated sites (e.g. adenosine C2). To investigate and quantify the effects of long-range (> 2 Å) 13C-13C dipolar interactions on RNA dynamics, we simulated adenosine C2 relaxation rates in uniformly [U-13C/15N]-ATP or selectively [2-13C]-ATP labeled RNAs. Our simulations predict non-negligible 13C-13C dipolar contributions from adenosine C4, C5, and C6 to C2 longitudinal (R1) relaxation rates in [U-13C/15N]-ATP labeled RNAs. Moreover, these contributions increase at higher magnetic fields and molecular weights to introduce discrepancies that exceed 50%. This will become increasingly important at GHz fields. Experimental R1 measurements in the 61 nucleotide human hepatitis B virus encapsidation signal ε RNA labeled with [U-13C/15N]-ATP or [2-13C]-ATP corroborate these simulations. Thus, in the absence of selectively labeled samples, long-range 13C-13C dipolar contributions must be explicitly taken into account when interpreting adenosine C2 R1 rates in terms of motional models for large RNAs.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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Quantifying the effects of long-range 13C-13C dipolar coupling on measured relaxation rates in RNA

Olenginski

Dayie

2021

J Biomol NMR

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“…This labeling pattern offered substantial sensitivity improvements and was used to develop a novel resonance assignment strategy [ 57 ]. Additional synthetic details, including a list of enzymes used, can be found in the original works [ 30 , 31 , 56 ], and the method has also been used in recent studies by our group [ 58 , 59 ]. This topic will also be the focus of an upcoming review.…”

Section: Stable Isotope Labeling Of Rna Building Blocksmentioning

confidence: 99%

Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies

et al. 2021

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RNA structural research lags behind that of proteins, preventing a robust understanding of RNA functions. NMR spectroscopy is an apt technique for probing the structures and dynamics of RNA molecules in solution at atomic resolution. Still, RNA analysis by NMR suffers from spectral overlap and line broadening, both of which worsen for larger RNAs. Incorporation of stable isotope labels into RNA has provided several solutions to these challenges. In this review, we summarize the benefits and limitations of various methods used to obtain isotope-labeled RNA building blocks and how they are used to prepare isotope-labeled RNA for NMR structure and dynamics studies.

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