Noncovalent and site-directed spin labeling of duplex RNA

Kamble, Nilesh R.; Gränz, Markus; Prisner, Thomas F.; Sigurdsson, Snorri Th.

doi:10.1039/c6cc08387k

Cited by 19 publications

(17 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The analysis of RNA structure and dynamics by EPR spectroscopy requires labeling of the samples with paramagnetic species. This is normally achieved by covalent attachment of persistent nitroxide radicals, although noncovalent labeling has become an interesting alternative . Due to the limited stability of nitroxides, postsynthetic attachment has some advantages.…”

Section: Introductionmentioning

confidence: 99%

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full‐Length TAR RNA and Investigation by In‐Line Probing and EPR Spectroscopy

Weinrich

Jaumann

Scheffer

et al. 2018

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2-nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full-length HIV-1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin-labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in-line probing.

show abstract

Section: Introductionmentioning

confidence: 99%

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full‐Length TAR RNA and Investigation by In‐Line Probing and EPR Spectroscopy

Weinrich

Jaumann

Scheffer

et al. 2018

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

show abstract

“…Ethylene glycol, phosphoric acid, LiOH and distilled water were purchased from Sigma‐Aldrich (Munich, Germany). Spin labels were prepared as previously described . Amicon Ultra centrifugal filters with a molecular weight cut‐off (MWCO) of 3 kDa were from Merck/Millipore (Darmstadt, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…[19 -23] There are examples of noncovalent spin-labeling of nucleic acids, such as the malachite green aptamer [24] and designed abasic sites in nucleic acid duplexes. [25][26][27][28][29] Of several purine-nitroxide derivatives that were recently prepared for noncovalent labeling (Figure 1, 1-5 and Ǵ), Ǵ (G-spin) was found to have high affinity to abasic sites in RNA opposite C, with an equilibrium dissociation constant (K d ) of 1.46 · 10 À 7 M at 20°C. [26] Here, we report the evaluation of purinenitroxides as spin labels for rationally designed purinebinding triple helices containing nucleotide gaps (I -III, Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Noncovalent Spin‐Labeling of DNA and RNA Triplexes

Kamble

Wolfrum

Halbritter

et al. 2020

Chemistry & Biodiversity

Self Cite

View full text Add to dashboard Cite

Studying nucleic acids often requires labeling. Many labeling approaches require covalent bonds between the nucleic acid and the label, which complicates experimental procedures. Noncovalent labeling avoids the need for highly specific reagents and reaction conditions, and the effort of purifying bioconjugates. Among the least invasive techniques for studying biomacromolecules are NMR and EPR. Here, we report noncovalent labeling of DNA and RNA triplexes with spin labels that are nucleobase derivatives. Spectroscopic signals indicating strong binding were detected in EPR experiments in the cold, and filtration assays showed micromolar dissociation constants for complexes between a guanine‐derived label and triplex motifs containing a single‐nucleotide gap in the oligopurine strand. The advantages and challenges of noncovalent labeling via this approach that complements techniques relying on covalent links are discussed.

show abstract

“…[17] However,t he binding of Å was highly flanking-sequenced ependent, showing full binding to only af ew sequences. Recently,w er eported the semi-flexible Ǵ (G-spin, Figure 1), [18] whichi saconjugate of guaninea nd an isoindoline-derived nitroxide radical. [16c] As eries of pyrimidinederived nitroxides was subsequently prepared and screened for binding to both DNA and RNA duplexes.…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, Å showed only partial binding to an abasic site of an RNA duplex. [18] To further explore the use of purine-derived nitroxides for noncovalent spin-labeling of abasic sites in duplex DNA and RNA, we have prepared five new spin labels (2-6,F igure 2). [16c] Only triazolelinked nitroxide 1 (Figure 1) showed complete bindingt o abasic sites in RNA, but it was not au seful spin label due to extensive nonspecific binding.…”

Section: Introductionmentioning

confidence: 99%

Purine‐Derived Nitroxides for Noncovalent Spin‐Labeling of Abasic Sites in Duplex Nucleic Acids

Kamble

Sigurdsson

2018

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

A series of purine-based spin labels was prepared for noncovalent spin-labeling of abasic sites of duplex nucleic acids through hydrogen bonding to an orphan base on the opposing strand and π-stacking interactions with the flanking bases. Both 1,1,3,3-tetramethylisoindolin-2-yloxyl and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were conjugated to either the C2- or C6-position of the purines, yielding nitroxide derivatives of guanine, adenine, or 2,6-diaminopurine. The isoindoline-derived spin labels showed extensive or full binding to abasic sites in RNA duplexes, whereas the TEMPO-derived spin labels showed limited binding. An adenine-derived spin label (5) bound fully at low temperature to abasic sites in both DNA and RNA duplexes when paired with thymine and uracil, respectively, complementing the previously described guanine-derived spin label Ǵ, which binds efficiently opposite cytosine. Compound Ǵ was also shown to bind to abasic sites in DNA-RNA hybrids, either in the DNA- or the RNA-strand. Ǵ showed only a minor flanking-sequence effect upon binding to abasic sites in RNA. When the abasic site was placed close to the end of the RNA duplex, the affinity of the spin label Ǵ was reduced; full binding was observed at the fourth position from the duplex end. In summary, spin labels 5 and Ǵ showed full binding to abasic sites in both DNA and RNA duplexes and are promising spin labels for structural studies of nucleic acids by pulsed EPR methods.

show abstract

Noncovalent and site-directed spin labeling of duplex RNA

Cited by 19 publications

References 49 publications

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full‐Length TAR RNA and Investigation by In‐Line Probing and EPR Spectroscopy

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full‐Length TAR RNA and Investigation by In‐Line Probing and EPR Spectroscopy

Noncovalent Spin‐Labeling of DNA and RNA Triplexes

Purine‐Derived Nitroxides for Noncovalent Spin‐Labeling of Abasic Sites in Duplex Nucleic Acids

Contact Info

Product

Resources

About