High-resolution measurement of long-range distances in RNA: pulse EPR spectroscopy with TEMPO-labeled nucleotides

Abstract

Distance measurements in RNAs by pulse EPR with TEMPO-labeled nucleotides allow for model free conversion of distances into base-pair separation.

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

“…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 .…”

“…Addition of thiols normally is required to keep ligase proteins active. Although spin labels 4 and 5 are less rigid than 2 or 3 and also cause some duplex destabilization, successful PELDOR studies have been published . Furthermore, if some decomposition is accepted, deoxynucleoside analogues of nitroxides 4 and 5 can be incorporated into DNA strands by specifically adapted phosphoramidite chemistry …”

“…Although spin labels 4 and 5 are less rigid than 2 or 3 and also cause some duplex destabilization,s uccessful PELDOR studies have been published. [5,26] Furthermore, if some decompositioni sa ccepted, deoxynucleoside analogues of nitroxides 4 and 5 can be incorporated into DNA strands by specifically adaptedp hosphoramidite chemistry. [40,43,46] To provide am ore general solution to the problemo fn itroxide instability,w eh ave developed ap rotection strategy that is based on the alkylation of hydroxylamines with light-sensitive groups ( Figure 2).…”

Measurement of Angstrom to Nanometer Molecular Distances with 19F Nuclear Spins by EPR/ENDOR Spectroscopy

“…[32] Subsequently, the C T label was introduced in one of the most energetically stable conformations compatible with hydrogen bonding using PyMOL, as predicted by previous DFT calculations of the C T energy surface. [31] The C T conformations were defined by two dihedral angles f 1 and f 2 ( Figure 4) and values of f 1 % 120 AE 75 and f 2 % 0 AE 60 were found within the energy minimum ( Figure S18), with a second, less broad minimum at f 1 % À60 AE 20 and f 2 % 0 AE 40. [31] These models allow for estimating NO- 19 F distances of approximately 12 and 14 in RNA1 and RNA2, respectively.…”

Advances in the synthesis of nitroxide radicals for use in biomolecule spin labelling

“…cytidine 113 can be prepared by reaction of electrophilic O 4chlorophenyl uridine 114 with amino-TEMPO 58 (Scheme 16). 52 This method affords exceptionally narrow distance distributions in DEER experiments on RNA duplexes containing a double spin labelled strand, by virtue of the rigidifying effect of base pairing coupled with the enforced positioning of the label in the major groove of the duplex. Nucleobase surrogates have been extensively developed and applied, showing this to be a versatile strategy that affords high quality EPR data.…”