An Integrated Spin-Labeling/Computational-Modeling Approach for Mapping Global Structures of Nucleic Acids

Abstract

The technique of site-directed spin labeling (SDSL) provides unique information on biomolecules by monitoring the behavior of a stable radical tag (i.e., spin label) using electron paramagnetic resonance (EPR) spectroscopy. In this chapter, we describe an approach in which SDSL is integrated with computational modeling to map conformations of nucleic acids. This approach builds upon a SDSL tool kit previously developed and validated, which includes three components: (i) a nucleotide-independent nitroxide probe, designated as R5, which can be efficiently attached at defined sites within arbitrary nucleic acid sequences; (ii) inter-R5 distances in the nanometer range, measured via pulsed EPR; and (iii) an efficient program, called NASNOX, that computes inter-R5 distances on given nucleic acid structures. Following a general framework of data mining, our approach uses multiple sets of measured inter-R5 distances to retrieve “correct” all-atom models from a large ensemble of models. The pool of models can be generated independently without relying on the inter-R5 distances, thus allowing a large degree of flexibility in integrating the SDSL-measured distances with a modeling approach best suited for the specific system under investigation. As such, the integrative experimental/computational approach described here represents a hybrid method for determining all-atom models based on experimentally-derived distance measurements.

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

“… 58 Molecular modeling, aiming at identifying all accessible conformations of R5 around its three rotatable bonds by taking into account possible clashes with the RNA backbone, is required for the interpretation of the measured frequencies. 25 A similar issue is encountered with the spin label attached to 4-thiouridine, where the linker consists of five rotatable bonds and Watson–Crick base-pairing is not preserved. 29 , 36 …”

“…Several examples for the application of PELDOR/DEER in studies of RNAs have been reported. 23 – 25 RNA secondary structures have been investigated in a hammerhead ribozyme 26 as well as in aptamers. 27 , 28 More recently the structure of a 70 kDa protein-RNA complex has been elucidated using an EPR-aided approach that combines distance constraints from EPR as well as NMR.…”

Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label

“…As a result, distances between a pair of nitroxides can be measured more precisely and can be explicitly correlated to the native structure. 37,38 …”

Nucleic Acid-Dependent Conformational Changes in CRISPR–Cas9 Revealed by Site-Directed Spin Labeling

“…A typical labeling reaction (~ 1 mL) contained 20 mM Tris-HCl (pH 7.5), 200 mM KCl, 5% (v/v) glycerol, 5 mM MgCl 2 , 0.5 mM TCEP, approximately 10 μM protein, and 10 molar excess of the R5p precursor (3-iodomethyl-1-oxyl-2,2,5,5-tetramethylpyrroline) prepared as previously described (33,34). The reaction mixture was incubated with consistent and smooth rocking at 4°C in dark overnight.…”