Probing Secondary Structures of Spin‐Labeled RNA by Pulsed EPR Spectroscopy

Abstract

The ability of RNA to interconvert between multiple conformational states is essential for the diversity of biological functions that have been discovered in the recent past. [1] For example, the correct operation of regulatory RNA elements, such as riboswitches, is based on the precise interplay of alternative RNA conformations.[2] Studying the molecular mechanisms of RNA function entails probing RNA-folding intermediates on the energy landscape. EPR spectroscopy, in particular, has been increasingly applied to obtain structural information on nucleic acids, including local conformational changes in RNA [3] and the identification of metal-ion binding sites.[4] Pulsed EPR techniques (PELDOR/ DEER) have been used to determine distances between paramagnetic centers in specifically modified RNA. [5] PELDOR should therefore be suitable for the detection of alternative RNA conformations that involve distinct changes in base-pairing patterns.The accessibility of spin-labeled RNA still poses the major challenge for the widespread applicability of powerful EPR techniques. Nitroxide radicals are the most commonly used type of paramagnetic labels for nucleic acids. Several methods have been reported for attaching nitroxide groups at internal positions at the ribose, the phosphate backbone, or at nucleobases, often by means of multiatom linkers that provide several unwanted degrees of rotational freedom.[6] Rigid nitroxide spin labels conjugated to the nucleobase or to nucleobase analogues have been reported for DNA.[7] Our RNA spin-labeling approach addresses the direct attachment of nitroxide labels onto RNA nucleobases, such that conformational changes can be directly detected by PELDOR (i.e., by the change in distance between two labeled nucleotides). The nucleobase spin labels used in this study are also designed to preserve the Watson-Crick base-pairing capability of labeled nucleotides and not to interfere with alternative base-pairing patterns in different RNA conformations.Here, we describe the installation of nitroxide spin labels on exocyclic amino groups of the RNA nucleobases guanine, adenine, and cytosine (Figure 1) with unprecedented efficiency, and we report on the evaluation of RNA secondary structures by pulsed double electron resonance spectroscopy.The convertible nucleosides [8] O 4 -(4-chlorophenyl)uridine, O 6 -(4-chlorophenyl)inosine, and 2-fluoroinosine were incorporated into RNA by solid-phase synthesis using commercially available, modified 2'-O-tert-butyldimethylsilylprotected building blocks 1-3 in combination with standard RNA 2'-O-triisopropylsilyloxymethyl-protected nucleoside phosphoramidites. In a postsynthetic modification step, the 4-chlorophenyl or fluoride leaving groups were displaced by

A dual-mode microwave resonator for double electron–electron spin resonance spectroscopy at W-band microwave frequencies

“…The W-band PELDOR data is consistent with previous experiments performed at X-band, which indicated a perpendicular component of the dipolar tensor of 1.6 MHz, i.e. a distance of 3.2 nm [24]. The Pake pattern, however, (Fig.…”

RNA dynamics: perspectives from spin labels

“…In another approach, nitroxide is attached to the target strand during solid-phase chemical synthesis, either via direct incorporation of a nitroxide containing phosphoroamidite 29 or via on-column derivatization. 30-32 Using this approach, one may incorporate “designer” nitroxides with sophisticated chemical structures (e.g., “C-spin” (Ç), 29 Figure 2, R3(ii)) to tune behaviors of spin labels with respect to the parent macromolecule, which is advantageous for extracting information on the macromolecule. On the other hand, this approach generally requires complex synthetic procedures, and certain analogues (e.g., Ç) have until now been incorporated into DNA strands only.…”

“…A number of RNA studies, primarily using model duplexes, 25, 31, 32, 36-38 have established methods for correlating measured inter-nitroxide distances to structures of the parent RNAs, thus establishing a basis for using SDSL distance measurements to map RNA global structures and to monitor conformational dynamics. With pulsed EPR, the upper limit of detectable distance is comparable to that measured using FRET, although most EPR measurements are carried out in frozen solution states as constrained by the short electron spin relaxation time.…”

“…With pulsed EPR, the upper limit of detectable distance is comparable to that measured using FRET, although most EPR measurements are carried out in frozen solution states as constrained by the short electron spin relaxation time. 12, 14 It is also notable that instead of a single distance value, EPR measurements yield the entire profile of distance distribution, which reveals the degree of structural ordering (or disordering) at given states of RNAs 39 or the presence of multiple RNA conformations 32, 40 (see later sections on the hammerhead ribozyme and riboswitches). This provides information on RNA conformational dynamics.…”

Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

“…Over the last decade PDS has been widely used to determine distances within doubly labeled oligonucleotide molecules in the range of 1.5-8 nm (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Further developments in PDS methods (3) enable even greater distances to be measured.…”