Site-Directed Spin Labeling of RNA with a Gem-Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability.

Ribonucleic acid function is governed by its structure, dynamics, and interaction with other biomolecules and influenced by the local environment. Thus, methods are needed that enable one to study RNA under conditions as natural as possible, possibly within cells. Site-directed spin-labeling of RNA with nitroxides in combination with, for example, pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy has been shown to provide such information. However, for in-cell measurements, the usually used gem-dimethyl nitroxides are less suited, because they are quickly reduced under in-cell conditions. In contrast, gem-diethyl nitroxides turned out to be more stable, but labeling protocols for binding these to RNA have been sparsely reported. Therefore, we describe here the bioconjugation of an azide functionalized gem-diethyl isoindoline nitroxide to RNA using a copper (I)-catalyzed azide–alkyne cycloaddition (“click”-chemistry). The labeling protocol provides high yields and site selectivity. The analysis of the orientation selective PELDOR data show that the gem-diethyl and gem-dimethyl labels adopt similar conformations. Interestingly, in deuterated buffer, both labels attached to RNA yield TM relaxation times that are considerably longer than observed for the same type of label attached to proteins, enabling PELDOR time windows of up to 20 microseconds. Together with the increased stability in reducing environments, this label is very promising for in-cell Electron Paramagnetic Resonance (EPR) studies.


Supporting Information
The azide functionalized gem-diethylisoindoline spin label 2 • (Figure 1b) was synthesized in six steps starting from N-benzylphtalimid with slight modification according to the synthesis reported by Haugland et al. [47]. In order to avoid handling the explosive diazotransfer reagent trifluoromethanesulfonyl azide, imidazole-1-sulfonyl azide hydrochloride was used following the protocol of . This led to a yield of 70% for the diazotransfer reaction as compared to the reported 87% for the trifluoromethanesulfonyl azide reaction [47]. Spin label 2 • was obtained as a yellow powder in an overall yield of 7%. Its identity and purity were confirmed by highperformance liquid chromatography, IR and EPR spectroscopy, as well as mass spectrometry ( Figure  S1). In the experimental high-resolution ESI (+) mass spectrum, a negligible amount of a species at 289.2023 m/z was detectable ( Figure S1a), which was assigned to the corresponding hydroxylamine ( Figure S1c) formed during the ESI measurement, because there was no additional peak in the HPLC analysis.  Figure S2. Experimental cw EPR spectrum of (a) A2 and (b) B2 overlaid with their double integral.  Figure S3. Experimental two-pulse ESEEM spectra of B2 at 50 K in dependence of the magnetic field.

PELDOR Data Analysis
The PeldorFit program [58], which is based on was used to analyze the orientation selectivity. The configuration file contains three main blocks of information filled in by the user: 1. Instrumental parameters of the PELDOR experiment; 2. Spectroscopic parameters of the involved spins which were obtained by simulating the experimental Q-band spectrum with easySpin [60] ( Figure S5). The parameter from the fit are collected in Table S4; 3. The parameters of Table S5 were used as fitting parameters assuming rhombic magnetic tensors for the spins.
The program fits the orientation selective time traces by means of a genetic algorithm and yields the geometric parameters r, ξ, φ, α, β, and γ of a simplified geometric model ( Figure  S6) and their distributions. The genetic algorithm was set to a maximal number of generations of 200 and a generation size of 192. The geometric parameters are optimized within the ranges given in Table S5 until the corresponding RSMD reached a minimum. The results are given in Table S6 and S7 including the 16 symmetry-related sets of parameters due to the invariance of the gand A-tensor towards inversion of their axes.

DNA Sequence
The DNA strands 5′ GGG TGX CTG GTA CCC 3′ and 5′ A GGG TAC CAG ACA CCC A 3′ were purchased from metabion.

Spin Labeling Reaction
The spin labeling was conducted in the same way as the labeling of the RNA (see Section 3.1.2.). Afterwards, the DNA was purified through reverse-phase high-performance liquid chromatography with an Agilent 1200 Series HPLC System (Agilent Technology, Santa Clara, CA, USA ) in combination with a Zorbax 300SB-C18 (4.6 mm × 150 mm) column (Agilent Technologies, Santa Clara, CA, USA). As the eluent was a 0.1 M aqueous solution of triethylammonium acetate (VWR Applichem), an increasing percentage of acetonitrile (VWR Chemicals) (8% to 20% over 14 min, then up to 80% acetonitrile for 15 min) was used.

DNA Sample Preparation
The labeled single DNA strand was mixed 1:1 with the unmodified DNA strand in phosphatebuffered saline solution (PBS; 137 mM sodium chloride (Carl Roth), 10 mM sodium hydrogen phosphate (Carl Roth), 2.7 mM potassium chloride (Carl Roth), 1.8 mM potassium dihydrogen phosphate (Carl Roth) pH 7.4). The mixture was denaturated for 5 min at 70 °C and incubated for at least 15 min at room temperature.