Nitroxide Sensing of a DNA Microenvironment: Mechanistic Insights from EPR Spectroscopy and Molecular Dynamics Simulations

The behavior of the nitroxide spin labels 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a) and 1-oxyl-2,2,5,5-tetramethylpyrroline (R5) attached at a phosphorothioate-substituted site in a DNA duplex is modulated by the DNA in a site- and stereospecific manner. A better understanding of the mechanisms of R5a/R5 sensing of the DNA microenvironment will enhance our capability to relate information from nitroxide spectra to sequence-dependent properties of DNA. Toward this goal, electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations were used to investigate R5 and R5a attached as R_<i>p</i> and S_<i>p</i> diastereomers at phosphorothioate _pSC₇ of d­(CTACTG_pSC₇Y₈TTAG). d­(CTAAAGCAGTAG) (Y = T or U). X-band continuous-wave EPR spectra revealed that the dT₈ to dU₈ change alters nanosecond rotational motions of R_<i>p</i>-R5a but produces no detectable differences for S_<i>p</i>-R5a, R_<i>p</i>-R5, and S_<i>p</i>-R5. MD simulations were able to qualitatively account for these spectral variations and provide a plausible physical basis for the R5/R5a behavior. The simulations also revealed a correlation between DNA backbone B_I/B_II conformations and R5/R5a rotational diffusion, thus suggesting a direct connection between DNA local backbone dynamics and EPR-detectable R5/R5a motion. These results advance our understanding of how a DNA microenvironment influences nitroxide motion and the observed EPR spectra. This may enable use of R5/R5a for a quantitative description of the sequence-dependent properties of large biologically relevant DNA molecules

Extending the spectrum of CLRN1‐ and ABCA4‐associated inherited retinal dystrophies caused by novel and recurrent variants using exome sequencing

Abstract Background Inherited retinal dystrophies (IRDs) are characterized by extreme genetic and clinical heterogeneity. There are many genes that are known to cause IRD which makes the identification of the underlying genetic causes quite challenging. And in view of the emergence of therapeutic options, it is essential to combine molecular and clinical data to correctly diagnose IRD patients. In this study, we aimed to identify the disease‐causing variants (DCVs) in four consanguineous Jordanian families with IRDs and describe genotype–phenotype correlations. Methods Exome sequencing (ES) was employed on the proband patients of each family, followed by segregation analysis of candidate variants in affected and unaffected family members by Sanger sequencing. Simulation analysis was done on one novel CLRN1 variant to characterize its effect on mRNA processing. Clinical evaluation included history, slit‐lamp biomicroscopy, and indirect ophthalmoscopy. Results We identified two novel variants in CLRN1 [(c.433+1G>A) and (c.323T>C, p.Leu108Pro)], and two recurrent variants in ABCA4 [(c.1648G>A, p.Gly550Arg) and (c.5460+1G>A)]. Two families with the same DCV were found to have different phenotypes and another family was shown to have sector RP. Moreover, simulation analysis for the CLRN1 splice donor variant (c.433+1G>A) showed that the variant might affect mRNA processing resulting in the formation of an abnormal receptor. Also, a family that was previously diagnosed with nonsyndromic RP was found to have Usher syndrome based on their genetic assessment and audiometry. Conclusion Our findings extend the spectrum of CLRN1‐ and ABCA4‐associated IRDs and describe new phenotypes for these genes. We also highlighted the importance of combining molecular and clinical data to correctly diagnose IRDs and the utility of simulation analysis to predict the effect of splice donor variants on protein formation and function