Investigation of Mg(2+)- and temperature-dependent folding of the hairpin ribozyme by photo-crosslinking: effects of photo-crosslinker tether length and chemistry

We have used photo-crosslinking to investigate the structure and dynamics of four-way junction hairpin ribozyme constructs. Four phenylazide photo-crosslinkers were coupled to 2′-NH(2)-modified U+2 in the substrate and irradiated at different Mg(2+) concentrations and temperatures. Consistent with the role of divalent metal ions in hairpin ribozyme folding, we observed more interdomain crosslinks in the presence of Mg(2+) than in its absence. In general, we observed intradomain crosslinks to nucleotides 2–11 and interdomain crosslinks to the U1A binding loop. Crosslinks to A26 and G36 in domain B were also observed when crosslinking was carried out at −78°C. In contrast to crosslinking results at higher temperatures (0, 25 and 37°C), similar crosslinks were obtained in the presence and absence of Mg(2+) at −78°C, suggesting Mg(2+) stabilizes a low-energy hairpin ribozyme conformation. We also evaluated the effects of photo-crosslinker structure and mechanism on crosslinks. First, most crosslinks were to unpaired nucleotides. Second, shorter and longer photo-crosslinkers formed crosslinks to intradomain locations nearer to and farther from photo-crosslinker modification, respectively. Finally, fluorine substitutions on the phenylazide ring did not change the locations of crosslinks, but rather decreased crosslinking efficiency. These findings have implications for the use of phenylazide photo-crosslinkers in structural studies of RNA

On the Limited Stability of BDPA Radicals

Post-print (lokagerð höfundar)1,3‐Bis(diphenylene)‐2‐phenylallyl (BDPA)‐based radicals are of interest as polarizing agents for dynamic nuclear polarization (DNP). For this purpose, a BDPA‐nitroxide biradical, employing a phosphodiester linkage, was synthesized. Contrary to what is commonly assumed, BDPA‐derived radicals were observed to have limited stability. Hence, the effects of various factors on the stability of BDPA radicals were investigated. Solvent polarity was found to play a significant role on degradation; a polar BDPA radical was observed to degrade faster in a non‐polar solvent, whereas non‐polar radicals were more unstable in polar solvents. The rate of decomposition was found to increase non‐linearly with increasing radical concentration; a 2‐fold increase in concentration led to a 3‐fold increase in the rate of degradation. Collectively, these results indicate that the dimerization is a significant degradation pathway for BDPA radicals and indeed, a dimer of one BDPA radical was detected by mass spectrometry.This work is funded by Icelandic Research Fund (163393-052) from the Icelandic Centre for Research (Rannis). We thank Dr. S. Jonsdottir for assistance with collection of the NMR and HRMS (ESI) data; Dr. Frederic Mentink-Vigier for help with simulations and for valuable suggestions; and members of the Sigurdsson research group for helpful discussions.Peer reviewe

Reduction Resistant and Rigid Nitroxide Spin-Labels for DNA and RNA

Post-print (lokagerð höfundar)Electron paramagnetic resonance (EPR) spectroscopy, coupled with site-directed spin labeling (SDSL), is a useful method for studying conformational changes of biomolecules in cells. To employ in-cell EPR using nitroxide-based spin labels, the structure of the nitroxides must confer reduction resistance to withstand the reductive environment within cells. Here, we report the synthesis of two new spin labels, EÇ and EÇm, both of which possess the rigidity and the reduction resistance needed for extracting detailed structural information by EPR spectroscopy. EÇ and EÇm were incorporated into DNA and RNA, respectively, by oligonucleotide synthesis. Both labels were shown to be nonperturbing of the duplex structure. The partial reduction of EÇm during RNA synthesis was circumvented by the protection of the nitroxide as a benzoylated hydroxylamine.The authors acknowledge financial support from the Icelandic Research Fund (173727-051). The authors thank Dr. S. Jonsdottir for assistance with collecting analytical data for structural characterization of new compounds and members of the Sigurdsson research group for helpful discussions.Peer reviewe

TEMPO-derived spin labels linked to the nucleobases adenine and cytosine for probing local structural perturbations in DNA by EPR spectroscopy

Publisher's version (útgefin grein)Three 2´-deoxynucleosides containing semi-flexible spin labels, namely TA, UA and UC, were prepared and incorporated into deoxyoligonucleotides using the phosphoramidite method. All three nucleosides contain 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) connected to the exocyclic amino group; TA directly and UA as well as UC through a urea linkage. TA and UC showed a minor destabilization of a DNA duplex, as registered by a small decrease in the melting temperature, while UA destabilized the duplex by more than 10 °C. Circular dichroism (CD) measurements indicated that all three labels were accommodated in B-DNA duplex. The mobility of the spin label TA varied with different base-pairing partners in duplex DNA, with the TA•T pair being the least mobile. Furthermore, TA showed decreased mobility under acidic conditions for the sequences TA•C and TA•G, to the extent that the EPR spectrum of the latter became nearly superimposable to that of TA•T. The reduced mobility of the TA•C and TA•G mismatches at pH 5 is consistent with the formation of TAH+•C and TAH+•G, in which protonation of N1 of A allows the formation of an additional hydrogen bond to N3 of C and N7 of G, respectively, with G in a syn-conformation. The urea-based spin labels UA and UC were more mobile than TA, but still showed a minor variation in their EPR spectra when paired with A, G, C or T in a DNA duplex. UA and UC had similar mobility order for the different base pairs, with the lowest mobility when paired with C and the highest when paired with T.We thank the Icelandic Research Fund and the University of Iceland Research Fund for financial support (141062-951). We thank Dr. Pavol Cekan for technical assistance, Dr. Sigridur Jonsdottir for NMR and MS analyses and the members of the Sigurdsson research group for helpful discussions.Peer reviewe

Nitroxide‐Derived N‐Oxide Phenazines for Noncovalent Spin‐Labeling of DNA

Post-print (lokagerð höfundar)Two o‐benzoquinone derivatives of isoindoline were synthesized for use as building blocks to incorporate isoindoline nitroxides into different compounds and materials. These o‐quinones were condensed with a number of o‐phenylenediamines to form isoindoline‐phenazines in high yields. Subsequent oxidation gave phenazine‐di‐N‐oxide isoindoline nitroxides that were evaluated for noncovalent and site‐directed spin‐labeling of duplex DNA and RNA that contained abasic sites. Although only minor binding was observed for RNA, the unsubstituted phenazine‐N,N‐dioxide tetramethyl isoindoline nitroxide showed high binding affinity and selectivity towards abasic sites in duplex DNA that contained cytosine as the orphan base.The authors acknowledge financial support from the Icelandic Research Fund (173727-051). The authors thank Dr S. Jonsdottir for assistance with collecting analytical data for structural characterization of new compounds, Dr. Thomas Halbritter for critically reading this manuscript, and the members of the Sigurdsson research group for helpful discussions.Peer reviewe

Site-directed spin labeling of 2′-amino groups in RNA with isoindoline nitroxides that are resistant to reduction

Post-print (lokagerð höfundar)Two aromatic isothiocyanates, derived from isoindoline nitroxides, were synthesized and selectively reacted with 2′-amino groups in RNA. The spin labels displayed limited mobility in RNA, making them promising candidates for distance measurements by pulsed EPR. After conjugation to RNA, a tetraethyl isoindoline derivative showed significant stability under reducing conditions.This work was supported by the Icelandic Research Fund (141062-051). S.S. and A.P.J. gratefully acknowledge doctoral fellowships provided by the University of Iceland. The authors thank Dr S. Jonsdottir for assistance with collecting analytical data for structural characterization of compounds prepared and members of the Sigurdsson research group for critical reading of the manuscript.Peer reviewe

Nitroxide-labeled pyrimidines for non-covalent spin-labeling of abasic sites in DNA and RNA duplexes

Post-print (lokagerð höfundar)Non-covalent and site-directed spin labeling gives easy access to spin-labeled nucleic acids for the study of their structure and dynamics by electron paramagnetic resonance (EPR) spectroscopy. In a search for improved spin labels for non-covalent binding to abasic sites in duplex DNA and RNA, ten pyrimidine-derived spin labels were prepared in good yields and their binding was evaluated by continuous wave (CW)-EPR spectroscopy. Most of the spin labels showed lower binding affinity than the previously reported label c towards abasic sites in DNA and RNA. The most promising labels were triazole-linked spin labels and a pyrrolocytosine label. In particular, the N1-ethylamino derivative of a triazole-linked uracil spin label binds fully to both DNA and RNA containing an abasic site. This is the first example of a spin label that binds fully through non-covalent interactions with an abasic site in RNA.We thank the Icelandic Research Fund (110035022) and the University of Iceland Research Fund for financial support. S. A. S. acknowledges a doctoral fellowship from the University of Iceland Research Fund. We thank Dr S. Jonsdottir for assistance in collecting analytical data for structural characterization of the compounds that were prepared and members of the Sigurdsson research group for critical reading of the manuscript.Peer reviewe

Benzoyl-Protected Hydroxylamines for Improved Chemical Synthesis of Oligonucleotides Containing Nitroxide Spin Labels

Post-print (lokagerð höfundar)Oligonucleotides containing nitroxide spin labels, used in biophysical studies of nucleic acids, are frequently prepared by chemical synthesis. However, during the synthesis of spin‐labeled oligonucleotides, the nitroxides are partially reduced to the corresponding amines. Here we report that a benzoylated hydroxylamine can be used as a protected form of the nitroxide to avoid this reduction. The benzoyl group is stable through the oligonucleotide synthesis and is readily removed under standard oligonucleotide deprotection conditions, yielding a hydroxylamine that is oxidized in situ to the nitroxide. This method was used to incorporate the rigid spin labels Ç and Çm into DNA and RNA oligonucleotides, respectively, including a doubly labeled 36‐nucleotide long DNAzyme. Enzymatic digestion of the spin‐labeled oligonucleotides and subsequent HPLC analysis showed that the nitroxides were intact. This protecting group strategy facilitates the high‐yielding synthesis of spin‐labeled DNA and RNA oligonucleotides using the phosphoramidite method.The authors acknowledge financial support by the Icelandic Research Fund (141062-051). The authors thank Dr S. Jonsdottir for assistance with collecting analytical data for structural characterization of new compounds and members of the Sigurdsson research group for helpful discussions.Peer reviewe