Three new dinitrosyl iron complexes LFe(NO)(2)(L = 2,2'-bipyridine (bipy)(1), 2,2',2''-terpyridine (terpy)(2) and 1,10-phenathroline (phen)(3)) were synthesized by the reaction of Fe(NO)(2)(CO)(2) with corresponding ligands in tetrahydrofuran. Complexes 1-3 were studied using IR, UV-vis, MS, NMR, and electrochemical techniques. Complexes 1 and 2 were also characterized using single crystal X-ray diffraction analysis. IR spectra of complexes 1-3 display two strong characteristic NO stretching frequencies (nu(NO)) in the region reflecting donor properties of the ligands. Cyclic voltammetry studies show two quasi-reversible one-electron reductions for all complexes. Electrochemical investigations using different concentrations show that an irreversible one-electron reduction at -1.85 V for complex 2 and -1.80 V for complex 3 are from solvated species. Single-crystal X-ray structural analysis reveals that complex 1 crystallizes in the triclinic P1 space group and the asymmetric unit consists of one Fe(NO)(2)(bipy) molecule with the two NO groups located on two sides of Fe(bipy) plane. Complex 2 crystallizes in monoclinic P21/n space group, and the asymmetric unit contains one Fe(NO)(2)(terpy) molecule, in which the NO groups are located on two sides of the plane consisted of Fe and two coordinated pyridyl rings, but almost parallel to the uncoordinated pyridyl ring. The crystal packings of both complexes 1 and 2 show intermolecular H-bonding and strong pi-pi stacking interactions.

The Tetrahymena group I intron recognizes its oligonucleotide substrate in a two-step process. First, a substrate recognition duplex, called the P1 duplex, is formed. The P1 duplex then docks into the prefolded ribozyme core by forming tertiary contacts. P1 docking controls both the rate and the fidelity of substrate cleavage and has been extensively studied as a model for the formation of RNA tertiary structure. However, previous work has been limited to studying millisecond or slower motions. Here we investigated nanosecond P1 motions in the context of the ribozyme using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A nitroxide spin label (R5a) was covalently attached to a specific site of the substrate oligonucleotide, the labeled substrate was bound to a prefolded ribozyme to form the P1 duplex, and X-band EPR spectroscopy was used to monitor nitroxide motions in the 0.1-50 ns regime. Using substrates that favor the docked or the undocked states, it was established that R5a was capable of reporting P1 duplex motions. Using R5a-labeled substrates it was found that the J1/2 junction connecting P1 to the ribozyme core controls nanosecond P1 mobility in the undocked state. This may account for previous observations that J1/2 mutations weaken substrate binding and give rise to cryptic cleavage. This study establishes the use of SDSL to probe nanosecond dynamic behaviors of individual helices within large RNA and RNA/protein complexes. This approach may help in understanding the relationship between RNA structure, dynamics, and function.

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.

This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron-electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes.

In site-directed spin labeling (SDSL), a nitroxide moiety containing a stable, unpaired electron is covalently attached to a specific site within a macromolecule, and structural and dynamic information at the labeling site is obtained via electron paramagnetic resonance (EPR) spectroscopy. Successful SDSL requires efficient site-specific incorporation of nitroxides. Work reported here presents a new method for facile nitroxide labeling at the 5' terminus of nucleic acids of arbitrary sizes. T4-polynucleotide kinase was used to enzymatically substitute a phosphorothioate group at the 5' terminus of a nucleic acid, and the resulting phosphorothioate was then reacted with an iodomethyl derivative of a nitroxide. The method was successfully demonstrated on both chemically synthesized and naturally occurring nucleic acids. The attached nitroxides reported duplex formation as well as tertiary folding of nucleic acids, indicating that they serve as a valid probe in nucleic acid studies.