Peter Z. Qin

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70 Å in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40 Å, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA–protein complexes.

Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

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.

Site-directed spin labeling studies on nucleic acid structure and dynamics

Publisher Summary This chapter summarizes the current site-directed spin labeling (SDSL) studies on nucleic acids, with discussions focusing on literature from the last decade. SDSL is useful in studying high molecular weight systems under physiological conditions. It has been particularly successful in studying systems (e.g., membrane proteins) that are difficult to investigate using other methods, such as X-ray crystallography and NMR spectroscopy. SDSL has been used to study nucleic acids, and data suggest that one can obtain unique structural and dynamic information about DNA and RNA at the level of individual nucleotides. The majority of nucleic acid SDSL studies have used one of two types of EPR measurements. Distance measurements between pairs of nitroxides provide direct structural constraints in nucleic acid systems. In addition, the mobility of a single-labeled nitroxide can be measured to yield structural and dynamic information at the labeling site. Because nucleic acids are different from proteins in the nature of the basic chemical constituents (4 nucleotides vs. 20 amino acids) and their secondary structural units (B-form/A-form doubled-stranded helix vs. α -helix/ β -sheet), SDSL of nucleic acids requires unique methodologies, particularly in the areas of nitroxide attachment and the correlation of the nitroxide behavior to that of the parent molecule.

STUDYING RNA USING SITE-DIRECTED SPIN-LABELING AND CONTINUOUS-WAVE ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY

In site-directed spin-labeling (SDSL), a stable nitroxide radical is attached to a specific location within a macromolecule and electron paramagnetic resonance (EPR) spectroscopy is used to interrogate the local environment surrounding the nitroxide. The SDSL strategy enables probing site-specific structural and dynamic features of RNA in solution without being limited by the size of the molecule, thus serving as a unique tool in biophysical studies of RNA. This chapter describes the use of continuous-wave (cw)-EPR to study dynamic features of RNAs as well as to monitor interactions between them. Various approaches for attaching nitroxide spin labels to nucleic acids are described, followed by detailed descriptions of cw-EPR spectral acquisition and processing procedures. Specific examples are subsequently used to illustrate analysis of EPR spectra, showing how information regarding the parent RNA can be extracted.

A facile method for attaching nitroxide spin labels at the 5′ terminus of nucleic acids

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.

Motions of the Substrate Recognition Duplex in a Group I Intron Assessed by Site-Directed Spin Labeling

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.

RNA Dynamics: Perspectives from Spin Labels

Dynamics are important and indispensible physical attributes that play essential roles in RNA function. RNA dynamics are complex, spanning vast timescales, and encompassing a large number of physical modes. The technique of site‐directed spin labeling (SDSL), which derives information on local structural and dynamic features of a macromolecule by monitoring a chemically stable nitroxide radical using electron paramagnetic resonance spectroscopy, has been applied to monitor intrinsic dynamics at defined structural states as well as to probe conformational transition dynamics of RNAs. The current state of SDSL studies of RNA dynamics is summarized here. Further development and application of SDSL promise to open up many more opportunities for probing RNA dynamics and connecting dynamics to structure and function. WIREs RNA 2012, 3:62–72. doi: 10.1002/wrna.104

G-quadruplex DNA targeted metal complexes acting as potential anticancer drugs

Although cisplatin and its analogues have been widely utilized as anticancer metallodrugs in clinics, their serious side effects and damage to normal tissues cannot be avoided because cisplatin kills cancer cells by attacking genomic DNA. Thus the design of metallodrugs possessing different actions of anti-cancer mechanism is promising. G-quadruplex nucleic acid, which is formed by self-assembly of guanine-rich nucleic acid sequences, has recently been considered as an attractive target for anticancer drug design. The basic unit of a G-quadruplex is a G-quartet, a planar motif generated from four guanine residues pairing together through Hoogsteen like hydrogen bonds. DNA G-quadruplex (G4) structures exist in the chromosomal telomeric sequences and the promoter regions of numerous genes, including oncogenetic promoters. Formation of G4 structures within the 3′-overhang of telomeric DNA can inhibit the telomerase activity, which is silent in normal cells but up-regulated in most cancer cells, thus significantly shortening telomeres and preventing cancer cell proliferation and immortalization. Intramolecular G4 structures formed within the oncogene promoter regions can effectively inhibit oncogenen transcription and expression. Thus rational design of small molecular ligands to selectively interact, stabilize or cleave G4 structures is a promising strategy for developing potent anti-cancer drugs with selective toxicity towards cancer cells over normal ones. This review will highlight the recent development of G4-interacting metal complexes, termed G4-ligands, discussing their binding modes with G-quadruplex DNA and their potential to serve as anticancer drugs in the medical field. Introduction to the international collaboration The collaboration between Prof. Zong-Wan Mao from Sun Yat-Sen University, P. R. China and Prof. Roland K. O. Sigel from the University of Zurich, Switzerland officially began in January, 2014. The international collaborative research project titled “Chemical Biology Research of New Metallodrugs for Cancer Therapy” is supported by the Science and Technology Program of Guangdong Provincial Government [20130501c]. With the rapid development of tumor molecular pharmacology, molecular targeted anti-tumor drugs have become a hot spot in the research of cancer therapy. This international collaborative research project combines the computer simulation and in vitro drug screening platform to design a series of metallodrugs that are systematic and have structural diversity, which can target specific nucleic acid structures (e.g. G-quadruplexes), key proteins (DNA topoisomerase, telomerase, CDK kinase) associated with the occurrence and development of tumor. With the advantages of both laboratories, the structural–functional relationship, interaction modes, co-crystallization, and mechanisms of action of these newly designed metallodrugs are intensively studied, and their in vitro and in vivo anti-tumor activities are comprehensively evaluated.

Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion

The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein–DNA interactions as previously observed, and the two dimers retain all the protein–protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.

Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H(T) and H(L)) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H(T) and H(L) stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.