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.

In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR–Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR–Cas9, which will provide key information for understanding the mechanism of CRISPR function.

Guanine-rich oligonucleotides can form a unique G-quadruplex (GQ) structure with stacking units of four guanine bases organized in a plane through Hoogsteen bonding. GQ structures have been detected in vivo and shown to exert their roles in maintaining genome integrity and regulating gene expression. Understanding GQ conformation is important for understanding its inherent biological role and for devising strategies to control and manipulate functions based on targeting GQ. Although a number of biophysical methods have been used to investigate structure and dynamics of GQs, our understanding is far from complete. As such, this work explores the use of the site-directed spin labeling technique, complemented by molecular dynamics simulations, for investigating GQ conformations. A nucleotide-independent nitroxide label (R5), which has been previously applied for probing conformations of noncoding RNA and DNA duplexes, is attached to multiple sites in a 22-nucleotide DNA strand derived from the human telomeric sequence (hTel-22) that is known to form GQ. The R5 labels are shown to minimally impact GQ folding, and inter-R5 distances measured using double electron–electron resonance spectroscopy are shown to adequately distinguish the different topological conformations of hTel-22 and report variations in their occupancies in response to changes of the environment variables such as salt, crowding agent, and small molecule ligand. The work demonstrates that the R5 label is able to probe GQ conformation and establishes the base for using R5 to study more complex sequences, such as those that may potentially form multimeric GQs in long telomeric repeats.

Nanodiamonds (NDs) are a new and attractive class of materials for sensing and delivery in biological systems. Methods for functionalizing ND surfaces are highly valuable in these applications, yet reported approaches for covalent modification with biological macromolecules are still limited, and characterizing behaviors of ND-tethered biomolecules is difficult. Here we demonstrated the use of copper-free click chemistry to covalently attach DNA strands at ND surfaces. Using site-directed spin labeling and electron paramagnetic resonance spectroscopy, we demonstrated that the tethered DNA strands maintain the ability to undergo repetitive hybridizations and behave similarly to those in solutions, maintaining a large degree of mobility with respect to the ND. The work established a method to prepare and characterize an easily addressable identity tag for NDs. This will open up future applications such as targeted ND delivery and developing sensors for investigating biomolecules.

The emerging Overhauser effect dynamic nuclear polarization (ODNP) technique measures the translational mobility of water within the vicinity (5–15 Å) of preselected sites. The work presented here expands the capabilities of the ODNP technique and illuminates an important, previously unseen, property of the translational diffusion dynamics of water at the surface of DNA duplexes. We attach nitroxide radicals (i.e., spin labels) to multiple phosphate backbone positions of DNA duplexes, allowing ODNP to measure the hydration dynamics at select positions along the DNA surface. With a novel approach to ODNP analysis, we isolate the contributions of water molecules at these sites that undergo free translational diffusion from water molecules that either loosely bind to or exchange protons with the DNA. The results reveal that a significant population of water in a localized volume adjacent to the DNA surface exhibits fast, bulk-like characteristics and moves unusually rapidly compared to water found in similar probe volumes near protein and membrane surfaces. Control studies show that the observation of these characteristics are upheld even when the DNA duplex is tethered to streptavidin or the mobility of the nitroxides is altered. This implies that, as compared to protein or lipid surfaces, it is an intrinsic feature of the DNA duplex surface that it interacts only weakly with a significant fraction of the surface hydration water network. The displacement of this translationally mobile water is energetically less costly than that of more strongly bound water by up to several kBT and thus can lower the activation barrier for interactions involving the DNA surface.

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 (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL 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.

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 Rp and Sp diastereomers at phosphorothioate pSC7 of d(CTACTGpSC7Y8TTAG). d(CTAAAGCAGTAG) (Y = T or U). X-band continuous-wave EPR spectra revealed that the dT8 to dU8 change alters nanosecond rotational motions of Rp-R5a but produces no detectable differences for Sp-R5a, Rp-R5, and Sp-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 BI/BII 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.

In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically inert unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented here examine the feasibility of probing local DNA structural and dynamic features using a class of nitroxide probes that are linked to chemically substituted phosphorothioate positions at the DNA backbone. Two members of this family, designated as R5 and R5a, were attached to eight different sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Measured X-band electron paramagnetic resonance (EPR) spectra, which report on nitroxide rotational motions, were found to vary depending on the location of the label (e.g., duplex center vs termini) and the surrounding DNA sequence. This indicates that R5 and R5a can provide information on the DNA local environment at the level of an individual nucleotide. As these probes can be attached to arbitrary nucleotides within a nucleic acid sequence, they may provide a means to “scan” a given DNA molecule in order to interrogate its local structural and dynamic features.

Site-directed spin labeling (SDSL) provides local structural and dynamic information about a macromolecule via electron paramagnetic resonance (EPR) spectroscopy measurements of a stable nitroxide radical attached to the macromolecule in a site-specific manner. SDSL has matured as a tool for studying proteins, especially in systems that are difficult to examine using methods such as X-ray crystallography or NMR spectroscopy1. SDSL studies of nucleic acids have also been reported2. Two major categories of measurements are used in SDSL: monitoring the rotational motions of a single nitroxide provides site-specific structural and dynamic information and measuring the distances between a pair of nitroxides yields direct structural data. Only inter-nitroxide distance measurements are discussed here.In SDSL, the distance between a pair of electron spins (i.e., the unpaired electron at the nitroxide) is determined by measuring the strength of the dipole–dipole interaction3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Inter-spin distances between 5 and 20 Å can be measured using continuous-wave (cw) EPR13, 14, 15, and distances between 20 and 70 Å have been measured using pulse EPR techniques16, 17, 18, 19, 20, 21, 22, 23, 24, 25. SDSL uses a pair of identical nitroxide probes, which simplifies the labeling procedure. Once the nitroxides are attached to the target molecule, the measurements are not limited by the molecular weight, which makes SDSL useful for studying high molecular weight systems. Nitroxides are generally smaller than other probes (e.g., chromophores), and therefore induce smaller structural perturbation as compared to other larger probes. It should be noted that the unpaired electron is predominantly localized at the nitroxyl group in a nitroxide26. This facilitates the interpretation of the measured distances.This protocol describes a tool-kit that uses SDSL distance measurements for mapping global structures and monitoring conformational changes of nucleic acids and protein/nucleic acid complexes. First, we describe a nitroxide probe, designated R5, that can be efficiently attached to any nucleotide position within a given sequence27. Second, we describe the use of a 4-pulse EPR technique, called double electron–electron resonance (DEER)28, to measure inter-nitroxide distances ranging from 20 to 70 Å. Finally, we describe an Internet-accessible program, called NASNOX-W29, which permits efficient evaluation of inter-R5 distances in nucleic acids.R5 is a 'flexible' probe—the nitroxide is attached via three rotatable single bonds in either one of the two phosphorothioate diastereomers that are introduced in a ~50/50 ratio during chemical synthesis of nucleic acids (Fig. 1)23, 25. We have measured experimentally inter-R5 distances between 20 and 50 Å in nucleic acid duplexes ranging from 12 to 68 bp23, 25. The measured distances can be reproduced theoretically using an efficient computer program (NASNOX) that properly accounts for the phosphorothiate diastereomers and the allowable R5 conformers23, 25, 29. In addition, R5 may also serve as a paramagnetic relaxing agent in NMR experiments to provide additional distance constraints. Preliminary studies also indicate that the dynamics of a single-labeled R5 provides information at the nucleotide level (A.P. and P.Z.Q., manuscript in preparation).For inter-R5 distances >20 Å, the mean distances computed by NASNOX have been shown to agree very well with those measured experimentally23, 25 and those computed by all atom molecular dynamics simulations29. The correlation between the s.d. of the distance distributions is less satisfying23, 25, in part due to DNA motion29.The biggest advantage of NASNOX is its speed—each R5 conformer distribution and the corresponding inter-R5 distances can be computed in seconds to minutes on a desktop PC. An Internet-accessible version of NASNOX, called NASNOX-W, is available at http://pzqin.usc.edu/NASNOX/. While the current NASNOX program is developed specifically for R5, the underlying principles should be applicable to other probes.The following protocol has been used to study DNA and RNA23, 25, 29. To facilitate the presentation, a dodecameric DNA duplex (Fig. 3a) is used as an example to illustrate the procedures. This DNA is designated as CS following its PDB id no. 1CS2 (ref. 47), and spin-labeling studies on this DNA have been reported23, 29.Steps 1–7, R5 precursor preparation and oligonucleotide labeling: 1–2 dSteps 8–11, purification of labeled oligonucleotides: 1–2 dSteps 12 and 13, DEER sample preparation: 1 dSteps 14–23, DEER data acquisition: 1–2 d. Data average time ranges from 6 to 24 h depending on the sample concentration and the range of the distance being measuredSteps 24–29, DEER data analysis: 1–2 hSteps 30–39, NASNOX-W analysis: <1 hTroubleshooting advice can be found in Table 3.One line is written for each conformer. The label number is specified in the order of definition in Steps 33–36. In the example shown in Figure 4, label 1 = nitroxide 1/O1P, label 2 = nitroxide 1/O2P, label 3 = nitroxide 2/O1P, label 4 = nitroxide 2/O2P. The conformer number specifies the particular set of (t1,t2,t3). With the 'fine search' and 'hydrophobic contact' switched off, the 'search result' is one of two options: 'clash' (the conformer is sterically disallowed) and 'conformer fit' (the conformer is acceptable). Invoking the 'fine search' option25, 29 leads to a third possibility: a 'modified fit' for conformers that are spatially close to an originally clashing conformer (in this case, the number of acceptable 'fine search' conformers is also written).Interpretation of the calculated inter-R5 distance in the 'data.add' file. The inter-R5 distances are output as follows:One line is written for each pair of label conformers. A label conformer is identified by the label number, the conformer number and the nucleotide attachment in parentheses. Distances are shown for measurements between nitrogen atoms (N–N), between oxygen atoms (O–O) and between the midpoints of the nitroxyl groups (the N–O bond; mNO–mNO) of each R5. For distances measured based on the conformers found using a 'modified fit' with the 'fine search' option, the number(s) of conformers found in the fine search is indicated at the end of the line.Interpretation of the overall inter-R5 distance in the 'data.add' file. The overall inter-R5 distance for the specified system is output as follows (last lines of the 'data.add' file):Overall N–N, O–O, mNO–mNO distances (defined above) are output, with the s.d. for each. The number of inter-R5 distances in the ensemble is shown in parentheses.

In this report, stereospecific structural and dynamic features in DNA are studied using the site-directed spin labeling technique. A stable nitroxide radical, 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), was attached postsynthetically to phosphorothioates that were chemically introduced, one at a time, at five sites of a DNA duplex. The two phosphorothioate diastereomers (Rp or Sp) were separated, and nitroxide rotational motions were monitored using electron paramagnetic resonance spectroscopy. The resulting spectra vary according to diastereomer identity and location of the labeling site, with Rp-R5a spectra effectively reporting on local DNA structural features and Sp-R5a spectra sensing variations in local DNA motions. This establishes Rp- and Sp-R5a as unique probes for investigating nucleic acids in a site- and stereospecific manner, which may aid studies of stereospecific DNA/protein interactions. In addition, weighted averages of individual Rp and Sp spectra match those of R5a attached to mixed diastereomers. This suggests that R5a linked to mixed diastereomers reports on the composite behaviors of Rp- and Sp-R5a and is useful in initial probing of the DNA local environment. This work advances understanding of R5a/DNA coupling, and is a key step forward in developing a nucleotide-independent spectroscopic probe for studying nucleic acids.

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.