**8.2. Nucleic acids**

EPR spectroscopy has been used broadly for investigating peculiarities of the structural organization and its transformation for DNA, RNA molecules, their models and different protein-nucleic acid complexes. A lot of works were published during last 45 years. Below, we will discuss only several of them relating to the topic of the chapter.

The study of DNA-dye interaction by the spin label method was carried out by Zavriev, et al., 1976. The binding of ethidium bromide and acriflavin dyes with DNA macromolecule modified with spin-labeled analogue of ethylene imine has been studied. These spin labels were shown to bind covalently to DNA, at the same time the number of the dye molecules bound to DNA was decreased without any changes of the binding constant. Analysis of EPR spectra of the samples in the frozen 50% water-glycerol mixture at 77 K for spin-labeled DNA has shown that addition of the dyes increased distances <r> between the labels, that was explained by the increase in DNA length upon formation of the complex with dye molecules. Structural data in the work were obtained from measuring d1/d values (Zavriev, et al., 1976).

The use of d1/d measurements for structural characterization of spin labeled DNA and RNA was also described in a review of EPR studies of the structure and dynamic properties of nucleic acids and other biological systems written by Kamzolova & Postnikova, 1981. When spin labels were attached to different sites of a macromolecule, the quantitative information could be obtained about conformational properties of these local regions and, as a result, about the functional behaviour of the systems.

A distance ruler for RNA using EPR and site-directed spin labeling (SDSL) has been suggested by Kim et al., 2004. The site-directed spin-labeled 10-mer RNA duplexes and HIV-1 TAR RNA motifs with various interspin distances were examined. HIV-1 TAR RNA is the binding site of the viral protein Tat, the trans-activator of the HIV-1 LTR. The long terminal repeat, LTR, regulates HIV-1 viral gene expression via its interaction with multiple viral and host factors. It is present at the 5'- end of all HIV-1 spliced and unspliced mRNAs in the nucleus as well as in the cytoplasm. SDSL was applied to RNA structural biology rather rare, despite an importance of knowledge of RNA structure and RNA-protein complex formation. As a model study for measuring distances in RNA molecules using continuous wave (CW) EPR spectroscopy, the spin labels were attached to the 2′-NH2 positions of appropriately placed uridines in the duplexes, and interspin distances were measured from both molecular dynamics simulations (MDS) and Fourier deconvolution method (FDM) developed by Rabenstein & Shin, 1995. The 10-mer duplexes had interspin distances in the range from 1.0 to 3.0 nm by MDS estimations; however, dipolar line broadening of the CW EPR spectra was observed for the RNAs with interspin distances of 1.0 to 2.1 nm and not for distances over 2.5 nm. Unfortunately, the authors did not use the d1/d method for the distance measurement, probably because this approach was described only in Russian language literature. Its application could add the necessary information to the subject, the more so the appropriate EPR spectra at low temperature were recorded. The conformational changes in TAR (transactivating responsive region) RNA in the presence and in the absence of different divalent metal ions were monitored by measuring distances between two nucleotides in the bulge region. The predicted interspin distances obtained from the FDM method and those from MDS calculations match well for both the model RNA duplexes and the structural changes predicted for TAR RNA. These results demonstrate that distance measurement using EPR spectroscopy is a potentially powerful method to help predict the structures of RNA molecules (Kim et al., 2004).

152 Nitroxides – Theory, Experiment and Applications

about the functional behaviour of the systems.

EPR spectroscopy has been used broadly for investigating peculiarities of the structural organization and its transformation for DNA, RNA molecules, their models and different protein-nucleic acid complexes. A lot of works were published during last 45 years. Below,

The study of DNA-dye interaction by the spin label method was carried out by Zavriev, et al., 1976. The binding of ethidium bromide and acriflavin dyes with DNA macromolecule modified with spin-labeled analogue of ethylene imine has been studied. These spin labels were shown to bind covalently to DNA, at the same time the number of the dye molecules bound to DNA was decreased without any changes of the binding constant. Analysis of EPR spectra of the samples in the frozen 50% water-glycerol mixture at 77 K for spin-labeled DNA has shown that addition of the dyes increased distances <r> between the labels, that was explained by the increase in DNA length upon formation of the complex with dye molecules. Structural data in the work were obtained from measuring d1/d values (Zavriev,

The use of d1/d measurements for structural characterization of spin labeled DNA and RNA was also described in a review of EPR studies of the structure and dynamic properties of nucleic acids and other biological systems written by Kamzolova & Postnikova, 1981. When spin labels were attached to different sites of a macromolecule, the quantitative information could be obtained about conformational properties of these local regions and, as a result,

A distance ruler for RNA using EPR and site-directed spin labeling (SDSL) has been suggested by Kim et al., 2004. The site-directed spin-labeled 10-mer RNA duplexes and HIV-1 TAR RNA motifs with various interspin distances were examined. HIV-1 TAR RNA is the binding site of the viral protein Tat, the trans-activator of the HIV-1 LTR. The long terminal repeat, LTR, regulates HIV-1 viral gene expression via its interaction with multiple viral and host factors. It is present at the 5'- end of all HIV-1 spliced and unspliced mRNAs in the nucleus as well as in the cytoplasm. SDSL was applied to RNA structural biology rather rare, despite an importance of knowledge of RNA structure and RNA-protein complex formation. As a model study for measuring distances in RNA molecules using continuous wave (CW) EPR spectroscopy, the spin labels were attached to the 2′-NH2 positions of appropriately placed uridines in the duplexes, and interspin distances were measured from both molecular dynamics simulations (MDS) and Fourier deconvolution method (FDM) developed by Rabenstein & Shin, 1995. The 10-mer duplexes had interspin distances in the range from 1.0 to 3.0 nm by MDS estimations; however, dipolar line broadening of the CW EPR spectra was observed for the RNAs with interspin distances of 1.0 to 2.1 nm and not for distances over 2.5 nm. Unfortunately, the authors did not use the d1/d method for the distance measurement, probably because this approach was described only in Russian language literature. Its application could add the necessary information to the subject, the more so the appropriate EPR spectra at low temperature were recorded. The conformational

we will discuss only several of them relating to the topic of the chapter.

**8.2. Nucleic acids** 

et al., 1976).

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe has been carried out also in (Cai et al., 2006). Analysis of electron spin dipolar interactions between pairs of nitroxides yields the internitroxide distance, which provides quantitative structural information. The PELDOR and NMR methods had enabled such distance measurements up to 7.0 nm in bio-molecules, thus opening up the possibility of SDSL global structural mapping. The study evaluated SDSL distance measurement using a nitroxide spin label that was attached, in an efficient manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. Radical pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 2.0 to 4.0 nm, were measured using PELDOR technique. The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable radical conformations in the NMR structure, and the accurate distance measurements was demonstrated. The method was proposed for global structural mapping of DNA and DNA–protein complexes (Cai et al., 2006).

The molecular chaperone DnaK recognizes and binds substrate proteins via a stretch of seven amino acid residues that is usually only exposed in unfolded proteins. The binding kinetics is regulated by the nucleotide state of DnaK, which alternates between DnaK and ATP (fast exchange) and DnaK and ADP (slow exchange). These two forms cycle with a rate mainly determined by the ATPase activity of DnaK and nucleotide exchange. The different substrate binding properties of DnaK were mainly attributed to changes of the position and mobility of a helical region in the C-terminal peptide-binding domain, the so-called LID (Popp et al., 2005). Authors investigated the nucleotide-dependent structural changes in the peptide-binding region and the question: could they induce structural changes in peptide stretches using the energy available from ATP hydrolysis. Model peptides contained two cysteine residues at varying positions and were derived from the structurally well-studied peptide NRLLLTG and labelled with spin probes. Measurements of distances between spin labels were carried out by EPR for free peptides or peptides bound to the ATP and ADPstate of DnaK, respectively. No significant change of distances between labels was observed, hence, no structural changes that could be sensed by the probes at the position of central leucine residues located in the center of the binding region occur due to different nucleotide states. It was concluded that the ATPase activity of DnaK is not connected to structural changes of the peptide-binding pocket but has an effect on the LID domain or other further remote residues (Popp et al., 2005).

A rigid, spin-labeled nucleoside was prepared using a convergent synthetic strategy that could also be applied for the synthesis of the corresponding ribonucleoside (Barhate et al., 2007). EPR spectroscopic analysis of a DNA that contained the rigid spin label verified its limited mobility within a DNA duplex. The rigid spin label had several advantages over previously reported spin labels for nucleic acids: a) distance measurements between two rigid spin labels can be done more accurate than between flexible nitroxides; b) possibility of determination of relative orientations of the two rigid labels, that thereby provided more detailed structural information; c) the nucleoside became fluorescent upon reduction of the nitroxide with a mild reducing agent, that was the first example of a spectroscopic probe that could be used for structural studies by both EPR and fluorescence spectroscopy. The dual spectroscopic activity of the spin label enabled the preparation of nucleic acids that contain a redox-active sensor in their structure. More detailed characterization of the new bifunctional spectroscopic probe and its application for the studies of the structure and dynamics of nucleic acids will be reported in due course (Barhate et al., 2007).

Schiemann et al., 2007, described the facile synthesis of the nitroxide spin-label 2,2,5,5 tetramethyl-pyrrolin-1-oxyl-3-acetylene, TPA, and its binding to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. They also have measured distance between two such spin-labels on RNA/DNA using PELDOR, and suggested to use this approach for studying global structure elements of oligonucleotides in frozen solutions at RNA/DNA amounts of ~10 nmol. The procedure suggested by authors should be applicable to RNA/DNA strands of up to ~80 bases in length and PELDOR yields reliably spin–spin distances up to ~6.5 nm (Schiemann et al., 2007).

Indeed, over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule under studying, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been already used as spin centres (Reginsson & Schiemann, 2011).

### **8.3. Biomembranes and lipid-protein complexes**

Native biological membranes and their chemical models such as vesicles and lipid doublelayered membranes (emulsions) as well as various lipid-protein complexes are a huge class of objects suitable for investigation by spin probe/label technique. Structural results obtained by EPR for the third group (lipid-protein complexes) were discussed in Section 8.1 of this review. Numerous articles were published during last 50 years concerning biomembranes but only very few were related to quantitative measurements of the local concentration of spin probes and interspin distances. The main problem in such studies of the structural organization of biological membranes is that when spin probes (nitroxide radicals) are penetrated into outer or inner layer of the double-layered membrane, they promptly become distributed in both layers of the membrane because of flip-flop transitions (Berliner, 1976, Kuznetsov, 1976, Likhtenshtein et al., 2008, Hemminga & Berliner, 2007, Webb, 2006). Therefore, it is usually quite difficult to distinguish between dipolar coupling of spins inside one lipid layer or between spins localized in two lipid monolayers.

## **9. Conclusion**

154 Nitroxides – Theory, Experiment and Applications

& Schiemann, 2011).

A rigid, spin-labeled nucleoside was prepared using a convergent synthetic strategy that could also be applied for the synthesis of the corresponding ribonucleoside (Barhate et al., 2007). EPR spectroscopic analysis of a DNA that contained the rigid spin label verified its limited mobility within a DNA duplex. The rigid spin label had several advantages over previously reported spin labels for nucleic acids: a) distance measurements between two rigid spin labels can be done more accurate than between flexible nitroxides; b) possibility of determination of relative orientations of the two rigid labels, that thereby provided more detailed structural information; c) the nucleoside became fluorescent upon reduction of the nitroxide with a mild reducing agent, that was the first example of a spectroscopic probe that could be used for structural studies by both EPR and fluorescence spectroscopy. The dual spectroscopic activity of the spin label enabled the preparation of nucleic acids that contain a redox-active sensor in their structure. More detailed characterization of the new bifunctional spectroscopic probe and its application for the studies of the structure and

dynamics of nucleic acids will be reported in due course (Barhate et al., 2007).

reliably spin–spin distances up to ~6.5 nm (Schiemann et al., 2007).

**8.3. Biomembranes and lipid-protein complexes** 

Schiemann et al., 2007, described the facile synthesis of the nitroxide spin-label 2,2,5,5 tetramethyl-pyrrolin-1-oxyl-3-acetylene, TPA, and its binding to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. They also have measured distance between two such spin-labels on RNA/DNA using PELDOR, and suggested to use this approach for studying global structure elements of oligonucleotides in frozen solutions at RNA/DNA amounts of ~10 nmol. The procedure suggested by authors should be applicable to RNA/DNA strands of up to ~80 bases in length and PELDOR yields

Indeed, over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule under studying, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been already used as spin centres (Reginsson

Native biological membranes and their chemical models such as vesicles and lipid doublelayered membranes (emulsions) as well as various lipid-protein complexes are a huge class of objects suitable for investigation by spin probe/label technique. Structural results obtained by EPR for the third group (lipid-protein complexes) were discussed in Section 8.1 of this review. Numerous articles were published during last 50 years concerning biomembranes but only very few were related to quantitative measurements of the local concentration of spin probes and interspin distances. The main problem in such studies of the structural organization of biological membranes is that when spin probes (nitroxide radicals) are penetrated into outer or inner layer of the double-layered membrane, they promptly become distributed in both layers of the membrane because of flip-flop transitions Measurement of distances or local concentrations in physical and macromolecular chemistry, solid state chemistry, molecular biology and biophysics is an important quantitative tool for investigating structure, spatial organization and conformational transitions in solids, solid solutions, polymers, biological macromolecules, and complex supramolecular systems using site-directed spin labeling and various techniques of EPR spectroscopy. Modern approaches of EPR such as high frequency/high field EPR, pulce technique and double resonances, dipolar EPR spectroscopy allow researchers determine not only interspin distances but also their relative 3-D orientation and the behaviour of these complex systems under their functioning using stable nitroxide radicals. Analysis of the results obtained shows that became a method making possible controlling quantitatively spatial structure and properties of chemical and biological systems in conditions the most close to natural. And this is very important for correct understanding of the mechanisms of these processes.

Scientific progress is irreversible. During the last forty years three generations of researchers were changed, and naturally the development of new modern methods of quantitative investigation is continuously in progress. These methods are usually correct, informative but very technically complex and need a lot of theoretical and computer calculations, i.e. take a lot of time. Therefore, such simple method as d1/d parameter for estimation distances **r** in the case of pairwise distribution of nitroxide radicals and local concentrations Cloc or mean local distances <r> at their random (chaotic) distribution of paramagnetic centres can provide valuable structural information at the beginning serious complex and long-time investigation. Measurements of d1/d values are simple and do not need much time especially considering important information provided by it. Evidently, d1/d parameter should be used in the distance or concentration intervals and experimental conditions in which it determination is correct.
