Mykhailo Azarkh

Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

Double electron-electron resonance (DEER) is an electron paramagnetic resonance (EPR) technique used to determine distance distributions in the nanometer range between spin labels by measuring their dipole-dipole interactions. Here we describe how in-cell DEER can be applied to spin-labeled DNA sequences to unravel their conformations in living cells by long-range distance measurements in cellula. As EPR detects unpaired electron spins only, diamagnetic molecules provide no background and do not reduce detection sensitivity of the specific signal. Compared with in-cell NMR spectroscopy, low concentrations of spin-labeled molecules can be used owing to the higher sensitivity of EPR per spin. This protocol describes the synthesis of the spin labels, their introduction in DNA strands, the injection of labeled DNA solutions in cells and the performance of in-cell EPR measurements. Completion of the entire protocol takes ∼20 d.

Long-Range Distance Determination in a DNA Model System inside Xenopus Laevis Oocytes by In-Cell Spin-Label EPR

Elucidating the structure and dynamics of biomacromolecules, for example, proteins, RNA, or DNA, is crucial for understanding their physiological function. Beside widespread techniques, such as NMR spectroscopy or crystallography, sophisticated electron paramagnetic resonance spectroscopy (EPR) methods in combination with site-directed spin labelingll ) are receiving increasing attention for this purpose. Spin-label EPR (SL-EPR) offers access to structural information on a length scale between approximately 0.5 and 10 nm, and enables the study of dynamics in the picoto microsecond rangeY-B) To date, SLEPR has been carried out in defined buffer solutions. However, recently an in-cell SL-EPR experiment investigating the conformation of ubiquitin was reported;(9) this suggests that the technique can be applied to nucleic acids as well, which is of particular importance since non-canonical DNA conformations are involved in many processes, such as regulatory functions. The development of analytical tools for elucidating native structures inside cells is an ongoing challenge to be addressed by in-cell spectroscopy.(10) Here, we describe long-range distance measurements on a DNA model system in oocytes of Xenopus laevis utilizing in-cell EPR. Compared to in-cell NMR spectroscopy, in-cell EPR has two main advantages. Low concentrations can be used because EPR is much more sensitive per spin than NMR spectroscopy, and since EPR only detects unpaired electron spins no background from diamagnetic molecules is observed. The latter is of particular importance since in contrast to structural studies of isolated macromolecules, in-cell experiments are often hampered by many different cellular components and thereby provide for high background signals. On the other hand, in-cell studies are of greater significance since, in contrast to pure solution experiments, a natural environment is provided, which can be crucial for observing the biologically relevant conformation of biomacromolecules.lll.l2) Double electron-electron resonance (DEER or PELDOR) is a pulsed, two-frequency EPR technique for determining distance distributions (from 1.5 up to 8 nm) between two paramagnetic centers by measuring their dipoledipole interaction. I131S) Diamagnetic biomacromolecules can be site-directly labeled with nitroxides.ll )

Intracellular Conformations of Human Telomeric Quadruplexes Studied by Electron Paramagnetic Resonance Spectroscopy

In-cell DEER (in-cell double electron electron resonance) is applied to investigate quadruplex formation of the human telomeric repeat d[AGGG(TTAGGG)(3)]. The initially unfolded DNA sequence forms a mixture of different quadruplex topologies upon injection into living cells. In addition, time-dependent distance measurements are carried out to monitor quadruplex folding.

Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes

Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis-Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.

Small-Molecule-Triggered Manipulation of DNA Three-Way Junctions

DNA three-way junctions are frequently used in nanoarchitectures. Ligand-dependent designs that provide well-characterized building blocks for structure-switching DNA nanodevices are presented.

A molecular quantum spin network controlled by a single qubit

Control of molecular spins and their readout with a solid-state qubit are described as a unit cell in a quantum spin network. Scalable quantum technologies require an unprecedented combination of precision and complexity for designing stable structures of well-controllable quantum systems on the nanoscale. It is a challenging task to find a suitable elementary building block, of which a quantum network can be comprised in a scalable way. We present the working principle of such a basic unit, engineered using molecular chemistry, whose collective control and readout are executed using a nitrogen vacancy (NV) center in diamond. The basic unit we investigate is a synthetic polyproline with electron spins localized on attached molecular side groups separated by a few nanometers. We demonstrate the collective readout and coherent manipulation of very few (≤ 6) of these S = 1/2 electronic spin systems and access their direct dipolar coupling tensor. Our results show that it is feasible to use spin-labeled peptides as a resource for a molecular qubit–based network, while at the same time providing simple optical readout of single quantum states through NV magnetometry. This work lays the foundation for building arbitrary quantum networks using well-established chemistry methods, which has many applications ranging from mapping distances in single molecules to quantum information processing.