Alexander A. Lomzov

Complementary-addressed site-directed spin labeling of long natural RNAs

Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron–electron resonance provided spin–spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.

2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis, Fluorescent Properties, and Duplex Stability

The synthesis and properties two series of new 2′‐O‐methyl RNA probes, each containing a single insertion of a 2′‐bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21‐fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5′‐side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3′‐side are important: CC, CG, and UC dinucleotide units on the 3′‐side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2′‐bispyrene‐labeled 2′‐O‐methyl RNA probes might be useful tools for detection of RNAs.

Oxidative damage to epigenetically methylated sites affects DNA stability, dynamics and enzymatic demethylation

Abstract DNA damage can affect various regulatory elements of the genome, with the consequences for DNA structure, dynamics, and interaction with proteins remaining largely unexplored. We used solution NMR spectroscopy, restrained and free molecular dynamics to obtain the structures and investigate dominant motions for a set of DNA duplexes containing CpG sites permuted with combinations of 5-methylcytosine (mC), the primary epigenetic base, and 8-oxoguanine (oxoG), an abundant DNA lesion. Guanine oxidation significantly changed the motion in both hemimethylated and fully methylated DNA, increased base pair breathing, induced BI→BII transition in the backbone 3′ to the oxoG and reduced the variability of shift and tilt helical parameters. UV melting experiments corroborated the NMR and molecular dynamics results, showing significant destabilization of all methylated contexts by oxoG. Notably, some dynamic and thermodynamic effects were not additive in the fully methylated oxidized CpG, indicating that the introduced modifications interact with each other. Finally, we show that the presence of oxoG biases the recognition of methylated CpG dinucleotides by ROS1, a plant enzyme involved in epigenetic DNA demethylation, in favor of the oxidized DNA strand. Thus, the conformational and dynamic effects of spurious DNA oxidation in the regulatory CpG dinucleotide can have far-reaching biological consequences.

A new approach to precise thermodynamic characterization of hybridization properties of modified oligonucleotides: Comparative studies of deoxyribo- and glycine morpholine pentaadenines.

The development of new derivatives and analogues of nucleic acids for the purposes of molecular biology, biotechnology, gene diagnostics, and medicine has been a hotspot for the last two decades. Methylenecarboxamide (glycine) morpholine oligomer analogues (gM) seem to be promising therapeutic candidates because of the ability to form sequence specific complexes with DNA and RNA. In this paper we describe new approaches to the determination of thermodynamic parameters for hybridization of tandem oligonucleotide complexes with the complementary template. It makes possible to determine changes in enthalpy and entropy corresponding to the binding of an individual oligomer with the template, and to the formation of cooperative contact at the helix-helix interface of two neighboring duplex fragments (in the nick). We have experimentally analyzed the series of model tandem complexes of different length at various oligomer concentrations, ionic strength, and pH. The analysis of thermodynamic parameters of complex formation for native and modified oligomers revealed higher Gibbs free energy values of hybridization and cooperative interaction of morpholine-containing complexes at the helix-helix interface under standard conditions (1M NaCl, pH7.2). Further comparative analysis of the hybridization properties of modified oligomers at ionic strength and pH allows us to determine the charge state of the morpholine backbone and the thermodynamic origin of the effects observed. It was found that the decrease in pH to 5.5 led to the protonation of internal morpholine nitrogens. The obtained results prove the veracity of the proposed model and the possibility to evaluate thermodynamic parameters of short native and modified oligomers with high accuracy.