Synthesis, Purification and Crystallization of Guanine-rich RNA Oligonucleotides

Guanine-rich RNA oligonucleotides display many novel structural motifs in recent crystal structures. Here we describe the procedures of the chemical synthesis and the purification of such RNA molecules that are suitable for X-ray crystallographic studies. Modifications of the previous purification methods allow us to obtain better yields in shorter time. We also provide 24 screening conditions that are very effective in crystallization of the guanine-rich RNA oligonucleotides. Optimal crystallization conditions are usually achieved by adjustment of the concentration of the metal ions and pH of the buffer. Crystals obtained by this method usually diffract to high resolution

Kinetics and mechanism of G-quadruplex formation and conformational switch in a G-quadruplex of PS2.M induced by Pb2+

DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV–Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG3TAG3CG3TTG2). UV-melting curves demonstrate that the Pb2+-induced G-quadruplex formed unimolecularly and the highest melting temperature (Tm) is 72°C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb2+ ions to PS2.M is two, suggesting that the Pb2+ ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb2+-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO3)2 to the Na+- or K+-induced G-quadruplexes, which may be attributed to the replacement of Na+ or K+ by Pb2+ ions and the generation of a more compact Pb2+–PS2.M structure. Comparison of the relaxation times shows that the Na+→Pb2+ exchange is more facile than the K+→Pb2+ exchange process, and the mechanisms for these processes are proposed

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays

The role of the preserved sequences of Dam methylase.

We have undertaken a site directed mutational analysis of two of the preserved regions in the amino acid sequence of Dam methylase in order to characterize their role. Mutations in region IV (sequence DPPY) abolish catalytic activity and greatly affect AdoMet crosslinking. Mutants in region III display a lowered specific activity with an unchanged AdoMet crosslinking capacity. We have also made a series of deletions both at the N and C terminal parts of the protein, which have been found to provide inactive enzyme. We discuss the significance of these results for the understanding of the functional properties of the enzyme

Essential tremor and cerebellar dysfunction: abnormal ballistic movements

Background: Clinical characteristics reminiscent of cerebellar tremor occur in patients with advanced essential tremor. Ballistic movements are known to be abnormal in cerebellar disease. The hypothesis was proposed that ballistic movements are abnormal in essential tremor, reflecting cerebellar dysfunction. Objective: To elucidate the role of the cerebellum in the pathophysiology of essential tremor. Methods: Kinematic parameters and the triphasic electromyographic (EMG) components of ballistic flexion elbow movements were analysed in patients assigned to the following groups: healthy controls (n = 14), pure essential postural tremor (ET(PT); n = 17), and essential tremor with an additional intention tremor component (ET(IT); n = 15). Results: The main findings were a delayed second agonist burst (AG(2)) and a relatively shortened deceleration phase compared with acceleration in both the essential tremor groups. These abnormalities were most pronounced in the ET(IT) group, which had additional prolongation of the first agonist burst (AG(1)) and a delayed antagonist burst (ANT). Conclusions: Abnormalities of the triphasic pattern and kinematic parameters are consistent with a disturbed cerebellar timing function in essential tremor. These abnormalities were most pronounced in the ET(IT) group. The cerebellar dysfunction in essential tremor could indicate a basic pathophysiological mechanism underlying this disorder. ET(PT) and ET(IT) may represent two expressions within a continuous spectrum of cerebellar dysfunction in relation to the timing of muscle activation during voluntary movements