Intrinsic contribution of the 2'-hydroxyl to RNA conformational heterogeneity

Canonical duplex RNA assumes only the A-form conformation at the secondary structure level while, in contrast, a wide range of noncanonical, tertiary conformations of RNA occur. Here, we show how the 2'-hydroxyl controls RNA conformational properties. Quantum mechanical calculations reveal that the orientation of the 2'-hydroxyl significantly alters the intrinsic flexibility of the phosphodiester backbone, favoring the A-form in duplex RNA when it is in the base orientation and facilitating sampling of a wide range of noncanonical, tertiary structures when it is in the O3' orientation. Influencing the orientation of the 2'-hydroxyl are interactions with the environment, as evidenced by crystallographic survey data, indicating the 2'-hydroxyl to sample more of the O3' orientation in noncanonical RNA structures. These results indicate that the 2'-hydroxyl acts as a "switch", both limiting the conformation of RNA to the A-form at the secondary structure level and allowing RNA to sample a wide range of noncanonical tertiary conformations.     

8-溴鸟苷探针测定生物样品中蛋白质含量的光谱法研究

研究了8-溴鸟苷(BG)与人血清白蛋白(HSA)体系的荧光猝灭光谱、三维荧光光谱,证明了两者可以发生相互作用,导致人血清白蛋白疏水微环境极性的改变和构象的变化;还分析了△λ值、pH值、离子强度等因素对该体系同步荧光强度的影响,选定了8-溴鸟苷测定生物样品中蛋白质含量的最佳实验条件;在最佳实验条件下,体系的同步荧光强度与...     

DBU induced formation of 8-bromoguanosine dimer with three hydrogen bonds between the GG− base pairs

Upon hemideprotonation of 8-bromoguanosine (8-BrG) at the N1 position, induced by DBU, the adduct [8-BrG][8-BrG]^?[DBU–H]⁺ was formed. Slow evaporation of the 8-BrG methanol solution, in the presence of 0.5 equiv of DBU, yielded two polymorphic structures (1 and 2), where a neutral [8-BrG] (A) and N1 deprotonated, anionic 8-bromoguanosine [8-BrG]^? (B) were joined together through three intermolecular hydrogen bonds involving O6, N1 and C2–NH₂ sites. Such pairing gave planar GG^? dimers as the basic motif of crystal packing in both polymorphs. Both neutral and deprotonated guanosine molecules in the structure of 1 had the ribose units in a syn conformation. In the structure of polymorph 2, the N1 deprotonated guanosine molecule (B) retained the syn glycosidic conformation, while the non-deprotonated guanosine molecule (A) adopted the natural anti conformation of the ribose unit with respect to the nucleobase. Ribose rings revealed different puckering; only those of deprotonated molecules 1B and 2B possessed the usual C2′-endo envelope conformation. Crystal packing in both structures was guided by the highly complex H-bonded pattern. The CSD was searched for related structures, which are discussed with reference to polymorphs 1 and 2. ¹H and ¹³C NMR spectroscopic evidence is provided showing that the three H-bonded adduct [8-BrG][8-BrG]^?[DBU–H]⁺ was also formed in the highly H-bond competitive DMSO solution.     

Assessment of conformational ensemble sizes necessary for specific resolutions of coverage of conformational space

The size of conformational ensembles required for regular coverage of the conformational space of druglike molecules was examined. Using the conformer generation program Omega, the number of regularly distributed conformers (NRC) of flexible compounds was determined as a function of the root-mean-square deviation (RMSD) resolution of coverage. A regression equation was developed predicting the NRC of a molecule as a function of RMSD. The model yielded R(2) of 0.91 for both training and test sets, which consisted of 3414 and 3352 compounds, respectively. Utilizing 14 504 ligands from the Protein Data Bank with experimentally determined 3-D conformations, the regression equation was applied to the estimation of the NRC and the success rate of reproduction of experimental conformations from a theoretical conformation ensemble as a function of RMSD and flexibility was explored.     

Oligonucleotide incorporation of 8-thio-2'-deoxyguanosine

[reaction: see text] 8-Thio-2'-deoxyguanosine (SdG) is a useful analogue of the abundant promutagen 8-oxo-2'-deoxyguanosine (OdG). Its synthesis and DNA incorporation using standard phosphoramidite chemistry is reported. To prevent oxidation during DNA synthesis, the sulfur was protected as a 2-(trimethylsilyl)ethyl sulfide. Subsequent treatment with TBAF yielded the desired 8-thiocarbonyl functionality. Melting studies with SdG revealed almost equal stabilities of SdG:dC and SdG:dA base pairs, lending insight into the base-pairing preferences of OdG.     

Inhibition of sulfur incorporation to transfer RNA by ultraviolet-A radiation in Escherichia coli

tRNA sulfurtransferase activity was assayed in Escherichia coli cell extracts obtained from bacterial suspensions exposed to a sub-lethal dose of ultraviolet-A radiation (fluence 148 kJ m(-2)) imparted at a low fluence rate (41 W m(-2)). We found that the irradiation reduced the enzymatic activity to one fourth of the control value, indicating that ultraviolet-A exposure inhibits the synthesis of 4-thiouridine, the most abundant thionucleoside in E. coli tRNA. Changes in the tRNA content of 4-thiouridine and its derived photoproduct 5-(4'-pyrimidin 2'-one) cytosine were studied in bacteria growing under ultraviolet-A irradiation. In these conditions the accumulation of photoproduct was limited, and the kinetics of this process was non-coincident with disappearance of 4-thiouridine. The results, which are compatible with the fact that ultraviolet-A induces an inhibition of the 4-thiouridine synthesis, suggest that the effect of radiation on tRNA modification is relevant to tRNA photo-inactivation in growing bacteria.     

Highly sequence specific RNA terminal labeling by DNA photoligation

We report the nonenzymatic terminal labeling of oligoribonucleotide (ORN) by using template-directed photoligation through 5-carboxyvinyl-2'-deoxyuridine ((CV)U) with high selectivity.     

An efficient thermally induced RNA conformational switch as a framework for the functionalization of RNA nanostructures

RNA offers a variety of interactions and dynamic conformational switches not available with DNA that may be exploited for the construction of nanomolecular structures. Here, we show how the RNA loop-loop, or "kissing", interaction can be used to construct specific circular RNA arrangements that are capable of thermal isomerization to alternative structures. We also show how this thermally induced structural rearrangement can be used to unmask a functional RNA structure, in this case, a peptide-binding RNA structure, the Rev-response element (RRE) of HIV, thereby acting as a functional peptide-binding switch. The relative ease with which the RRE could be engineered into the RNA substrates suggested that a variety of functional RNA structures may be introduced. In addition, the structural rearrangement was extremely efficient, showing that the "kissing" complexes described in this study may provide a useful framework for the construction of functional RNA-based nanostructures, as well as aid in our understanding of the way RNA functions in biological systems.     

Evidence for the induction of a conformational change of trypsin by a specific substrate at pH 8.0

A method is described that permits the covalent attachment of enzymes to solid matrices in the presence of saturating levels of a specific substrate under conditions where the enzyme is optimally active. Bovine trypsin immobilized on glass and agarose in the presence of saturating levels of N-α-benzoyl-L-arginine ethyl ester differs in a number of respects from the enzyme bound in the absence of substrate. Trypsin bound in the presence of substrate (ES form) is attached to porous glass through three more side chains than the enzyme coupled in the absence of substrate (E form). The rate of inactivation by iodoacetamide for the ES form is much greater than that for the E form. The two enzyme forms differ also in their reactivity with TLCK and DFP, active site-directed inhibitors. The pH dependences of the ES and the E forms bound to agarose differ; the pH optima are 9.0 and 8.5 for the ES and E forms, respectively. The thermal denaturation of the agarose-trypsins occurs in two distinct steps. The inactivation rate of the ES form is significantly slower than the E form in both stages. Also, for the hydrolysis of both N-α-benzoyl-L-arginine ethyl ester and N-α-benzoyl-Larginine amide, the K_m values for the ES form were lower than the K_m values for the E form. Our data suggest that a substrate-induced conformatonal change occurs with trypsin, and that this conformation is stabilized when the enzyme is bound by multiple attachments to a solid support. These findings support the “induced-fit” theory in general and lend support to the interpretation of the activation of trypsin by methyl guanidine that is part of a specific substrate (Inagami, T., and Hatano, H. [1969] J. Biol. Chem. 244, 1176).     

Probing conformational variations at the ATPase site of the RNA helicase DbpA by high-field electron-nuclear double resonance spectroscopy

The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein's ATPase site in solution. Specifically, we substituted the essential Mg(2+) cofactor in the ATPase active site for paramagnetic Mn(2+) and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn(2+) interactions with the nucleotide phosphates through the (31)P hyperfine couplings and the coordination by protein residues through (13)C hyperfine coupling from (13)C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the (13)C and the (31)P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn(2+) ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn(2+) cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the protein's ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn(2+) ion. Finally, a competitive Mn(2+) binding site was found for single-stranded RNA construct.     

Stereochemistry of oligomers in stereospecific and conformational specific ionic polymerizations

Low molecular weight oligomers of methyl methacrylate (MMA) and perhaloacetaldehyde were studied as to their composition, end groups and stereochemistry. The oligomerization of methyl methacrylate can be directed toward almost pure isotactic or toward almost syndiotactic oligomers depending upon the intitiating system used. Perhaloacetaldehydes showed a stereospecificity which depended upon the size and bulkiness of the side group in the polymer chain. There is no dependence of the sterospecificity based on the initiating system and only isotactic polymers could be obtained which are not only stereospecific but also conformationally specific giving conformational specific isotactic 4/1 helices.     

Synthesis of 8-bromo-, 8-methyl- and 8-phenyl-dATP and their polymerase incorporation into DNA

dATP derivatives bearing Br, Me or Ph groups in position 8 were prepared and tested as substrates for DNA polymerases to show that 8-Br-dATP and 8-Me-dATP were efficiently incorporated, while 8-Ph-dATP was a poor substrate due to its bulky Ph group.