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The preparation of two complete series of RNA nucleoside phosphoramidities, each having an appropriately protected amine or thiol functional group is presented. The new phosphoramidites would be particularly valuable for enabling site-specific introduction of biophysical probes and constraints into RNA.

In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2', 3E-cyclic phosphate and a 5'-hydroxyl group. In addition to providing interesting new examples of nucleic acid catalysts, deoxyribozymes that ligate RNA are anticipated to be a valuable practical complement to protein-mediated splint ligation from preparation of site-specifically modified RNAs.

Research has been conducted on the GAAA tetraloop-receptor motif of the intron RNA P4-P6 domain. Results indicate the existance of the differences between the thermodynamic and kinetic roles of this motif in Mg (super)2+ -dependent folding of P4-P6 tetraloop.

Research has been conducted on the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA. The study of this domain has been conducted via stopped-flow fluorescence.

RNA tertiary folding has been monitored by fluorescence of covalently and site-specifically attached pyrene, made possible by availability of 2'-amino-2'-deoxynucleoside phosphoramidites. This kind of internal labeling gives a much wider array of possible derivatization sites vs only one 5'-end and one 3'-end per molecule. Pyrene was chosen because the fluorescence intensity depends especially strongly it it on environment when attached to nucleic acids. A single site-specifically tethered chromophore can report formation of global structure of a large RNA molecule and make it possible to monitor the equilibrium progress and the real-time kinetics of RNA tertiary folding.

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier [K.sup.+] channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by [G.sub.[Beta][Gamma]] subunits, but instead shows constitutive activation, and (ii) is no longer a [K.sup.+]-selective channel but conducts [Na.sup.+] as well. The present experiments on weaverGIRK2 (wv-GIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular [Na.sup.+] concentration. In particular, manipulations that decrease intracellular [Na.sup.+] produce a component of [Na.sup.+]-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of [Na.sup.+] influx through the wvGIRK2 channel, leading to additional [Na.sup.+] activation. We also show that the wvGIRK2 channel is permeable to [Ca.sup.2+], providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known [K.sup.+] channels.

Ab initio computations on pyridine and pyridinium analogues of m-xylylene show that the neutral heterocycle is essentially similar to benzene as a ferromagnetic coupling unit, while the cationic pyridiniums act differently. A protonated pyridine functions either as a ferromagnetic coupling unit or an antiferromagnetic coupling unit, depending on the substitution pattern. Bis(trimethylenemethane) analogues of the pyridine and pyridinium biradicals are studied by electron paramagnetic resonance spectroscopy to test the theoretical predictions.

The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the α subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding., In the study of membrane-bound receptor, channel, and transporter proteins, classical pharmacology has defined highly specific agonists and antagonists, and quantitative structure-activity studies have generated many hypotheses concerning ligand-receptor interactions. [...]

Many different but closely related perceptual processes all contribute to human visual perception. Many of these processes change slowly as a result of the normal aging process; some perceptual processes are also impaired by dementing illnesses such as Alzheimer's disease. A study was conducted to evaluate the changes in motion perception that occur with normal aging and to compare these changes with any change which might be present in patients with Alzheimer's disease. The perception of motion was evaluated using dots on a computer screen which moved under program control. Ninety-five normal volunteers, ranging in age from 25 to 80, were tested. Also tested were 20 patients with Alzheimer's disease who ranged in age from 62 to 82. It was found that motion thresholds increase with normal aging. That is, the smallest amount of motion which is visually detectable increases with age, and, in those 70 to 80, this threshold is about twice as large as for subjects under 30. Ophthalmological examinations were used to confirm that the observed differences in motion perception were not due to changes in the eyes themselves, but rather to differences in the processing of motion information within the visual perceptual systems. When the results from the Alzheimer's patients were compared with the healthy volunteers of comparable age, a significant deficit was found among the Alzheimer's disease patients. Furthermore, the deficit in motion detection was correlated with the severity of dementia. Motion detection threshold testing may prove useful in the evaluation of patients suspected of having Alzheimer's disease in its earliest stages. (Consumer Summary produced by Reliance Medical Information, Inc.)

The article explains the optimization of 10MD5, a DNA-hydrolyzing deoyribozyme, achieved by the compromises taking place among pH tolerance, site specificity and sequence tolerance. The compound is shown to be highly sequence, as well as site-specific.

An ensemble and single-molecule fluorescence resonance energy transfer (FRET) study was performed on three orthogonally labeled variants of the xpt guanine riboswitch aptamer domain. The data obtained elucidated the biological role of guanine as stabilizing factor for the globally folded aptamer domain conformation at physiologically relevant [Mg.sup.2+] concentrations ([less than]1 mM), while much higher [Mg.sup.2+] concentrations are required to induce aptamer folding event in absence of guanine.

An energetically substantial stabilization of the RNA tertiary structure is obtained by the strategic attachment of a DNA constraint that has selectively stabilized the folded conformation of the RNA. The results have shown that significant stabilizations are seen when various attachment sites are used on the P4-P6 RNA and energetically nontrivial RNA stabilization is introduced without needing extensive contacts to the RNA itself.

In vitro selection is used for identifying new deoxyribozymes that has catalyzed the Diels-Alder reaction between anthracene and maleimide substrates. The comparison of both rate constants and rate enhancements has shown that DAB22 has catalyzed the Diels-Alder reaction as efficiently as 39M49, thus showing that deoxyribozymes are as efficient as the ribozymes for catalysis of C-C bond formation.

The synthesis of a thymidine phosphoramidite that has the 5'-tethered aldehyde masked as a 1,2-diol and a convenient protocol developed for the oxidative cleavage of the 1,2-diol to the aldehyde using NaIO4 is described. Both the procedures would find applicability in attaching DNA to biomolecules.

The role of the leaving group in the 7S11-catalyzed reaction by altering the 5'-phosphorylation state and the length of the second RNA substrate is comprehensively examined. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide playing the role of the 5'-exon in the first step.

A brief report on Zn(super 2 +)-dependent deoxyribozymes that ligate RNA is presented. The DNA enzymes are identified by in vitro selection and ligate RNA at 1 mM Zn(super 2 +) and 23 Celsius, pH 7.9 that is faster.

The 7S11 DNA enzyme mediates the nucleophilic attack of an adenosine 2'-hydroxyl group at a 5'-triphosphate, forming 2',5'-branched RNA in a reaction that resembles the first step of in vivo RNA splicing. The 7S11 characterization experiments that have two important implications for nucleic acid chemistry and biochemistry are described.

The results related to natural selection, in which abrupt environmental variation can provide a rapid change in selection pressure. The new approach to obtain 3'-5' selective RNA ligase deoxyribozymes is particularly important for ongoing selection efforts. Linear 3'-5' RNA linkages are an important practical objective because the native backbone is desirable in site-specifically modified ribozymes assembled by ligation.

A study is conducted to obtain deoxyribozymes that instead create linear 3'-5'-linked RNA, a strategically modified the selection approach is proposed by embedding the nascent ligation junction within an RNA:DNA duplex region. These RNA ligase deoxyribozymes are the first that create native 3'-5' RNA linkages, which are highly elusive via other selection approaches.

Characterization of DNA enzymes with respect to their branch forming activity is discussed on the basis the type of branched RNA synthesized by it. The results support the utility of deoxyribozymes in creating synthetic 2', 5' branched RNAs for investigation of group II intron splicing and other biochemical reactions.

Branched and lariat RNA can be synthesized by deoxyribozymes that have significant potential for convenient and practical generalization is demonstrated. Efforts are being made to expand the scope of branched and lariat RNA formation by the new deoxyribozymes, including variation of loop size and composition for the lariats.

The evolution of deoxyribozymes with RNA ligase activity is reported. The deoxyribozymes that emerged from the process ligate RNA with highly selective formation of 2'-5' phosphodiester linkages.