Arrays of immobilized oligonucleotides--contributions to nucleic acids technology.

The interactions of nucleic acids technology and the technology of arrayed nucleic acids are described, showing the interdependence of nucleic acids chemistry, surface chemistry, (micro-) technology and the requirements of bio-medical applications. The methods and problems of the production of large numbers of oligonucleotides as well as the methods of arraying oligonucleotides are highlighted. The basic approaches, in-situ synthesis and postsynthetic immobilization, are described with a special emphasis on the postsynthetic immobilization of ready-made oligonucleotides on support materials. Techniques for the detection of nucleic acids interactions on arrays are outlined in brief.

Pressure ulcers: collaboration in wound care. Is there a reasonable approach?

Pressure ulcers represent a significant impact on utilization of healthcare beds, cost for the insurer, and adverse emotional impact for the family. With this in mind, one must address in an effective fashion a method of objective assessment using the current methodology to deal with risk factors in mobility, disease states, and nutrition, as well as give significant attention to prevention protocols. One should adhere to a schedule of turning the patient, sleep surfaces, and appropriate skin care while recognizing the impact of each. Finally, treatment modalities should be undertaken, not only with cost-effective issues in mind, but with ease and convenience for the healthcare provider and the least discomfort for the patient as well. One can focus on debridement by way of enzymatic, autolytic, or surgical methods, recognizing surgery as the least effective for our goals in the management of this patient. Assessment and, finally, reassessment using the available scales will allow us to provide effective healthcare in a therapeutic fashion.

Refinement of the solution structure of the RNA-DNA hybrid 5'-[r(GCA)d(TGC)]2. Combined use of nuclear magnetic resonance and restrained molecular dynamics.

The solution conformation of the self-complementary RNA-DNA hybrid hexamer 5'-[r(GCA)d(TGC)]2 is investigated by NMR spectroscopy and restrained molecular dynamics. The 1H-NMR spectrum is assigned in a sequential manner using two-dimensional homonuclear Hartmann-Hahn and nuclear Overhauser enhancement spectroscopy. From the latter a set of 178 approximate interproton distance restraints are determined and used as the basis of a structure refinement by restrained molecular dynamics. Eight independent calculations are carried out, four from a classical A-type geometry and four from a classical B-type one. Convergence is achieved to very similar A-type structures with an average atomic root mean square difference between them of 1.0 +/- 0.2 A. The converged structures exhibit variations in helical parameters similar to those found previously for the analogue RNA hexamer 5'-r(GCAUGC)2 [(1988) Biochemistry 27, 1735-1743].

Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics.

The solution structure of the self-complementary dodecamer 5'd(CGCGPATTCGCG)2, containing a purine-thymine base pair within the hexameric canonical recognition site GAATTC for the restriction endonuclease EcoRI, is investigated by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Nonexchangeable and exchangeable protons are assigned in a sequential manner. A set of 228 approximate interproton distance restraints are derived from two-dimensional nuclear Overhauser enhancement spectra recorded at short mixing times. These distances are used as the basis for refinement using restrained molecular dynamics in which the interproton distance restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight calculations are carried out, four starting from classical A-DNA and four from classical B-DNA. In all cases convergence to very similar B-type structures is achieved with an average atomic root mean square (rms) difference between the eight converged structures of 0.7 +/- 0.2 A, compared to a value of 6.5 A for that between the two starting structures. It is shown that the introduction of the purine-thymine mismatch does not result in any significant distortion of the structure. The variations in the helical parameters display a clear sequence dependence. The variation in helix twist and propeller twist follows Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. Overall the structure is straight. Closer examination, however, reveals that the central 5 base pair steps describe a smooth bend directed toward the major groove with a radius of curvature of approximately 38 A, which is compensated by two smaller kinks in the direction of the minor groove at base pair steps 3 and 9. These features can be explained in terms of the observed variation in roll and slide.

Refinement of the solution structure of the ribonucleotide 5'r(GCAUGC)2: combined use of nuclear magnetic resonance and restrained molecular dynamics.

The solution structure of the self-complementary hexamer 5'r(GCAUGC)2 is investigated by means of nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton resonances are assigned in a sequential manner, and a set of 110 approximate interproton distance restraints are derived from the two-dimensional nuclear Overhauser enhancement spectra. These distances are used as the basis of a structure refinement by restrained molecular dynamics in which the experimental restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight restrained molecular dynamics simulations are carried out, four starting from a structure with regular A-type geometry and four from one with regular B-type geometry. The atomic root mean square (rms) difference between the initial structures is 3.2 A. In the case of all eight simulations, convergence is achieved both globally and locally to a set of very similar A-type structures with an average atomic rms difference between them of 0.8 +/- 0.2 A. Further, the atomic rms differences between the restrained dynamics structures obtained by starting out from the same initial structures but with different random number seeds for the assignment of the initial velocities are the same as those between the restrained dynamics structures starting out from the two different initial structures. These results suggest that the restrained dynamics structures represent good approximations of the solution structure. The converged structures exhibit clear sequence-dependent variation in some of the helical parameters, in particular helix twist, roll, slide, and propellor twist.(ABSTRACT TRUNCATED AT 250 WORDS)

2'(3')-O-glycyl oligoribonucleotides with sequences of the 3'-terminus of glycyl-tRNA: chemical synthesis and properties in partial reactions of protein biosynthesis.

Specific syntheses of 2'(3')-O-aminoacyl oligoribonucleotides C-C-A-Gly (12), C-C-A(AcGly) (7), U-C-C-A-Gly (17), and C-U-C-C-A-Gly (19), which are the 3'-terminal sequences of Escherichia coli Gly-tRNA (or AcGly-tRNA, respectively) are described. Compounds 12, 17, and 19 were synthesized by employing the benzotriazolyl phosphotriester approach with the following protection groups on the components: benzoyl for the heterocyclic amino groups, 2-chlorophenyl group for internucleotide phosphate protection, dimethoxytrityl and levulinoyl groups for blocking of the 5'-hydroxyl, methoxytetrahydropyranyl group for protection of the 2'-hydroxyl functions, and N-(benzyloxycarbonyl)orthoglycinate as the masked aminoacyl group simultaneously protecting the 2',3'-cis diol group of the 3'-terminal adenosine moiety. The fully protected tri-, tetra-, and pentanucleotides were obtained via 5'-extension of di- and trinucleotide blocks after prior selective removal of the 5'-O-levulinoyl group with hydrazine. The blocked derivatives 11, 16, and 18 were totally deprotected by reactions with NH4OH, H+, and H2/Pd to yield the target compounds 12, 17, and 19 in good yields. C-C-A(AcGly) (7) was synthesized according to a stepwise procedure via activation of preformed diesters with (mesitylenesulfonyl)tetrazole. C-C-A-Gly (12), U-C-C-A-Gly (17), and C-U-C-C-A-Gly (19) were all acceptor substrates in the peptidyltransferase reaction with the Ac-Phe-tRNA-70S ribosome-poly(U) system. All three models also promoted EF-Tu-70S ribosome GTP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

New methodology for the synthesis of 2'(3')-O-aminoacyl oligoribonucleotides related to the 3'-terminus of aa-tRNA.

A new synthetic approach to the title compounds is reported. It is based on the phosphotriester methodology and uses a unique combination of protecting groups (Fmoc and Ph for the aglycons; DMT, CPTr and Mthp for the carbohydrate hydroxy functions; 2-CIPh for the phosphodiester bonds; BPOC for amino acid). This enables selective deprotection of the blocked oligonucleotide intermediates in two steps (oximate and acid treatments) to yield the 2'(3')-O-aminoacyl oligoribonucleotides suitable for biochemical investigations.

Chemical synthesis of r(GCAUGC) for high-resolution 1H-NMR studies.

The chemical synthesis of a short RNA duplex, to be used for 500 MHz 1H-NMR experiments, is presented. The target oligonucleotide was constructed in a blockwise fashion according to the benzotriazolyl phosphotriester approach in solution. The CPTr group, 4,4',4"-tris(4,5-dichlorophthalimido)trityl, was used for temporary protection of the 5'-OH; this blocking group was removed by buffered hydrazine without harming any other protecting group.