Biomedical subjects

E LeProust

Publications and source records attributed to E LeProust.

4 recordsLinked to original sources

PubMed2001

A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids.

Oligonucleotide microarrays or oDNA chips are effective decoding and analytical tools for genomic sequences and are useful for a broad range of applications. Therefore, it is desirable to have synthesis methods of DNA chips that are highly flexible in sequence design and provide high quality and general adoptability. We report herein, DNA microarray synthesis based on a flexible biochip method. Our method simply uses photogenerated acid (PGA) in solution to trigger deprotection of the 5'-OH group in conventional nucleotide phosphoramidite monomers (i.e. PGA-gated deprotection), with the rest of the reactions in the synthesis cycle the same as those used for routine synthesis of oligonucleotides. The complete DNA chip synthesis process is accomplished on a regular DNA synthesizer that is coupled with a UV-VIS projection display unit for performing digital photolithography. Using this method, oDNA chips containing probes of newly discovered genes can be quickly and easily synthesized at high yields in a conventional laboratory setting. Furthermore, the PGA-gated chemistry should be applicable to microarray syntheses of a variety of combinatorial molecules, such as peptides and organic molecules.

DNA↗

PubMed2001

Characterization of oligodeoxyribonucleotide synthesis on glass plates.

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5'-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using (32)P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5'-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.

Electrophoresis, Polyacrylamide Gel↗

PubMed1997

An efficient and economic site-specific deuteration strategy for NMR studies of homologous oligonucleotide repeat sequences.

We describe herein the use of a 2H-labeling strategy to achieve specific assignments of considerably overlapped cross peaks in the 1H-NMR spectrum of a DNA trinucleotide repeat sequence. Our strategy focuses on site-specific 2H-labeling of base moieties to simplify the NMR spectral regions which contain the major portion of the structural information. To achieve efficient preparation of 2H8- or 2H6-labeled DNA and RNA nucleosides and nucleotides, the existing synthetic and purification procedures were significantly improved. Our experiments demonstrate that pyrimidine H6 deuteration reactions may be carried out using non-deuterated base reagents with DMSO-d6 as a 2H donor. These reactions are simple and economic to perform and produce base deuterated nucleosides and nucleotides in high yield. The 2H-labeled residues have been incorporated into oligonucleotides with minor modifications of the existing reaction conditions. Using the homologous CGG repeat sequence, d(CGG)5, as an example, the effectiveness of the site-specific base deuteration strategy is demonstrated. In the otherwise extensively overlapped spectra of d(CGG)5, 2H-labeling has permitted unambiguous identification of a sequential connectivity at a central CG step and confirmation of several other NOE assignments. This information is critical for elucidation of the structure and the folding of the CGG repeat sequences and will contribute to the intensive effort to understand the mechanisms of triplet expansion, which has been implicated in the development of a number of hereditary neurodegenerative diseases. In addition to the two dimensional spectral simplification in a key spectral region using site-specific 2H8/2H6-labeling, the potential applications of the prescribed strategy in homonuclear three dimensional experiments are also discussed.

DNA↗

PubMedDate not supplied

Digital light-directed synthesis. A microarray platform that permits rapid reaction optimization on a combinatorial basis.

Solution reactions using photogenerated reagents (Gao, X.; Yu, P.; LeProust, E.; Sonigo, L.; Pellois, J. P.; Zhang, H. J. Am. Chem. Soc. 1998, 120, 12698) are a potentially powerful means for combinatorial parallel synthesis of addressable molecular microarrays. In this report, we demonstrate that this chemistry permits combinatorial screening of reaction conditions on a microarray platform. Using this method of optimization and our reaction apparatus, efficient photogenerated acids and reaction conditions suitable for removal of the acid labile protection group on 5'-O of nucleotides are identified in a short period of time. The chemistry platform demonstrated opens new avenues for rapid, simultaneous investigation of multiple reactions using different reagents and reaction parameters directly on a solid support (e.g., a glass plate). The combinatorial screening method described may be extended to include general organic reactions employing photogenerated and conventional reagents as well as a microarray reaction device. This should be especially valuable for efficient synthesis of addressable organic compound libraries.

Chemistry, Organic↗