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Concept

Synthèse d'oligonucléotide

La synthèse d'oligonucléotide est la synthèse chimique de fragments relativement courts d'acide nucléique avec une structure définie. Principe La technique est largement utilisée dans les laboratoires. Elle permet d'obtenir un accès inédit ou peu couteux à des oligonucléotides avec la séquence de nucléotides désirée. Le procédé utilise comme building block des nucléosides de type désoxyadénosine (dA), la thymidine (T), la désoxycytidine (dC) et la désoxyguanosine (dG) pour l'ADN et de type adénosine (A), la thymidine (T), la cytidine (C) et la guanosine (G) pour l'ARN sous forme de phosphoramidite. La technique a pour point de départ un support solide sur lequel est greffé le premier nucléotide. Une fois la synthèse terminée, l'oligonucléotide va être séparé du support solide par un clivage chimique. Pour obtenir l'oligonucléotide désiré, la synthèse s'effectue selon plusieurs cycles de synthèse. À chaque cycle, un nucléotide est incorporé sur la chaine oligonucléotidique

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Novel irreversible covalent BTK inhibitors discovered using DNA-encoded chemistry

Sevan Mleh Habeshian, Ying Zhang

Libraries of DNA-Encoded small molecules created using combinatorial chemistry and synthetic oligonucleotides are being applied to drug discovery projects across the pharmaceutical industry. The majority of reported projects describe the discovery of reversible, i.e. non-covalent, target modulators. We synthesized multiple DNAencoded chemical libraries terminated in electrophiles and then used them to discover covalent irreversible inhibitors and report the successful discovery of acrylamide- and epoxide-terminated Bruton's Tyrosine Kinase (BTK) inhibitors. We also demonstrate their selectivity, potency and covalent cysteine engagement using a range of techniques including X-ray crystallography, thermal transition shift assay, reporter displacement assay and intact protein complex mass spectrometry. The epoxide BTK inhibitors described here are the first ever reported to utilize this electrophile for this target.

PERGAMON-ELSEVIER SCIENCE LTD2021

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SV40 activates transcription from the transferrin receptor promoter by inducing a factor which binds to the CRE/AP-1 recognition sequence

Peter Martin Beard, Nicole Paduwat

During the course of lytic infection by simian virus 40 (SV40), expression of both the viral late genes and certain host cellular genes is induced. The promoter of the cellular transferrin receptor (TR) gene contains a DNA sequence which is similar to the AP-1- and AP-4-binding region in SV40 which has been implicated in the control of the viral late promoter. Expression of TR is needed for cells to enter S-phase and is therefore expected to be important for the SV40 lytic cycle. Here we show that the level of TR mRNA in vivo was increased by SV40 infection. A factor which activates transcription from the TR promoter in vitro was specifically induced in SV40-infected cells. Gel mobility shift assays with an oligonucleotide comprising this part of the TR promoter showed three nucleoprotein complexes to be formed with proteins from CV-1 cells. Following SV40 infection, one of the complexes was increased ten-fold. Formation of this complex was specifically reduced by competition with the phorbol ester-responsive element of the collagenase gene, implying that the factor is a member of the AP-1/Jun/Fos family. Cross-linking of the complex by ultraviolet light showed major DNA-binding components to be proteins of about 55 kD and 47 kD. Removal of this factor by adding the oligonucleotide to in vitro transcription reactions with the TR promoter, abolished the activation of TR transcription. The factor which binds to the TR promoter co-sedimented with SV40 chromosomes extracted late in infection. This suggests that similar transcriptional regulatory proteins are involved in controlling transcription from both the SV40 and the TR promoters, and that the virus can use a common mechanism to induce viral and host cellular transcription.

1991

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Optimized methods for the enzymatic ligation of oligoribonucleotides and analogues

Frédéric David Meylan

The preparation of oligoribonucleotides is of great interest for structural and functional investigations in biomolecular chemistry and biology. Relevant RNAs are relatively long and contain often modifications. Two methods for their preparation are currently available: automated chemical synthesis and in vitro transcription. The chemical synthesis allows the preparation of relatively short (< 60mers) pure RNA sequences containing modified nucleotides. In contrast, in vitro transcription is appropriate for the synthesis of long RNA sequences but does not allow the incorporation of modifications. An elegant solution to this dilemma is offered by enzymatic ligations of RNA fragments with T4-DNA ligase, which is template-dependent and therefore highly selective. Here, we present a comprehensive study of parameters, which govern the effectiveness of this ligation. The influence of secondary structures present within templates were studied in detail. That allowed to design a new type of hybrid template, containing both DNA and 2'-OMeRNA nucleotides, which were particularly efficient (Chapter 3.2). Ligation reactions with structured substrates and different types of oligonucleotides were carried out, leading to a better comprehension of the enzyme mechanism. In this context, we found very strong indications for a different recognition of the donor and acceptor substrates by the enzyme (Chapter 3.2 and 3.3). Optimization of the enzymatic ligations with different substrates or templates has showed the importance of secondary structures for design of a successful reaction (Chapter 3.2 and 3.3). The lessons drawn from these preliminary experiments have been employed for the reliable preparative synthesis of natural oligoribonucleotides containing modifications (Chapter 3.4) and the preparation of long RNA sequences (Chapter 3.5).

EPFL2006

Chargement

Oligonucléotide

Les oligonucléotides sont de courts segments de chaines d'acides nucléiques (ARN ou ADN) de quelques dizaines de nucléotides. Ils sont en général obtenus par synthèse chimique, sous forme de simple b

Analogue d'acide nucléique

Les analogues d'acides nucléiques sont des composés qui sont structurellement similaires aux ARN et ADN présents dans la nature. Ils ont des utilisations pour la recherche en médecine et en biologi

Acide nucléique peptidique

L’acide nucléique peptidique (ANP), ou Peptide Nucleic Acid (PNA) en anglais, est une molécule aux bases similaires à l'ADN ou à l'ARN mais qui se différencie par son squelette (backbone en anglais).

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BIO-471: Cancer biology I

The course covers in detail molecular mechanisms of cancer development with emphasis on cell cycle control, genome stability, oncogenes and tumor suppressor genes.

MICRO-560: BioMEMS

This course covers the main applications of micro devices for life science and biomedical applications. The course is organized by application topic. It is also covering the basic physical, biological, chemical, technological concepts, which are presented as transversal introductory section

BIO-205: Cellular and molecular biology I

The course covers the regulation of gene expression, which translates the information contained in the genome into function, by adjusting the levels and activities of mRNAs and proteins to the needs of specific cells, tissues and environments. A particular emphasis is given on experimental methods.