Search

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here, using a candidate-based shRNA screen, we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1-SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells, neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2, for which an FDA-approved small molecule inhibitor, EPZ6438 (also known as tazemetostat), is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately, EZH2 inhibitors do not efficiently cross the blood-brain barrier, limiting their therapeutic use for FXS. Recently, antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons, and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively, our results establish EZH2 inhibition in general, and EZH2 ASOs in particular, as a therapeutic approach for FXS. [ABSTRACT FROM AUTHOR]

The ability to metabolically label DNA in a way that produces a latent change from one nucleobase to another would create asignal that can beamplified by PCR--this in turn would allow studies of newly synthesized DNA using high-throughput sequencing. To function as an amplifiable metabolic label, a nucleotide analogue would need to be taken up by cells and incorporated into cellular DNA; after purification of DNA, it could be converted into a different nucleobase with a different base pairing pattern. We selected 4-thiothymidine (4sT) as a candidate metabolic label: 4sT is readily taken up by a large number of polymerases in vitro, and we present a method that allows 4sT to be converted into 5-methyl-2'-deoxycytidine (5mC) after incorporation into DNA. Encouraged by these results, we treated cells with 4sT nucleoside; however, we found that 4sT is not incorporated into DNA in bacterial, yeast, or mammalian cells to useful levels under the conditions we tested. A phosphorodiamidate prodrug of 4sTMP was successfully synthesized but did not measurably improve incorporation into cellular DNA. Key words: metabolic labelling, convertible nucleobase, nucleoside prodrug, 4-thiothymidine. La capacite de realiser le marquage metabolique de l'ADN de maniere a produire la transformation latente d'une base nucleique en une autre pourrait permettre de generer un signal amplifiable par PCR, ce qui, par consequent, permettrait d'etudier de nouveaux ADN synthetises par sequençage a haut debit. Pour fonctionner comme un marqueur metabolique amplifiable, un analogue de nucleotide doit pouvoir penetrer dans les cellules et s'incorporer dans l'ADN cellulaire, puis apres purification de l'ADN, doit pouvoir etre transforme en une autre base nucleique dont l'appariement differe. Nous avons choisi la 4-thiothymidine (4sT) comme marqueur metabolique potentiel, car elle est facilement integree in vitro par un grand nombre de polymerases. Nous presentons une methode qui permet de transformer la 4sT en 5-methyl-2'-deoxycytidine (5mC) apres incorporation dans l'ADN. Encourages par ces resultats, nous avons traite des cellules avec le nucleoside 4sT. Cependant, nous avons observe que, dans les conditions evaluees, la 4sT n'est pas incorporee dans l'ADN de cellules de bacteries, de levures et de mammiferes dans une mesure suffisante pour etre utile. Nous avons reussi a synthetiser un promedicament de la 4sTMP a base de phosphorodiamidate, mais nous ne sommes pas parvenus a ameliorer de façon mesurable l'incorporation dans l'ADN cellulaire. [Traduit par la Redaction] Mots-cles : marquage metabolique, base nucleique convertible, promedicament a base de nucleoside, 4-thiothymidine., Introduction Metabolic labeling of cellular biopolymers has opened new frontiers in fields from proteomics (1) to RNA biology (2,3) to glycobiology. (4,5) In this family of approaches, cells or organisms [...]

Abstract: The development of arabinonucleosides and oligoarabinonucleotides is described, focusing especially on 2'-deoxy-2'-fluoroarabinonucleosides (araF-N) and -oligonucleotides (2T-ANA). In addition to their chemical and enzymatic synthesis, we discuss various properties of [...]

Six 2-Y-substituted oxacyclohexane and thiacyclohexane heterocycles (Y = F, OMe, NHMe) were examined using DFT molecular orbital calculations. Natural bond orbital (NBO) analysis of the total energy behaviour yielded the orbital-interaction factors contributing to the conformational equilibria. The dipole moments of the optimized systems were used to estimate the electrostatic contributions to the anomeric effect. The primary determinant of the X-C-Y anomeric effect was found to be the orbital interaction components associated with the combined endo- and exo-anomeric effects acting in concert in the axial conformers. Electrostatic interactions made a contribution to the observed conformer stabilization in all cases, but did not account for the relative magnitudes of the energy differences among conformers of homologous molecules. In the case of the methylamino substituent, accentuated steric interactions in the axial conformer precluded stabilization by the exo-anomeric interaction and consequently, the net endo/exo anomeric stabilization did not dominate the conformational equilibria. Key words: anomeric effect, component analysis, natural bond orbital analysis, electrostatic, steric, and orbital interaction effects. Six oxacyclohexane et thiacyclohexane heterocycles (substitues en position 2 par F, OMe, NHMe) ont ete examines par des calculs DFT des orbitales moleculaires. Des analyses des orbitales naturelles des liens (<< NBO >>) du comportement global de l'energie a donne les contributions des interactions des orbitales aux equilibres conformationnels. Les moments dipolaires des systemes optimizes ont ete utilizes pour estimer les contributions electrostatiques a l'effet anomere. La cause determinante primaire de l'effet anomere X-C-Y s'est montre les elements d'interactions orbitalaires associes aux effets anomeres endo et exo, qui agissent ensemble dans les conformeres axiaux. Les interactions electrostatiques ont contribue a la stabilization du conformere observe dans tous les cas, mais n'ont pas pu expliquer les magnitudes relatives des differences de l'energie entre les conformeres des molecules homologues. Dans le cas du substituant methylamino, des interactions steriques accentuees dans le conformere axial ont interdit la stabilization par l'interaction anomere exo et par consequent, la stabilization anomere endo/exo net n'a pas domine les equilibres conformationnels. Mots cles : effet anomere, analyse des composants, analyse des orbitales naturelles des liens, effets electrostatiques, effets steriques, effets des interactions orbitalaires., Introduction The anomeric effect (1, 2) has challenged the imagination and provoked the actions of both experimental and theoretical chemists alike (3). This effect, in its generalized form, refers to [...]

An improved synthesis of 2'-deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine is described. Conformational analysis of 2'-deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine showed that it adopted north and south conformations with a 77% preference for the north, this is in sharp contrast to the southeast conformer that dominates the conformational equilibrium of its 4'-oxygen congener.

Biomolecules contain tremendous amounts of information; this information is "written" and "read" through their chemical structures and functions. A change in the information of a biomolecule is a change in the physical properties of that molecule--a change in the molecule itself. It is impossible to separate the information contained in biomolecules from their structure and function. For molecules such as DNA and RNA, new information can be incorporated into the sequence of the molecules when that new sequence has favorable structural and functional properties. New biological information can arise by natural processes, mediated by the interactions between biomolecules and their environment, using the inherent relationship between structure and information. This fact has important implications for the generation of new biological information and thus the question of origins. [ABSTRACT FROM AUTHOR]

Glioblastoma is the most common primary malignant brain tumor in adults, with a median survival of just over 1 year. The failure of available treatments to achieve remission in patients with glioblastoma (GBM) has been attributed to the presence of cancer stem cells (CSCs), which are thought to play a central role in tumor development and progression and serve as a treatment‐resistant cell repository capable of driving tumor recurrence. In fact, the property of “stemness” itself may be responsible for treatment resistance. In this study, we identify a novel long noncoding RNA (lncRNA), cancer stem cell‐associated distal enhancer of SOX2 (CASCADES), that functions as an epigenetic regulator in glioma CSCs (GSCs). CASCADES is expressed in isocitrate dehydrogenase (IDH)‐wild‐type GBM and is significantly enriched in GSCs. Knockdown of CASCADES in GSCs results in differentiation towards a neuronal lineage in a cell‐ and cancer‐specific manner. Bioinformatics analysis reveals that CASCADES functions as a super‐enhancer‐associated lncRNA epigenetic regulator of SOX2. Our findings identify CASCADES as a critical regulator of stemness in GSCs that represents a novel epigenetic and therapeutic target for disrupting the CSC compartment in glioblastoma. [ABSTRACT FROM AUTHOR]