A modular spring-loaded actuator for mechanical activation of membrane proteins.

How cells respond to mechanical forces by converting them into biological signals underlie crucial cellular processes. Our understanding of mechanotransduction has been hindered by technical barriers, including limitations in our ability to effectively apply low range piconewton forces to specific mechanoreceptors on cell membranes without laborious and repetitive trials. To overcome these challenges we introduce the Nano-winch, a robust, easily assembled, programmable DNA origami-based molecular actuator. The Nano-winch is designed to manipulate multiple mechanoreceptors in parallel by exerting fine-tuned, low- piconewton forces in autonomous and remotely activated modes via adjustable single- and double-stranded DNA linkages, respectively. Nano-winches in autonomous mode can land and operate on the cell surface. Targeting the device to integrin stimulated detectable downstream phosphorylation of focal adhesion kinase, an indication that Nano-winches can be applied to study cellular mechanical processes. Remote activation mode allowed finer extension control and greater force exertion. We united remotely activated Nano-winches with single-channel bilayer experiments to directly observe the opening of a channel by mechanical force in the force responsive gated channel protein, BtuB. This customizable origami provides an instrument-free approach that can be applied to control and explore a diversity of mechanotransduction circuits on living cells.

Development of specific dengue virus 2'-O- and N7-methyltransferase assays for antiviral drug screening.

Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'OMe)-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA(2'OMe)-RNA74, by ligation of two RNA fragments. Then, we use GpppA(2'OMe)-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA74 in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA. We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.

Glycosylation reaction via a mild and efficient one-pot reaction using doped natural phosphate with iodine as catalyst.

Several alpha/beta-D-ribonucleosides were synthesized in good yields under mild conditions by N-glycosylations of acetyl 2,3,5-tri-O-benzoyl-beta-D-ribofuranose with silylated nucleobases in acetonitrile using NP doped with iodine as catalyst.

A glutaric acid ester as carrier system for sustained delivery of lamuvidine (3TC) dimers.

We report the synthesis of homo and heterodimers of 3TC conjugates. All new dimers were screened for their ability to inhibit HIV-1 in MT4 cell line and were compared to AZT alone and showed marked antiviral activity.

High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions.

Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.

Homo and heterodimers of ddI, d4T and AZT: influence of (5'-5') thiolcabonate-carbamate linkage on anti-HIV activity.

New homo and heterodimers of ddI, d4T and AZT with (5-5) thiolcarbonate-carbamate linkages have been prepared with the aim of testing them against wild type and NNRTI resistant HIV mutants. The prepared dimers showed a low activity in comparison to the parent drug.

Natural phosphate doped with KI in HMDS: a mild and efficient reagent for alkylation and glycosylation of nucleobases.

Several D-ribonucleosides are prepared from 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranoside and trimethylsilylated nucleobases under mild conditions by using natural phosphate doped with KI as catalyst.

Chemoenzymatic syntheses of homo- and heterodimers of AZT and d4T, and evaluation of their anti-HIV activity.

Homo- and heterodimers of AZT and d4T, possessing carbonate and carbamate linkers, have been synthesized with the aim to enhance the antiviral activity of their components. Homo- and heterodimer carbamates showed weak anti-HIV activity. On the other hand, dinucleoside carbonates showed marked antiviral activity.

Synthesis of new homo and heterodimers of 2',3'-dideoxyinosine (ddI) using ester linkages.

A series of new homo and heterodimers of ddI has been synthesized. A glutarate diester spacer was used to covalently couple ddI onto ddI, AZT or d4T.

Synthesis and biological evaluation of some acyclic 4,6-disubstituted 1H-pyrazolo[3,4-d]pyrimidine nucleosides.

The chemical synthesis and biological evaluation of some acyclic alpha-[6-(1'-carbamoylalkylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylamide nucleosides are described.

Imidazolethyl-phosphoramidate alpha-oligonucleotides.

Alpha-ODNs conjugated to imidazole groups via phosphoramidate internucleosidic linkages were synthesized. The presence of the imidazolethyl-phosphoramidate linkage improved the affinity of alpha-ODNs for their nucleic acid targets.

Use of MALDI-TOF mass spectrometry to monitor solid-phase synthesis of oligonucleotides.

MALDI-TOF mass spectrometry has been used to characterize solid-supported oligonucleotides containing natural and non-natural and non-nucleoside moieties and a variety of internucleosidic linkages including phosphate and phosphite triesters and H-phosphonate diesters. This technique was used to follow the reactions involved in oligonucleotide synthesis; this enabled direct control of the elongation and optimization of the coupling process.

Synthesis and biological evaluation of some acyclic alpha-(1H-pyrazolo-[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides.

The chemical synthesis of some acyclic alpha-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides (10-12)a-c is described. The treatment of IH-pyrazolo[3,4-d]pyrimidin-4-thione 1 with compounds 2a-c gave, regioselectively, ethyl alpha-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylates 3a-c, respectively. These heterocycles were alkylated, separately, with alkylating agents 4, 5 and 6 to give, regioselectively, the N1-acyclic nucleosides (7-9)a-c which were deprotected to afford the desired products (10-12)a-c. All synthetic compounds were characterized on the basis of their physical and spectroscopic properties. The products (10-12)a-c were evaluated for their inhibitory effects against the replication of HIV-1 (III(B)), HIV-2 (ROD), various DNA viruses, a variety of tumor-cell lines and M. tuberculosis. No marked biological activity was found.

Synthesis and biological evaluation of some 4-substituted 1-[1-(4-hydroxybutyl)-1,2,3-triazol-(4 &5)-ylmethyl]-1H-pyrazolo- 13,4-dipyrimidines.

The synthesis of 1-[1-(4-hydroxybutyl)-1,2,3-triazol-(4 and 5)-ylmethyl]-1H-pyrazolo[3,4-d]pyrimidines 11a,b, 12a,b and 13-17 as carboacyclic nucleosides is described. The compounds 8a,b were condensed, separately, with compound 7 via 1,3-dipolar cycloaddition reaction to afford, after separation and deprotection. 1,4-regioisomers 11a,b and 1,5-regioisomers 12a,b. The deprotected carboacyclic nucleosides 11a served as precursor for the preparation of 4-amino 13. 4-methylamino 14, 4-benzylamino 15, 4-methoxy 16 and 4-hydroxy 17 analogues. All deprotected carboacyclic nucleosides were evaluated for their inhibitory effects against the replication of HIV-1(III), HIV-2(ROD), various DNA viruses, a variety of tumor-cell lines and tuberculosis. No marked biological activity was found.

Kinetics study of the biotransformation of an oligonucleotide prodrug in cells extract by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. The oligonucleotide prodrug was fully metabolized to yield the T12 phosphorothioate likely through a carboxyesterase mediated mechanism.

Alpha-oligonucleotides with anionic phosphodiester and cationic phosphoramidate linkages enhanced stability of DNA triple helix.

Synthesis of several alpha-oligonucleotides containing both anionic phosphodiester and cationic N-(dimethylaminopropyl)phosphoramidate internucleosidic linkages is described. Their ability to form triple helix with dsDNA was evaluated at various pH by UV melting experiments.

Pro-oligonucleotide synthesis using allyl and allyloxycarbonyl protections: direct MALDI-TOF MS analysis on solid support.

The solid-support synthesis of pro-oligonucleotide heteropolymer chimeras had been performed with allyloxycarbonyl group (AOC) for the protection of nucleobases and of allyl and S-acetyl-2-thioethyl (MeSATE) for phosphate protections to respectively generate phosphodiester and MeSATE phosphotriester linkages.

Synthesis of new homo and heterodinucleosides containing the 2',3'-dideoxynucleosides AZT and D4T.

The synthesis of new dinucleosides of AZT and D4T is described.

Synthesis of some acyclonucleosides alpha-(pyrazolo[3,4-d]pyrimidin-4-ylthio)alkylamides.

The synthesis of some acyclic alpha-(pyrazolo[3,4-d]pyrimidin-4-ylthio)alkylamide nucleosides is described.

Step-by-step control by MALDI-TOF MS of an oligonucleotide synthesis on standard CPG solid-support.

MALDI-TOF mass spectrometry was used to monitor DNA solid-phase synthesis on long-chain alkylamine controlled-pore glass (LCAA-CPG) with a standard succinyl linker between the solid support and the growing oligonucleotide.