ABSTRACT
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.
Subject(s)
Mechanotransduction, Cellular , Membrane Proteins , DNA , Focal Adhesion Protein-Tyrosine Kinases , Mechanoreceptors/physiology , Stress, MechanicalABSTRACT
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.
Subject(s)
Dengue Virus/enzymology , Dengue/virology , Drug Evaluation, Preclinical/methods , Enzyme Assays/methods , Methyltransferases/analysis , Viral Nonstructural Proteins/analysis , Antiviral Agents/pharmacology , Dengue/drug therapy , Dengue Virus/drug effects , Dengue Virus/genetics , Dengue Virus/metabolism , Enzyme Inhibitors/pharmacology , Humans , Methyltransferases/antagonists & inhibitors , Methyltransferases/genetics , Methyltransferases/metabolism , RNA Caps/genetics , RNA Caps/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
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.
Subject(s)
Ribonucleosides/chemical synthesis , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Catalysis , Glycosylation , Iodine , Methods , Organosilicon Compounds , Phosphates , Ribonucleosides/chemistryABSTRACT
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.
Subject(s)
Anti-HIV Agents/administration & dosage , Lamivudine/analogs & derivatives , Anhydrides/chemistry , Anti-HIV Agents/chemistry , Cell Line , Dimerization , Drug Carriers/chemical synthesis , Drug Carriers/chemistry , Drug Delivery Systems , Drug Design , Glutarates/chemistry , HIV-1/drug effects , Humans , Lamivudine/administration & dosage , Lamivudine/chemistryABSTRACT
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.
Subject(s)
Methyltransferases/metabolism , Oligoribonucleotides/biosynthesis , RNA Cap Analogs/biosynthesis , Adenosine/metabolism , Chromatography, High Pressure Liquid , Cytidine Triphosphate/metabolism , DNA Primase , Guanine/metabolism , Humans , Oligoribonucleotides/isolation & purification , Oligoribonucleotides/metabolism , RNA Cap Analogs/chemistry , RNA Cap Analogs/isolation & purification , Templates, GeneticABSTRACT
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.
Subject(s)
Anti-HIV Agents/pharmacology , Antiviral Agents/chemistry , Carbamates/chemistry , Didanosine/chemistry , HIV/metabolism , Stavudine/chemistry , Sulfhydryl Compounds/chemistry , Zidovudine/chemistry , Antiviral Agents/pharmacology , Dimerization , HIV/genetics , HIV Infections/drug therapy , Models, Chemical , Molecular Conformation , MutationABSTRACT
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.
Subject(s)
Molecular Biology/methods , Phosphates/chemistry , Alkylation , Catalysis , Chromatography , Glycosides/chemistry , Glycosylation , Hydrocarbons/chemistry , Indicators and Reagents/chemistry , Methane/analogs & derivatives , Methane/chemistry , Models, Chemical , Nucleosides/chemistry , Potassium Iodide/chemistryABSTRACT
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.
Subject(s)
Anti-HIV Agents/chemical synthesis , Fungal Proteins/chemistry , Lipase/chemistry , Stavudine/chemical synthesis , Zidovudine/chemical synthesis , Anti-HIV Agents/pharmacology , Cell Line , Dimerization , HIV Infections/drug therapy , HIV Infections/virology , HIV-1/drug effects , HIV-2/drug effects , Humans , Stavudine/pharmacology , Virus Replication/drug effects , Zidovudine/pharmacologyABSTRACT
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.
Subject(s)
Didanosine/analogs & derivatives , Didanosine/chemistry , Anti-HIV Agents/chemistry , Didanosine/chemical synthesis , Dimerization , Esters , Indicators and Reagents , Molecular Conformation , Stavudine/chemistry , Zidovudine/chemistryABSTRACT
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.
Subject(s)
Antiviral Agents/chemical synthesis , Nucleosides/chemical synthesis , Nucleosides/pharmacology , Pyrazoles/chemistry , Pyridines/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cell Line , Cell Survival/drug effects , Cytomegalovirus/drug effects , Herpesvirus 3, Human/drug effects , Humans , Indicators and Reagents , Models, Molecular , Molecular Structure , Mycobacterium tuberculosis/drug effects , Nucleosides/chemistry , Pyrazoles/chemical synthesis , Pyridines/chemical synthesis , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Amides , Imidazoles , Oligodeoxyribonucleotides/chemical synthesis , Phosphoric Acids , Base Sequence , Binding Sites , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/chemistryABSTRACT
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.
Subject(s)
Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Base Sequence , Oligonucleotide Array Sequence AnalysisABSTRACT
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.
Subject(s)
Nucleosides/chemical synthesis , Pyrazoles/chemistry , Pyrimidines/chemistry , Animals , Antiviral Agents/pharmacology , Chlorocebus aethiops , Drug Screening Assays, Antitumor , Microbial Sensitivity Tests , Nucleosides/chemistry , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Tumor Cells, Cultured , Vero CellsABSTRACT
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.
Subject(s)
Nucleosides/chemistry , Nucleosides/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Cell Line , Fibroblasts/metabolism , HIV/metabolism , HeLa Cells , Humans , Models, Chemical , Nucleosides/chemical synthesis , Pyrimidines/chemical synthesis , Temperature , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Oligonucleotides/pharmacokinetics , Prodrugs/pharmacokinetics , Pyrimidine Nucleotides/pharmacokinetics , Thymidine/analogs & derivatives , Biotransformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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.
Subject(s)
Amides/chemistry , DNA/chemistry , Oligonucleotides/chemistry , Phosphoric Acids/chemistry , Cross-Linking Reagents/chemistry , Hydrogen-Ion Concentration , Nucleic Acid Conformation , Nucleic Acid Hybridization , Oligonucleotides/chemical synthesisABSTRACT
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.
Subject(s)
Oligonucleotides/chemical synthesis , Prodrugs/chemical synthesis , Oligonucleotides/chemistry , Organophosphates/chemical synthesis , Organophosphates/chemistry , Organophosphorus Compounds/chemistry , Prodrugs/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
The synthesis of new dinucleosides of AZT and D4T is described.
Subject(s)
Dideoxynucleosides/chemical synthesis , Stavudine/analogs & derivatives , Zidovudine/analogs & derivatives , Anti-HIV Agents/chemical synthesis , Stavudine/chemical synthesis , Zidovudine/chemical synthesisABSTRACT
The synthesis of some acyclic alpha-(pyrazolo[3,4-d]pyrimidin-4-ylthio)alkylamide nucleosides is described.
Subject(s)
Pyrazoles/chemical synthesis , Pyrimidine Nucleosides/chemical synthesis , HIV-1/drug effects , HIV-2/drug effects , Humans , Pyrazoles/pharmacology , Pyrazoles/toxicity , Pyrimidine Nucleosides/pharmacology , Pyrimidine Nucleosides/toxicity , Virus Replication/drug effectsABSTRACT
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.
