RESUMO
A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/genética , Desoxirribonucleosídeos/química , Fosfatos de Dinucleosídeos/química , Polímeros/síntese química , Adenina/química , Adenina/metabolismo , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Sequência de Bases , Citosina/química , Citosina/metabolismo , DNA/química , DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleosídeos/genética , Desoxirribonucleosídeos/metabolismo , Fosfatos de Dinucleosídeos/genética , Fosfatos de Dinucleosídeos/metabolismo , Guanina/química , Guanina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Reação em Cadeia da Polimerase , Polímeros/metabolismo , Uracila/química , Uracila/metabolismoRESUMO
Succinyl-cycloSal-phosphate triesters of ribo- and 2'-deoxyribonucleosides were attached to aminomethyl polystyrene as an insoluble solid support and reacted with phosphate-containing nucleophiles yielding nucleoside di- and triphosphates, nucleoside diphosphate sugars, and dinucleoside polyphosphates in high purity after cleavage from the solid support. Here, reactive cycloSal-phosphate triesters were used as immobilized reagents that led to a generally applicable method for the efficient synthesis of phosphorylated biomolecules and phosphate-bridged bioconjugates.
Assuntos
Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , Indicadores e Reagentes/química , Açúcares de Nucleosídeo Difosfato/química , Açúcares de Nucleosídeo Difosfato/síntese química , Nucleosídeos/química , Nucleosídeos/síntese química , Nucleotídeos/química , Nucleotídeos/síntese química , Poliestirenos/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , FosforilaçãoRESUMO
Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a major target for currently approved anti-HIV drugs. These drugs are divided into two classes: nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). This study illustrates the synthesis and biochemical evaluation of a novel bifunctional RT inhibitor utilizing d4T (NRTI) and a TMC-derivative (a diarylpyrimidine NNRTI) linked via a poly(ethylene glycol) (PEG) linker. HIV-1 RT successfully incorporates the triphosphate of d4T-4PEG-TMC bifunctional inhibitor in a base-specific manner. Moreover, this inhibitor demonstrates low nanomolar potency that has 4.3-fold and 4300-fold enhancement of polymerization inhibition in vitro relative to the parent TMC-derivative and d4T, respectively. This study serves as a proof-of-concept for the development and optimization of bifunctional RT inhibitors as potent inhibitors of HIV-1 viral replication.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Primers do DNA , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/isolamento & purificação , Desenho de Fármacos , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/isolamento & purificação , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Espectrometria de Massas , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/isolamento & purificação , Polietilenoglicóis/farmacologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos , Difração de Raios XRESUMO
An environmental, estrogen-like substance, bisphenol A (BPA), is the monomer for the production of polycarbonate plastics used in baby bottles, dental sealants, and as a major component of epoxy resin for the lining of food cans. The oxidation of BPA leads to the reactive electrophilic BPA-o-3,4-quinone (BPA-Q), which can damage DNA and may be implicated in cancer initiation. BPA-Q reacts in vitro with 2'-deoxyguanosine 5'-phosphate (dGMP) and 2'-deoxyadenosine 5'-phosphate (dAMP) but not with 2'-deoxycytidine-5'-phosphate and 2'-deoxythymidine 5'-phosphate. In aqueous acetic acid, BPA-Q also reacts with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) but not with 2'-deoxycytidine and 2'-deoxythymidine. The reactions are accompanied by loss of the modified base (depurination). We determined the structures of the modified bases by primarily tandem mass spectrometry. In mixtures of deoxynuclesides and deoxynucletides treated with BPA-Q, reactions occur more readily with dGMP/dG followed by dAMP/dA. With calf thymus DNA, significant apurinic sites must be produced because we detected the BPA-Q-guanosine adduct in the incubation mixture. We also found that BPA-Q reacts readily with glutathione (GSH) under acidic or neutral conditions, and we characterized the BPA-Q-GSH conjugate with tandem mass spectrometry (MS/MS). The results are consistent with a mechanism of carcinogenesis whereby BPA-Q, formed in vivo and not adequately detoxified by reactions with GSH, reacts with DNA, causing depurination. The adducts reported will also be appropriate references for identification of BPA-Q adducts in environmental and biological systems.