RESUMO
Synthesis of aminomethyl and bis-aminomethylfluorescein derived energy transfer terminators is described.
Assuntos
Fluoresceína/química , Fluoresceínas/síntese química , Análise de Sequência de DNA , Transferência de Energia , Fluoresceínas/químicaRESUMO
Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.
Assuntos
Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/farmacologia , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Fc/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos/imunologia , Humanos , Concentração de Íons de Hidrogênio , Macaca fascicularisRESUMO
A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.
Assuntos
DNA/química , Desoxirribonucleotídeos/química , Análise de Sequência de DNA/métodos , Corantes/química , DNA/genética , DNA/metabolismo , Eletroforese/métodos , Mutação , Reprodutibilidade dos Testes , Taq Polimerase/genética , Taq Polimerase/metabolismoRESUMO
A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Didesoxinucleosídeos/química , Lisina/análogos & derivados , Nucleotídeos/metabolismo , Análise de Sequência de DNA/métodos , Sequência de Bases , Cromatografia Líquida de Alta Pressão/métodos , Corantes/química , DNA/química , DNA/genética , Lisina/química , Dados de Sequência Molecular , Estrutura Molecular , Nucleotídeos/química , Nucleotídeos/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Synthesis of novel piperidinyl linker based ET cassettes and terminators is described These novel terminators are evaluated in the DNA sequencing experiments using thermostable DNA polymerase.
Assuntos
DNA Polimerase Dirigida por DNA/química , Piperidinas/química , Análise de Sequência de DNA/instrumentação , DNA/química , Transferência de Energia , Corantes Fluorescentes/farmacologia , Temperatura Alta , Indicadores e Reagentes/farmacologia , Modelos Químicos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Análise de Sequência de DNA/métodos , Espectrometria de FluorescênciaRESUMO
Fluorescence resonance energy transfer (FRET) dye labeled cassettes and terminators with one or more donor dyes (fluorescein) and acceptor dye (rhodamine dyes) with benzofuran or tyrosine linker moieties were synthesized. These terminators were evaluated for their energy transfer and DNA sequencing potential using thermostable DNA polymerase.
Assuntos
DNA/química , Tirosina , Sequência de Bases , Benzofuranos , Transferência Ressonante de Energia de Fluorescência/métodos , Regiões Terminadoras GenéticasRESUMO
A four-color set of negatively charged, single dye as well as energy transfer dye labeled-ddNTPs were synthesized and evaluated in combination with a novel polymerase in a "direct-load" DNA sequencing, obviating the laborious and time consuming post-reaction work-up.
Assuntos
Corantes , DNA/química , Desoxirribonucleotídeos/síntese química , Didesoxinucleosídeos/síntese química , Sequência de Bases , Transferência de Energia , Indicadores e ReagentesRESUMO
Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.