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1.
Protein Sci ; 8(12): 2806-12, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10631998

RESUMO

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.


Assuntos
Fator 2 de Crescimento de Fibroblastos/química , Oligonucleotídeos/química , Fosfatase Alcalina/química , Sequência de Aminoácidos , Bromodesoxiuridina , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas , Fator 2 de Crescimento de Fibroblastos/efeitos da radiação , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Oligonucleotídeos/efeitos da radiação , Fragmentos de Peptídeos/química , Fosfodiesterase I , Diester Fosfórico Hidrolases/química , Radiossensibilizantes , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos da radiação , Tripsina/química , Raios Ultravioleta
2.
J Biotechnol ; 81(2-3): 167-78, 2000 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-10989176

RESUMO

High sensitivity and specificity of two modified ssDNA aptamers capable of photocross-linking recombinant human basic fibroblast growth factor (bFGF((155))) were demonstrated. The aptamers were identified through a novel, covalent, in vitro selection methodology called photochemical systematic evolution of ligands by exponential enrichment (PhotoSELEX). The aptamers exhibited high sensitivity for bFGF((155)) comparable with commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF((155)) under prevailing test conditions. The aptamers exquisitely distinguished bFGF((155)) from consanguine proteins, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). A commercially viable diagnostic system incorporating PhotoSELEX-evolved aptamers capable of simultaneous quantification of a large number of analyte molecules is also described. Such a system benefits from covalent bonding of aptamer to target protein allowing vigorous washing with denaturants to improve signal to noise.


Assuntos
Bromodesoxiuridina/química , DNA de Cadeia Simples/química , Fator 2 de Crescimento de Fibroblastos/análise , Sequência de Bases , DNA de Cadeia Simples/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Fator 2 de Crescimento de Fibroblastos/química , Dados de Sequência Molecular , Fotoquímica , Sensibilidade e Especificidade
3.
Bioconjug Chem ; 9(5): 573-82, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9736491

RESUMO

Nuclease-resistant aptamers identified from randomized nucleic acid libraries represent a novel class of drug candidates. Aptamers are synthesized chemically and therefore can be readily modified with functional groups that modulate their properties. We report here on the preparation, initial characterization, and functional properties of a nuclease-resistant vascular endothelial growth factor (VEGF) aptamer anchored in liposome bilayers through a lipid group on the aptamer. While the high-affinity binding to VEGF is maintained, the plasma residence time of the liposome-anchored aptamer is considerably improved compared with that of the free aptamer. The lipid group attachment and/or liposome anchoring leads to a dramatic improvement in inhibitory activity of the aptamer toward VEGF-induced endothelial cell proliferation in vitro and vascular permeability increase and angiogenesis in vivo.


Assuntos
Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/efeitos dos fármacos , Lipossomos/metabolismo , Linfocinas/metabolismo , Oligonucleotídeos/farmacologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Microscopia Eletrônica , Estrutura Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Tamanho da Partícula , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Ribonuclease T1/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
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