RESUMEN
The importance of chemically modified and surface immobilized nucleic acids has inspired the development of a wide variety of complementary techniques for covalent oligonucleotide preparation and immobilization. We are developing technology based on the use of a Diels-Alder reaction for accomplishing the covalent modification of oligonucleotides. Reported herein is preliminary progress toward the establishment of robust reagents for introducing the reactive functionality, as well as studies employing the BIACORE system to demonstrate surface immobilization by the method.
Asunto(s)
Oligonucleótidos/química , Oligonucleótidos/síntesis química , Propiedades de SuperficieRESUMEN
In an effort to offer complementary technology for covalent biomolecule modification (bioconjugation), we have developed a method that exploits the aqueous acceleration of Diels--Alder reactions for this purpose. Three different diene phosphoramidite reagents have been synthesized that enable diene modification of synthetic oligonucleotides prepared by the phosphoramidite method. Clean and efficient Diels--Alder cycloaddition of these diene oligonucleotides with maleimide dieneophiles was carried out, and the labeled oligonucleotide bioconjugates were characterized by HPLC and electrospray mass spectrometry. Dieneophile stoichiometry, temperature, and pH are all parameters that were shown to influence the efficiency of the process.
Asunto(s)
Oligonucleótidos/síntesis química , Biotinilación , Cumarinas/química , Fluoresceína/química , Oligonucleótidos/química , TemperaturaRESUMEN
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.
Asunto(s)
Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/efectos de los fármacos , Liposomas/metabolismo , Linfocinas/metabolismo , Oligonucleótidos/farmacología , Animales , Permeabilidad Capilar/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Humanos , Microscopía Electrónica , Estructura Molecular , Neovascularización Fisiológica/efectos de los fármacos , Tamaño de la Partícula , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Ribonucleasa T1/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.