Optimization of SELEX: Comparison of different methods for monitoring the progress of in vitro selection of aptamers

Autor(es)

Mencin, Nina ^{[1, 2]} ; Smuc, Tina ^[1] ; Vranicar, Marko ^[1] ; Mavri, Jan ^[1] ; Hren, Matjaz ^[1] ; Galesa, Katja ^[1] ; Krkoc, Peter ^[1] ; Ulrich, Henning ^[3] ; Solar, Borut ^[1]

Afiliação do(s) autor(es)

^[1] Ctr Excellence Biosensors Instrumentat & Proc Con, Lab Bioinstrumentat, Solkan 5250 - Slovenia

^[2] Instrumentat Technol, Solkan 5250 - Slovenia

^[3] Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-01498 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Pharmaceutical and Biomedical Analysis; v. 91, p. 151-159, MAR 25 2014.
• Citações Web of Science: 20

Resumo

Oligonucleotide aptamers are selected from libraries typically comprising up to 1015 different sequences by an iterative process of binding, separation, amplification and purification, called SELEX. During this process, the diversity of the oligonucleotide pool decreases until, presumably, only sequences with highest binding affinities towards chosen targets remain. This selection technique is time-consuming, laborintensive and expensive. Though well posed in principles, the SELEX procedure is noise sensitive, due to amplification of unspecific-binding sequences, and it is not surprising that aptamer selection is often not successful in practice. In view of that, a follow-up of the progress of selection during its course with simple yet reliable methods is necessary. In this paper, we describe five independent assays to estimate the sequence complexity of SELEX pools including qualitative restriction fragment length polymorphism analysis, melting curve analysis, quantitative fluorescence intensity measurements of bound ssDNA, real time PCR quantification and pool dissociation constant analysis during the progress of aptamer selection against streptavidin. Properties and features of each method are discussed and compared. Pool dissociation constant analysis and sequencing serve as reference methods. (C) 2014 Published by Elsevier B.V. (AU)

• Processo FAPESP: 12/50393-6 - Aptâmeros para descoberta de marcadores diagnósticos e inibidores terapêuticos em doenças infecciosas
• Beneficiário: Alexander Henning Ulrich
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 12/50880-4 - Células-tronco: dos papéis de receptores de cininas e purinas às aplicações terapêuticas
• Beneficiário: Alexander Henning Ulrich
• Modalidade de apoio: Auxílio à Pesquisa - Temático