Optimization of SELEX: comparison of different methods for monitoring the progress of in vitro selection of aptamers.
Oligonucleotide aptamers are selected from libraries typically comprising up to 10(15) 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, labor-intensive 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.
Instrumentation Technologies, Velika pot 22, 5250 Solkan, Slovenia; Laboratory for Bioinstrumentation, Centre of Excellence for Biosensors, Instrumentation and Process Control (CO BIK), Velika pot 22, 5250 Solkan, Slovenia. Electronic address: nina.mencin@i-tech.si.
Nina
N
Mencin
Laboratory for Bioinstrumentation, Centre of Excellence for Biosensors, Instrumentation and Process Control (CO BIK), Velika pot 22, 5250 Solkan, Slovenia.
Tina
T
Šmuc
Laboratory for Bioinstrumentation, Centre of Excellence for Biosensors, Instrumentation and Process Control (CO BIK), Velika pot 22, 5250 Solkan, Slovenia.
Marko
M
Vraničar
Laboratory for Bioinstrumentation, Centre of Excellence for Biosensors, Instrumentation and Process Control (CO BIK), Velika pot 22, 5250 Solkan, Slovenia.
Jan
J
Mavri
Laboratory for Bioinstrumentation, Centre of Excellence for Biosensors, Instrumentation and Process Control (CO BIK), Velika pot 22, 5250 Solkan, Slovenia.
Matjaž
M
Hren
Laboratory for Bioinstrumentation, Centre of Excellence for Biosensors, Instrumentation and Process Control (CO BIK), Velika pot 22, 5250 Solkan, Slovenia.
Katja
K
Galeša
Laboratory for Bioinstrumentation, Centre of Excellence for Biosensors, Instrumentation and Process Control (CO BIK), Velika pot 22, 5250 Solkan, Slovenia.
Peter
P
Krkoč
Departamento de Bioquímica, Instituto de Química, Universidade de Saõ Paulo, Saõ Paulo, Brazil.
Henning
H
Ulrich
Laboratory for Bioinstrumentation, Centre of Excellence for Biosensors, Instrumentation and Process Control (CO BIK), Velika pot 22, 5250 Solkan, Slovenia.
Borut
B
Šolar
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D052156:SELEX Aptamer Technique / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
D052157:Aptamers, Nucleotide; D009841:Oligonucleotides; D019809:Streptavidin
Aptamer; Aptamer pool evolution; Evolution monitoring; SELEX; Selection progress