Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing.
We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K(d) < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have approximately 3-8-fold higher affinity and approximately 2-4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.
Cho
Minseon
M
Department of Mechanical Engineering, University of California, Santa Barbara, CA 93106, USA.
Xiao
Yi
Y
Nie
Jeff
J
Stewart
Ron
R
Csordas
Andrew T
AT
Oh
Seung Soo
SS
Thomson
James A
JA
Soh
H Tom
HT
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D000077214:Becaplermin; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D007700:Kinetics; D044085:Microfluidics / Q000379:methods*; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020574:Proto-Oncogene Proteins c-sis; D015203:Reproducibility of Results; D052156:SELEX Aptamer Technique / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin