Investigating the Influences of Random-Region Length on Aptamer Selection Efficiency Based on Capillary Electrophoresis–SELEX and High-Throughput Sequencing

For
aptamer selection, the random-region length of an ssDNA library
was generally taken in a relatively arbitrary fashion, which may lead
to failure for unsuitable target binding. Herein, we coupled high-efficiency
capillary electrophoresis (CE)–SELEX and high-throughput sequencing
(HTS) to investigate the influences of random-region length. First,
one round of selection against programmed cell death-ligand 1 (PD-L1)
was performed using ssDNA libraries with random-region lengths of
15, 30, 40, and 60 nt, respectively. A good correlation was observed
between candidates’ random-region lengths and dissociation
constant (Kd), in which the longer sequences
presented higher affinity, and the picked Seq 60–1 after one
round notably presented a similar affinity toward a reported aptamer
through eight rounds. Molecular dynamics (MD) simulation suggested,
for PD-L1, the long sequence could supply more noncovalent bonds including
hydrogen bonds, electrostatic interactions, and hydrophobic interactions
to form a stable protein/aptamer complex. Besides, four other proteins
with selective binding performances validated the importance of random-region
length. To further investigate how random-region length affects the
selection efficiency, a mixed library with random-region lengths ranging
from 10 to 50 nt was employed for six rounds of selection against
Piezo2. Sequence variations were tracked by HTS, showing the preferential
evolution and PCR uncertainty with even higher impact were the main
causes. This study suggested random-region length plays a crucial
factor, and a mixed library with different random-region sequences
can be a worthy choice for increasing the speed of high-affinity aptamer
selection. Moreover, the PCR process should be given particular attention
in aptamer selection.