Bioinformatic analysis of the contribution of primer sequences to aptamer structures

Abstract

Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust.

Fig. 1

Cartoon schematic of the anatomy of an aptamer sequence. Top: Aptamer sequences consist of a random region (black rectangle) that is flanked by two constant regions (gray rectangles). The random region undergoes most evolution during in vitro selection, whereas the constant regions serve for amplifying the aptamers during the selection process. Bottom: Comparison of different folded strcutures used in the present study. This structure of the random region (black) is robust to the absence of constant regions or the presence of randomized constant regions (gray)

Fig. 2

(A) Distribution of the number of aptamers in the database at each pool size. (B) Distribution of the number of aptamers in the database at each constant region length. In both (A) and (B), we only include aptamers in which the entire sequence (random and constant regions) is contained in the Aptamer Database (Lee et al. 2004)

Fig. 3

The relative interference caused by constant regions (A) over the range of pool sizes, (B) across the range of constant region sizes, and (C) by the individual constant regions. In (A) and (B), the dark lines (stars) show the interference caused by the true constant regions, whereas the light lines (squares) show the interference from randomized constant regions. In (C), the dark lines (stars, circles) depict the interference caused by the true constant regions, and the light lines (squares, triangles) depict interference caused by randomized constant regions. We normalized the distance to the length of the random region to facilitate comparison across the range of pool sizes. Error bars depict one standard error about the mean

Fig. 4

A regression of upstream constant region-induced interference on downstream constant region-induced interference. The slope of the regression line is 0.55 (p < 2.2 × 10⁻¹⁶)

Fig. 5

Contribution of random regions to thee thermostability of aptamer structures. The Boltzmann probability of the ground-state structure for each true aptamer is compared to the Boltzmann probabilities of 100 sequences in which the random region is shuffled. Data points below the zero line represent aptamers in which the randomized sequences are more thermostable, on average, than the true aptamer sequences. Error bars depict one standard error about the mean