Isolation of high-affinity GTP aptamers from partially structured RNA libraries

Abstract

Aptamers, RNA sequences that bind to target ligands, are typically isolated by in vitro selection from RNA libraries containing completely random sequences. To see whether higher-affinity aptamers can be isolated from partially structured RNA libraries, we selected for aptamers that bind GTP, starting from a mixture of fully random and partially structured libraries. Because stem-loops are common motifs in previously characterized aptamers, we designed the partially structured library to contain a centrally located stable stem-loop. We used an off-rate selection protocol designed to maximize the enrichment of high-affinity aptamers. The selection produced a surprisingly large number of distinct sequence motifs and secondary structures, including seven different aptamers with K(d)s ranging from 500 to 25 nanomolar. The engineered stem-loop was present in the three highest affinity aptamers, and in 12 of 13 independent isolates with a single consensus sequence, suggesting that its inclusion increased the abundance of high-affinity aptamers in the starting pool.

Figure 1

Design of the initial libraries. Engineered (A) and random (B) libraries, both 124 bases long with 64 bases between the constant primer binding regions, were used to generate the starting RNA. The random library contains 64 random bases, whereas the engineered library contains 52 random bases with a 4 bp stem and stable UUCG tetraloop in the middle.

Figure 2

(A) Progress of the selection over 10 rounds. Filled squares represent the fraction still bound to the column after 6 column volumes of washing. Open triangles represent the fraction specifically eluted after surviving the full washing and the pre-elution, if used. Arrows indicate the rounds where stringency was increased, either by more extensive washing or more pre-elution (see Table 1 for details). (B) Profile of round 7, expressed as percentage of total counts in each fraction. (C) Profile of round 10, illustrating the shift toward more stable complexes. In round 7, most of the bound RNA eluted in the first pre-elution fraction, indicating a fast k_off, whereas by round 10 less than half of the bound aptamers eluted in that fraction.

Figure 3

(A) Binding curve of clone 9-4, as measured by ultrafiltration assays. The y axis is the fraction bound, whereas the x axis is the concentration of aptamer in the assay. The concentration at which the curve intersects the horizontal line representing one half bound (0.5) is the K_d, in this case 25 nM. (B) Biacore trace of clone 9-4, the aptamer with the lowest off-rate. RNA was allowed to bind to the chip for 6 min (horizontal bar), then the chip was washed with selection buffer at 25 μl/min. The signal from the bound aptamer peaked at 40 response units (RU) above baseline, and after 1 h it had dropped by 6 RU, or 15%. This result corresponds to a k_off of 0.0027 min⁻¹, or 0.16 h⁻¹. The off-rate was similar when washed with buffer containing 5 mM GTP (not shown).

Figure 4

Alignment of unique Class I sequences. The top 12 derive from the engineered library, whereas 10-32 is the lone class representative from the random library. The box contains the fixed 12-base central hairpin, with one or two extra base pairs in several of the sequences (as expected by chance). Bases that form Watson–Crick or G-U wobble pairs in the stems are underlined. The recognition loops are under the two heavy bars; bold text denotes 100% conserved bases, regular capital letters are semivariant bases (i.e., two species are allowed at that position), and lowercase are fully variable. The last base in the top strand of the outer stem, marked by an asterisk (*), is always a G or C, so it can also be considered semivariant.

Figure 5

Sequences of the highest-affinity RNA aptamers, in order of their measured binding constants (see Table 2), and their proposed secondary structures. The lengths were determined by end mapping, and the secondary structures based on the assumption that complementary termini form a duplex, and that the engineered hairpin is formed whenever present. G (10-6) has a potential 4-base stem within the binding loop, and A (9-4) has a number of possible structures, so no particular structure is presented. C, a Class I aptamer, is the best characterized structure, because 13 independent variants emerged from the selection. The variable positions in the binding loop (see Fig. 4) are denoted by lowercase letters.