In vitro selection of l-DNA aptamers that bind a structured d-RNA molecule

Abstract

The development of structure-specific RNA binding reagents remains a central challenge in RNA biochemistry and drug discovery. Previously, we showed in vitro selection techniques could be used to evolve l-RNA aptamers that bind tightly to structured d-RNAs. However, whether similar RNA-binding properties can be achieved using aptamers composed of l-DNA, which has several practical advantages compared to l-RNA, remains unknown. Here, we report the discovery and characterization of the first l-DNA aptamers against a structured RNA molecule, precursor microRNA-155, thereby establishing the capacity of DNA and RNA molecules of the opposite handedness to form tight and specific 'cross-chiral' interactions with each other. l-DNA aptamers bind pre-miR-155 with low nanomolar affinity and high selectivity despite the inability of l-DNA to interact with native d-RNA via Watson-Crick base pairing. Furthermore, l-DNA aptamers inhibit Dicer-mediated processing of pre-miRNA-155. The sequence and structure of l-DNA aptamers are distinct from previously reported l-RNA aptamers against pre-miR-155, indicating that l-DNA and l-RNA interact with the same RNA sequence through unique modes of recognition. Overall, this work demonstrates that l-DNA may be pursued as an alternative to l-RNA for the generation of RNA-binding aptamers, providing a robust and practical approach for targeting structured RNAs.

Figure 1.

Mirror image in vitro selection of cross-chiral DNA aptamers for pre-miR-155. (A) Sequences and secondary structures of pre-miR-155 and truncated variant pre-miR-155t. For consistency, numbering of nucleotides is maintained upon truncation. (B) Chemical structure of 5-aminoallyl-2′-deoxyuridine (5AdU). (C) Schematic representation of the in vitro selection method used to isolate modified DNA aptamers. For unmodified aptamers, Lib.dT is directly applied to the selection step.

Figure 2.

Sequence and binding affinity of cross-chiral DNA aptamers. (A) Sequences of full-length aptamers T9 and AdU5 and their truncated variants. Blue Ts indicate the position of 5AdU and primer-binding sites are underlined. Guanosine residues shown to be involved in G-quadruplex formation are colored red (see Figure 4). (B) Saturation plots for binding of T9t to either pre-miR-155t or pre-miR-155 (asterisk) having the opposite chirality. (C) Saturation plots for binding of AdU5t to either pre-miR-155t or pre-miR-155 (asterisk) having the opposite chirality. K_d values reported as mean ± S.D. (n = 3). (D) Chemical structure of 5-aminoallyl-dU phosphoramidite (l-5AdU-CEP).

Figure 3.

CD spectrum of d-T9t (A) and d-AdU5t (B) aptamers. All CD spectrum were obtained with 9 μM aptamer in the presence of a buffer containing 20 mM NaCl, 25 mM Tris (pH 7.6) and either 50 mM KCl (K⁺, shown in black solid line) or LiCl (Li⁺, shown in red solid line) at 23°C. CD spectra recorded under no salt condition were prepared in TE buffer (shown in dashed black line).

Figure 4.

Characterization of G-quadruplex formation in d-T9t (A) and d-AdU5t (B) aptamers. Each aptamer was annealed in the presence of LiCl, KCl or KCl plus l-pre-miR-155t and subjected to DMS footprinting. The nucleotide sequence of each aptamer is shown to the right of the gel. Protected guanosine residues are colored red and 5AdU is represented by blue Ts. Treatment of d-AdU5t with formic acid (G/A) and DMS resulted in cleavage at 5AdU residues (asterisks). M = untreated control.

Figure 5.

Cross-chiral DNA aptamers bind selectively to the loop domain of pre-miR-155. (A) In-line probing analysis of pre-miR-155t in the presence and absence of the indicated l-aptamer. Residues that underwent significant conformational changes (i.e. protection from hydrolysis) in the presence of excess l-aptamer are boxed in red. Partial digestion by ribonuclease T1 (cleavage after G residues) is shown to the left (T1). Uncropped gel images are shown in Supplementary Figures S11a and b. (B) The extent of pre-miR-155 protection differs between aptamers. Fold change was determined by dividing the fraction cleaved in the absence of the aptamer by that in its presence (i.e. greater fold change corresponds to greater protection). Asterisk indicates increased hydrolysis in the presence of the aptamer. (C) Binding of excess l-T9t and l-AdU5t to 5′-[³²P]-pre-miR-155t or modified variants. All binding reactions contain 3 μM aptamer, 1 nM 5′-[³²P]-pre-miR-155t (or variant), 5 mM MgCl₂, 50 mM KCl, 20 mM NaCl, and 25 mM Tris (pH 7.6). Variant M1 is mouse pre-miR-155. M = 5′-[³²P]-pre-miR-155t (or variant) control.

Figure 6.

Cross-chiral l-DNA aptamers inhibit Dicer-mediated cleavage of pre-miR-155. Percent Dicer activity (relative to a no l-aptamer control) was plotted as a function of l-aptamer concentration. Each data point represents the mean ± S.D. (n = 3). The data were fit to a four-parameter sigmoidal dose-response model and IC₅₀ values were determined.