This work demonstrates the utility of peptoid microarrays for discovering new ligands for specific RNAs. Previous work has employed the microarray format as a quantitative analytical tool to profile the relative affinities and specificities of known RNA-binding antibiotics (36–38
) and to find improvements to peptide sequences that bind to an RNA required for packaging HIV (46
). Recently, Labuda and co-workers reported a screen of peptoids in a microarray format to find improved inhibitors of a group I intron (47
). These efforts used libraries of 4–120 different RNA-binding compounds. To discover new ligands for a novel, pre-defined RNA target, we have used the microarray platform as a qualitative tool to screen a relatively large library of 7680 different compounds. As a qualitative screen, it is akin to bead-based library screens, in which a library of compounds on beads is screened for binding to a fluorescently labeled biomolecule, and hit compounds are identified by visual brightness of individual beads with the use of a fluorescent microscope (25
). However, having the library elements arrayed on a surface allows systematic consideration of each, which is difficult in the bead-based format. Also, the ability to perform replicate experiments allows confirmation of reproducibility and specificity prior to hit identification and solution-phase testing.
The miR-21 precursor hairpin is a novel target, without previously known small molecule ligands, and very few sequence-specific ligands are known for any RNA hairpin loop. Thus, we had no a priori expectations for the affinities that library compounds with specificity for the target RNA would have. For this initial test of the approach, our goal was to discover positive and specific molecular interactions that can be subsequently utilized toward making ligands of high affinity and specificity. We chose the peptoid scaffold for our library, in part, because these subsequent steps are facilitated by the ease with which peptoids can be modified. We attempted to bias our library toward a baseline affinity of Kd ~1–10 mM by including arginine in every library element. The affinity of the diglycine linker was fortuitous, since we had no prior expectation that it would contribute to binding. Together, the arginine and glycine components of the library contribute 4.9 kcal/mol (calculated from the Kd) to the free energy of association of the library elements to the target RNA, with a weak specificity for the target RNA over the control.
Most of the array features gave no indication of binding the labeled target RNA, but 12% of them were fluorescent after its application, some with SNR greater than 100. Most of these features displayed similar signal to noise with the control hairpin. The compounds rich in alkyl amines that were predominant among the peptoids with high signal to noise are likely to have high affinity for the RNA because the amines are substantially protonated, creating an electrostatic attraction for the polyanionic RNA. While some of these compounds may have specificity for the target RNA that could be revealed by screening under more stringent conditions and by rigorous quantitation of the fluorescent signal, a large nonspecific component of binding is likely to interfere with optimization of the specific interactions. Therefore, we focused our attention on hit compounds that reproducibly showed no interaction with the control hairpin.
Both of the compounds to which this requirement narrowed our focus had affinity for the target RNA in solution 15- to 20-fold higher than the affinity of a compound that did not bind the labeled target on the array (compound 3
). The affinity of compound 3
is comparable to that of the arginine and linker alone (4
). Thus, the array screen was successful in identifying compounds from the library that are better ligands than one that shows no interaction on the array. The variable peptoid components of the lead compounds identified contribute ~20-fold to the affinity over the arginine and glycines alone, corresponding to ~1.6 kcal/mol of additional free energy of binding. Furthermore, peptoids 1
share common features that are not found in other library elements: an aromatic heterocycle at the N-terminus and an oxygen three atomic centers from the backbone in the second position. These similarities suggest involvement of specific molecular interactions in the binding of the RNA, supporting the conclusion that the microarray screen successfully identified library elements that form positive, specific interactions with the target. Though the signal to noise ratio of compound 1
, near the threshold of being visible, was significantly lower than that of compound 2
, the solution phase affinities of these two compounds for the target were similar, indicating that fluorescent signal on the array is influenced by factors other than solution-phase affinity, such as linker effects, well known in the interactions of surface-bound ligands with RNA (36
Though the specificities of the initial lead compounds are modest, the 4.7-fold specificity of 1 is significant in light of the similarity between the target and control hairpins and the paucity of ligands reported in the literature with appreciable sequence specificity for RNA hairpin loops. This difference is all the more significant in light of the fact that it is primarily due to the sequence order within the five-nucleotide loop, the loop sequence composition being held constant, with little dependence on other differences between the target and control hairpins. Furthermore, much of this specificity can be attributed to distinct functional elements of the ligand. The terminal pyridine and the hydroxyethyl side chain contribute the majority of the free energy of association that is specific to the target RNA.
Although these groups contribute specific interactions to the complex, they do not necessarily optimize those interactions. The potential for enhancing these contacts suggests an avenue for improvement of the lead compound, by screening a series of derivatives in which a critical group is incrementally varied. This approach was immediately productive when applied to the terminal pyridine of 1
. By simply varying the position at which the pyridine is linked to the backbone, the affinity and specificity were both increased. The affinities of the resulting compounds for the target RNA are comparable to the affinity of DGCR8, the double-stranded RNA-binding component of the microprocessor complex, for primary miRNAs (50
). This is true for compound 13
in the presence of physiologically relevant concentrations of mono- and divalent cations. Development of further optimized ligands by testing conservative variations of the hydroxyethyl side chain is in progress.
The process followed to discover ligand 13 illustrates a systematic approach to the development of ligands for specific, pre-defined RNA molecules. The screen of a library of potential ligands is the first step, aimed at discovery of specific interactions. The library is designed so that its members not only have desirable qualities for biologically active compounds (cell permeability, limited size, biological stability), but are also easily modified in the subsequent steps, when the structural elements of the lead compounds that contribute specific affinity are identified and varied to find improvements.
Work is underway to test the effects of the ligands found here on miRNA processing in cell culture and in cell-free miRNA processing assays and to explore the broader biological effects of these compounds. Though cleavage of miRNA precursors by Drosha does not depend on specific loop sequences, mutations that stabilize an internal conformation of the loop diminish or abolish cleavage (51
). Thus, a ligand that recognizes, thereby stabilizing, a hairpin conformation might similarly inhibit processing. This inhibition could be realized even if the bound conformation is different from the most stable conformation in the absence of ligand. The dependence of binding on loop sequence, the binding-induced change of 2-ap fluorescence when the fluorescent probe is in the hairpin but not in the stem, and the conformational changes within the loop revealed by Mg2+
-mediated cleavage indicate that the binding site for the compounds identified in this study includes the loop. A small molecule that binds to a miRNA precursor might also interfere with endogenous factors that interact with the hairpin loop to stimulate cleavage by Drosha (13
) or with processing steps that follow Drosha cleavage, including removal of the loop or intracellular localization (53
). In any of these cases, a compound that specifically interferes with maturation of miR-21 will be a useful tool for understanding miRNA regulation and could be of therapeutic value.