**7. Targeting RRE Conformations: the HIV Epitranscriptome.**

Over several decades, post-transcriptional chemical modifications that impact RNA metabolism, function, and localization have been recorded, the most common associated with tRNA and ribosomal RNA. Of the >100 post-transcriptional modifications identified to date, N6-methyladenosine (m6A) has been the most extensively studied with respect to its importance in health and disease [66]. Although the functional role remains to be fully elucidated, a role for post-transcriptional modification of the retroviral RNA genome was first proposed from studies by Kane et al. [67]. Three recent studies have established m6A modification of the HIV-1 genome [68–70], of which Lichinchi et al. [69] have suggested that methylation of conserved adenosines in RRE SL-II (A7877 and A7883) enhanced Rev binding and influenced nuclear export of viral RNA.

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Of possible roles ascribed to post-transcriptional modification, alterations to RNA structure to upor downregulate interactions with either host or viral proteins would seem likely [71]. An extension of this notion would be the potential to develop small molecules that specifically recognized m6A-induced structural alterations within the RNA genome. As a step in this direction, a small molecule microarray (SMM) strategy was developed to allow facile and rapid screening for RNA-binding chemotypes [72–74]. The SMM strategy is outlined in Figure 8A, wherein a short, structured RNA (40–60 nt), appended with a fluorophore, is flowed over a library of covalently immobilized small molecules. Ligands interacting with the RNA are recorded as a fluorescent signal (Figure 8B). To determine whether differential recognition of modified RNA could be achieved, unmethylated and m6A-modified RREs were screened. As indicated in Figure 8C, chemotypes specific for SL-IIB and m6A SL-IIB were identified, in addition to a third class that recognized both RNAs. Although recent study of Chu et al. [75] employing several biochemical and biophysical strategies concluded that the stability, structure, and dynamics of RRE SL-IIB are only marginally affected by m6A modification, SMM screening implies that these might be sufficient for selective ligand recognition. While high throughput screening strategies for RNA are in their infancy, data reported here suggests the notion of targeting viral epitranscriptomes should not be overlooked.

**Figure 8.** Screening for small molecules that recognize m6A-modifed HIV-1 RRE SL-IIB. (**A**) A synthetic RNA fragment harboring the m6A modifications reported in SL-IIB is labeled with Cy5 on its 3- terminus. (**B**) Labeled SL-II RNA was flowed over microtiter plates containing covalently immobilized small molecules. Binding of RRE SL-II RNA to candidate ligands was recorded via a fluorescence signal. (**C**) HTS screening suggests specificity of small molecules for unmethylated (left) and methylated SL-IIB (right). The central panel highlights a ligand that recognizes both SL-II forms.
