Defining small molecule-RNA fold interactions and studying features in small molecules that bind RNA. An in silico analysis of the AstraZeneca corporate collection (2M compounds) generated a diverse compound subset comprised of 1967 members with RNA-binding potential based on chemical similarity with previous SMIRNAs deposited in the Inforna server. These compounds were then studied for binding to libraries of 3-dimensional RNA folds. The RNA motif libraries comprise a unimolecular hairpin that displays various structural elements, including 32 (32 ILL; n = 1024 unique RNAs), 33 (33 ILL; n = 4096 unique RNAs), and 43 (43 ILL; n = 16384 unique RNAs) internal loops (ILs) respectively (Fig. ). To facilitate studying the binding interactions of the RNA motif and AstraZeneca small molecule libraries, we used an approach dubbed AbsorbArray, where the small molecules are absorbed onto agarose-coated microarrays allowing for spatial encoding. The arrays were then probed for binding to radiolabeled RNA motif libraries in the presence of 100-fold excess of unlabeled competitor oligonucleotides that mimic the constant regions in the library (1-5), DNA (6,7), and tRNA (8) (Fig. ). Due to the combinatorial nature of a library-versuslibrary screen, this experiment probed 43 million RNA fold-small molecule binding interactions simultaneously. Of the 1967 small molecules studied, 23 selectively bound the unique 3-dimensional folds displayed by the RNA libraries providing a hit rate of 1.2%.