Studying the features in RNA folds that bind small molecules. To identify the RNA 3-dimensional folds that bind each small molecule, bound RNAs were excised from the corresponding position on the microarray surface, amplified via RT-PCR, and sequenced. To define high affinity and selective or privileged interactions, we used a bioinformatics analysis tool previously developed in our laboratory. Coined high throughput-structure activity relationships through sequencing, HiT-StARTS calculates the enrichment of a particular SMIRNA-bound RNA fold in 2DCS sequencing data as compared to the sequencing data derived for the starting RNA motif library; and then calculates the statistical significance of that enrichment reported as a Z-score (Zobs). This approach allows precise definition of: (i) the number of RNA folds that each small molecule binds selectively; (ii) the relative affinity of each of the selected interactions; and (iii) a rich source of RNA fold-small molecule binding partners that can be used to design small molecules that target three dimensionally folded RNAs by using Inforna. We have previously shown that the RNA 3-dimensional motifs that bind small molecules avidly have a Zobs > 8 and that the relative affinity of the selected interactions directly correlates with Zobs. Hence, small molecules with higher Zobs are more fit for binding a SMIRNA than those with lower scores. In summary, these studies defined an encyclopedia of 41,000 RNA fold-small molecule interactions that were deposited into Inforna, an increase of 20-fold over the current Inforna server (Table ). Interestingly, some small molecules only bound members of certain RNA libraries while some bound members from all three. For example, 43 ILL bound 18 compounds (n = 3 unique) while 32 ILL bound 16 (n = 1 unique) and 33 ILL bound 15 small molecules (n = 1 unique). Perhaps, 43 ILL bound a larger subset of the small molecules because of the greater diversity in the RNA motif library. A detailed analysis of the types of RNA folds that bind small molecules is presented in the Supporting Information, including a LOGOS analysis (Figs. S2 to S6). Some interesting observations include that single nucleotide bulges bind a variety of small molecules (Table and Fig. and D); randomized regions with additional base pairs, and higher GC pairing appear more frequently in RNAs selected to bind small molecules than those that do not bind; and U-rich internal loops have lower small molecule binding capacity.