Using Selection to Identify and Chemical Microarray to Study the RNA Internal Loops Recognized by 6′‐N‐Acylated Kanamycin A

Abstract

Herein, we describe our initial steps towards identifying the RNA secondary structure motifs that are recognized by small molecules. We selected members of an RNA 3x3 internal loop motif library that bind kanamycin A, an RNA-binding aminoglycoside antibiotic, by using only one round of selection. A small internal-loop library was chosen because members are likely to be present in other larger, biologically relevant RNAs. We have identified several internal loops of various size and base composition that kanamycin A prefers to bind. The highest affinity structures are two 5'-UU/3'-CU 2x2 internal loops closed by AU pairs. Binding is specific for the selected internal loops with the highest affinities, since binding to the RNA cassette used to display the library or to DNA is >150-fold weaker. Enzymatic mapping experiments also confirm binding of kanamycin A to the internal loops. This method lays the foundation for finding RNA secondary structure elements that bind small molecules and for interrogating factors affecting RNA-ligand interactions. Information from these and subsequent studies will: 1) facilitate the rational and modular design of drugs or probes that bind target RNAs with high affinity, provided the secondary structure of the target is known and 2) give insight into the potential bystander RNAs that aminoglycosides bind.

New trends in the use of aminoglycosides

“…90–92 6’- N -hexynoylated KAN A was also investigated and demonstrated to improve proper splicing of the pre-mRNA transcripts. Upon further development, 6’- N -hexynoylated KAN A conjugated to d -Arg 9 was found to improve cellular permeability and localization in DM type 1 cell culture and animal model, signifying their application as a potential therapeutic option.…”

Folding and Finding RNA Secondary Structure

“…Structure in the target mRNA is an important consideration in designing siRNAs (Lu and Mathews 2007;Shao et al 2007;Tafer et al 2008) and determining microRNA targets Long et al 2007). Additionally, the Disney group is using small-molecule microarray methods to deduce the basis for matching potential drugs with RNA motifs that bind them strongly Disney and Childs-Disney 2007). Microarray methods based on short, chemically modified oligonucleotides (Kierzek et al 2008;Kierzek et al 2009) are also providing insight into the rules that govern oligonucleotide binding to structured RNAs, which should facilitate design of nucleic acid based therapeutics.…”

Methods to enable the design of bioactive small molecules targeting RNA

“…We employ a small molecule library-vs.-RNA library screen, named 2-Dimensional Combinatorial Screening (2DCS), to select the RNAs that bind each small molecule with the highest affinity. 2DCS uses a solid surface functionalized with RNA-binding small molecules, 35, 36 which is incubated with a labeled RNA library that contains a small, randomized region in the pattern of a discrete secondary structural element (such as a 3×3 nucleotide internal loop (3×3 ILL); Figure 1). The randomized region is kept intentionally small such that RNA motifs displayed in the library have a high probability of occurring in cellular RNAs.…”

“…K-alkyne binds the DM2 motif with nanomolar affinity, similar to affinities of the 2×2 pryimidine-rich RNA internal loops selected to bind via 2DCS. 35 …”