New discoveries in RNA biology underscore a need for chemical tools to clarify their roles in pathophysiological mechanisms. In certain cancers, synthesis of the let-7 microRNA tumor suppressor is blocked by an RNA binding protein (RBP) Lin28, which docks onto a conserved sequence in let-7 precursor RNA molecules and prevents their maturation. Thus, the Lin28/let-7 interaction might be an attractive drug target, if not for the well-known difficulty in targeting RNA-protein interactions with drugs. Here, we describe a protein/RNA FRET assay using a GFP-Lin28 donor and a black-hole quencher (BHQ)-labeled let-7 acceptor, a fluorescent protein/quencher combination which is rarely used in screening despite favorable spectral properties. We tested 16 000 molecules and identified N-methyl-N-[3-(3-methyl[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide, which blocked the Lin28/let-7 interaction, rescued let-7 processing and function in Lin28-expressing cancer cells, induced differentiation of mouse embryonic stem cells, and reduced tumor-sphere formation by 22Rv1 and Huh7 cells. A biotinylated derivative captured Lin28 from cell lysates consistent with an on-target mechanism in cells, though the compound also showed some activity against bromodomains in selectivity assays. The Lin28/let-7 axis is presently of high interest not only for its role as a bistable switch in stem-cell biology but also because of its prominent roles in numerous diseases. We anticipate that much can be learned from the use of this first reported small molecule antagonist of Lin28, including the potential of the Lin28/let-7 interaction as a new drug target for selected cancers. Furthermore, this approach to assay development may be used to identify antagonists of other RBP/RNA interactions suspected to be operative in pathophysiological mechanisms.

A chemical method for the post‐synthetic labeling of pre‐miRNAs on solid support using easily accessible reagents was developed. The procedure was employed to generate a library of 31 pre‐microRNAs carrying labels commonly used in chemical biology, including Cy3, trioxalen, biotin, and BHQ‐1.

The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson–Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation.

Modified oligonucleotides bearing multiple functional labels are valuable tools in RNA biology. Efficient synthetic access to singly modified short DNAs and RNAs has been developed in the past years and paved the way to a first generation of oligonucleotide tools. Here, we describe an efficient procedure for the site-specific hetero bis-labeling of long RNAs. We exemplified the method with the preparation of Cy3/Cy5 pre-microRNAs labeled at selected internal sites as probes for microRNA maturation.

Determining atomic-level characteristics of molecules on two-dimensional surfaces is one of the fundamental challenges in chemistry. High-resolution nuclear magnetic resonance (NMR) could deliver rich structural information, but its application to two-dimensional materials has been prevented by intrinsically low sensitivity. Here we obtain high-resolution one- and two-dimensional 31 P NMR spectra from as little as 160 picomoles of oligonucleotide functionalities deposited onto silicate glass and sapphire wafers. This is enabled by a factor >10 5 improvement in sensitivity compared to typical NMR approaches from combining dynamic nuclear polarization methods, multiple-echo acquisition, and optimized sample formulation. We demonstrate that, with this ultrahigh NMR sensitivity, 31 P NMR can be used to observe DNA bound to miRNA, to sense conformational changes due to ion binding, and to follow photochemical degradation reactions.