RNA-Ligand Interactions Quantified by Surface Plasmon Resonance with Reference Subtraction.

Structured RNAs bind ligands and are attractive targets for small-molecule drugs. A wide variety of analytical methods have been used to characterize RNA-ligand interactions, but our experience is that most have significant limitations in terms of material requirements and applicability to complex RNAs. Surface plasmon resonance (SPR) potentially overcomes these limitations, but we find that the standard experimental framework measures notable nonspecific electrostatic-mediated interactions, frustrating analysis of weak RNA binders. SPR measurements are typically quantified relative to a non-target reference channel. Here, we show that referencing to a channel containing a non-binding control RNA enables subtraction of nonspecific binding contributions, allowing measurements of accurate and specific binding affinities. We validated this approach for small-molecule binders of two riboswitch RNAs with affinities ranging from nanomolar to millimolar, including low-molecular-mass fragment ligands. SPR implemented with reference subtraction reliably discriminates specific from nonspecific binding, uses RNA and ligand material efficiently, and enables rapid exploration of the ligand-binding landscape for RNA targets.

Discovery of a large-scale, cell-state-responsive allosteric switch in the 7SK RNA using DANCE-MaP.

7SK is a conserved noncoding RNA that regulates transcription by sequestering the transcription factor P-TEFb. 7SK function entails complex changes in RNA structure, but characterizing RNA dynamics in cells remains an unsolved challenge. We developed a single-molecule chemical probing strategy, DANCE-MaP (deconvolution and annotation of ribonucleic conformational ensembles), that defines per-nucleotide reactivity, direct base pairing interactions, tertiary interactions, and thermodynamic populations for each state in RNA structural ensembles from a single experiment. DANCE-MaP reveals that 7SK RNA encodes a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium shifts in response to cell growth and stress and can be targeted to modulate expression of P-TEFbresponsive genes. Our study reveals that RNA structural dynamics underlie 7SK function as an integrator of diverse cellular signals to control transcription and establishes the power of DANCE-MaP to define RNA dynamics in cells.

Synthesis of Enantiomerically Pure 6-Substituted-Piperazine-2-Acetic Acid Esters as Intermediates for Library Production.

The piperazine heterocycle is broadly exploited in FDA-approved drugs and biologically active compounds, but its chemical diversity is usually limited to ring nitrogen substitutions, leaving the four carbon atoms underutilized. Using an efficient four-step synthesis, chiral amino acids were transformed into 6-substituted piperazine-2-acetic acid esters as diastereomeric mixtures whose cis and trans products could be chromatographically separated. From six amino acids (both antipodes), a complete matrix of 24 monoprotected chiral 2,6-disubstituted piperazines was obtained, each as a single absolute stereoisomer in multigram quantities. These diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as scaffolds for parallel library synthesis or intermediates for the production of novel piperazine compounds.