A naphthyridine carbamate dimer (NCD) is a synthetic ligand for DNA containing a CGG/CGG sequence. Although NCD can bind selectively and tightly to a CGG/CGG sequence, the highly cooperative 2:1 binding mode has hampered precise analysis of the binding. We describe herein the synthesis of a series of naphthyridine tetramers consisting of two NCD molecules connected with various linkers to seek a ligand that binds to a CGG/CGG sequence exclusively with a 1:1 stoichiometry. Among the tested ligands, NCTB and Z-NCTS, which have linker moieties with restricted conformational flexibility [biphenyl and (Z)-stilbene linker, respectively], gave the exclusive formation of a 1:1 ligand–CGG/CGG complex. The (Z)-stilbene linker in Z-NCTS was designed to have pre-organized conformation appropriate for the binding and, in fact, resulted in the highest binding affinity. Thermodynamic parameters obtained by isothermal titration calorimetry indicated that the stronger binding of Z-NCTS was attributed to its lower entropic cost. The present study provides not only a novel 1:1 binding ligand, but also valuable feedback for subsequent molecular design of DNA and RNA binding ligands.

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The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non-structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand-assisted formation of loop-loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G-G mismatches in double-stranded DNA, we successfully demonstrated the formation of both inter- and intra-molecular NCT6-assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)3 in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly-labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6-assisted loop-loop complex. Förster resonance energy-transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.

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Voller Schleifen: Die Wechselwirkung einer Reihe von fehlpaarenden Molekülen mit Haarnadelschleifen‐DNA, die eine d(CGG)3‐Sequenz in der Schleife enthält, wird beschrieben (siehe Bild). Native Polyacrylamid‐Gelelektrophorese von Haarnadelschleifen‐DNA zeigt, dass das neu synthetisierte fehlpaarende Molekül, eine tetramere Form von N‐Methoxycarbonyl‐1,8‐naphthyridin, an der Bildung eines Schleifen‐Schleifen‐Komplexes zwischen zwei DNA‐Haarnadelschleifen beteiligt ist.