Formation of a Ligand‐Assisted Complex of Two RNA Hairpin Loops

Abstract

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.

Restoration of Ribozyme Tertiary Contact and Function by Using a Molecular Glue for RNA

“…The mixture of HHR1 and HHRS′ was subjected to native gel electrophoresis, which produced a new band of yellow color with mobility between HHR1 and HHRS′ alone (Figure b, lane 4). The yellow color indicates the presence of both HHR1 and HHRS′ in the band, which shows the formation of the HHR1–HHRS′ complex . In the case of the wild‐type sequence, the wtHHR–HHRS′ complex band migrated faster than that of HHR1 (Figure b, lane 3).…”

“…Among a series of naphthyridine derivatives that bind to DNA/RNA G–G mismatches, Z ‐NCTS is the most effective for the HHR1 activation (Supporting Information, Figure S5). The chemical structures of the linker that connects naphthyridines have a significant effect on the selectivity of targeting particular RNA structures.…”

Molecular Glue for RNA: Regulating RNA Structure and Function through Synthetic RNA Binding Molecules

“…[15] NCTn speciesa re naphthyridine tetramers, each consisting of two NCD molecules connected by af lexible aliphatic alkyl linker of n methylene units (n = 5, 6, 7, and 8). [24,25] NCTnsw ere not effective in the activation of HHR1, butc ould induce complex formation in two RNA hairpin loops, as well as pseudoknot formation in particularR NAs, depending on n. [24,25] NCTnsf unction as molecular glue for the specific RNAs. Conversely,( Z)-NCTS was not effective in inducing these RNA structures (unpublished data).…”

Restoration of Ribozyme Tertiary Contact and Function by Using a Molecular Glue for RNA

“…HHR2 and HHR3 lack one of the GG sequences in an internal loop in stem Iand in ahairpin loop in stem II, respectively.Instead, they have awild-type sequence Z-NCTS recognized the UGG/AGG site in HHR1 and the resulting complex gained RNA-cleaving activity.T o confirm that the HHR1 activation is due to tertiary contact at the loop-loop region induced by Z-NCTS,t he structural change of HHR1 was analyzed by native PAGE (Figure 4). [24] In the case of the wild-type sequence,t he wtHHR-HHRS' complex band migrated faster than that of HHR1 (Figure 4b,l ane 3). [4] HHR1 and the uncleavable HHRS (HHRS')were labeled at the 5' terminus with different fluorophores,Cy3 (colored red in Figure 4b)a nd Alexa Fluor 488 (colored green), respectively.T he mixture of HHR1 and HHRS' was subjected to native gel electrophoresis,w hich produced an ew band of yellow color with mobility between HHR1 and HHRS' alone ( Figure 4b,lane 4).…”

“…Among as eries of naphthyridine derivatives that bind to DNA/RNAG -G mismatches, [21,23,24] Z-NCTS is the most effective for the HHR1 activation (Supporting Information, Figure S5). Thechemical structures of the linker that connects naphthyridines have as ignificant effect on the selectivity of targeting particular RNAstructures.…”