@article {104897, title = {HT-SELEX-based identification of binding pre-miRNA hairpin-motif for small molecules.}, journal = {Mol Ther Nucleic Acids}, volume = {27}, year = {2022}, month = {2022 Mar 08}, pages = {165-174}, abstract = {

Selective targeting of biologically relevant RNAs with small molecules is a long-standing challenge due to the lack of clear understanding of the binding RNA motifs for small molecules. The standard SELEX procedure allows the identification of specific RNA binders (aptamers) for the target of interest. However, more effort is needed to identify and characterize the sequence-structure motifs in the aptamers important for binding to the target. Herein, we described a strategy integrating high-throughput (HT) sequencing with conventional SELEX followed by bioinformatic analysis to identify aptamers with high binding affinity and target specificity to unravel the sequence-structure motifs of pre-miRNA, which is essential for binding to the recently developed new water-soluble small-molecule CMBL3aL. To confirm the fidelity of this approach, we investigated the binding of CMBL3aL to the identified motifs by surface plasmon resonance (SPR) spectroscopy and its potential regulatory activity on dicer-mediated cleavage of the obtained aptamers and endogenous pre-miRNAs comprising the identified motif in its hairpin loop. This new approach would significantly accelerate the identification process of binding sequence-structure motifs of pre-miRNA for the compound of interest and would contribute to increase the spectrum of biomedical application.

}, issn = {2162-2531}, doi = {10.1016/j.omtn.2021.11.021}, author = {Mukherjee, Sanjukta and Murata, Asako and Ishida, Ryoga and Sugai, Ayako and Dohno, Chikara and Hamada, Michiaki and Krishna, Sudhir and Nakatani, Kazuhiko} } @article {97217, title = {Cyclic mismatch binding ligands interact with disease-associated CGG trinucleotide repeats in RNA and suppress their translation.}, journal = {Nucleic Acids Res}, year = {2021}, month = {2021 Aug 06}, abstract = {

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.

}, issn = {1362-4962}, doi = {10.1093/nar/gkab669}, author = {Konieczny, Patryk and Mukherjee, Sanjukta and Stepniak-Konieczna, Ewa and Taylor, Katarzyna and Niewiadomska, Daria and Piasecka, Agnieszka and Walczak, Agnieszka and Baud, Anna and Dohno, Chikara and Nakatani, Kazuhiko and Sobczak, Krzysztof} } @article {25257, title = {Design and Synthesis of Cyclic Mismatch-Binding Ligands (CMBLs) with Variable Linkers by Ring-Closing Metathesis and their Photophysical and DNA Repeat Binding Properties.}, journal = {Chemistry}, volume = {23}, year = {2017}, month = {2017 Aug 22}, pages = {11385-11396}, abstract = {

Cyclophane-containing bis(2-amino-1,8-naphthyridine) moieties attached to variable linkers at the C2-position (linker B) were synthesized as cyclic mismatch-binding ligands (CMBLs). Ring-closing metathesis (RCM) is used as a key step for the introduction of double bonds at the linker B. Decreasing the size of the linker of the substrate, formation of the RCM products with an increasing trans/cis (E/Z) ratio was observed with moderate to high overall yields. Concentration-dependent fluorescence spectra were observed for CMBLs with longer linkers (n=3), whereas concentration-independent spectra were observed for CMBLs with shorter linkers (n=2 and/or 1) with a marked exception of the E-alkene 6 a. Concomitant changes in the absorption as well as in the fluorescence spectra were also observed for the CMBLs with an increasing hydrophobicity of the solvent. Absorption and fluorescence spectra of the CMBLs in solutions containing 99-100 \% methanol resembled to that of the monomer. The binding behavior of these CMBLs with repeat DNA structures was investigated by using a surface plasmon resonance (SPR) assay and circular dichroism (CD) spectra. The cyclic E-alkenes 1 a (n=3) and 3 a (n=2) show an orthogonal binding relationship with d(CCTG)9 and d(CAG)9 . However, the selectivity for the cyclic Z-alkenes increased with decreasing the length of the linker from compound 2 b (n=3) to compound 7 b (n=1). These compounds display a large molecular diversity, which allowed the tuning of the binding affinity and selectivity of the CMBLs by varying the linkers towards various biologically significant repeat DNA structures.

}, issn = {1521-3765}, doi = {10.1002/chem.201702064}, author = {Mukherjee, Sanjukta and Dohno, Chikara and Nakatani, Kazuhiko} }