| Autor: |
Surynt P; Division of Biophysics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093, Warsaw, Poland.; Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland. j.jemielity@cent.uw.edu.pl., Wojtczak BA; Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland. j.jemielity@cent.uw.edu.pl., Chrominski M; Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland. j.jemielity@cent.uw.edu.pl., Panecka-Hofman J; Division of Biophysics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093, Warsaw, Poland., Kwapiszewska K; Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224, Warsaw, Poland., Kalwarczyk T; Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224, Warsaw, Poland., Kubacka D; Division of Biophysics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093, Warsaw, Poland., Spiewla T; Division of Biophysics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093, Warsaw, Poland., Kasprzyk R; Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland. j.jemielity@cent.uw.edu.pl., Holyst R; Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224, Warsaw, Poland., Kowalska J; Division of Biophysics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093, Warsaw, Poland., Jemielity J; Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland. j.jemielity@cent.uw.edu.pl. |
| Abstrakt: |
The trimethylguanosine (TMG) cap is a motif present inter alia at the 5' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions in vitro and, potentially, in vivo , particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2'- O -position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar K D (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by ∼50-fold for the corresponding 3'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement ( F m / F 0 ) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions. |
