Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar KD

Lu, Xiaocen; Passalacqua, Luiz F. M.; Nodwell, Matthew; Kong, Kristen Y. S.; Caballero-García, Guillermo; Dolgosheina, Elena V.; Ferré-D’Amaré, Adrian R.; Britton, Robert; Unrau, Peter J.

doi:10.1093/nar/gkae493

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar K_D

Journal Article · Mon Jul 01 00:00:00 EDT 2024 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkae493· OSTI ID:2471008

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Simon Fraser Univ., Burnaby, BC (Canada)
National Institutes of Health (NIH), Bethesda, MD (United States)

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer–ligand complex with a remarkable set of properties: Its unprecedented K_D of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-05CH11231; AC02-06CH11357

OSTI ID:: 2471008

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 14 Vol. 52; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar KD

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Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar K_D