Biology subjects

Siwik, S. H.

Publications and source records attributed to Siwik, S. H..

3 recordsLinked to original sources

bioRxiv2026

Structure of a dopamine-binding RNA aptamer reveals metal-mediated ligand recognition

The selection of small-molecule binding RNA aptamers enables the creation of ligand-responsive RNA tools, yet most aptamers fail to operate reliably across diverse environments. Scaffolded selection addresses this limitation by preserving the tertiary architecture of a riboswitch scaffold while driving the evolution of a new ligand-binding pocket. Using the xpt purine riboswitch aptamer, we previously generated dopamine-binding aptamers. Here, we report the crystal structures of two representative variants in their apo and dopamine-bound states to define how they recognize ligand while maintaining scaffold integrity. The structures demonstrate that scaffolded selection enforces global fold conservation and retains the defining tertiary interactions of the parental riboswitch. Local remodeling, triggered by deletions acquired during selection, rewires the three-way junction to build extensive interaction networks that host the dopamine-binding pocket. A deeply buried potassium ion anchors ligand recognition by coordinating the dopamine hydroxyl group while the RNA engages the catechol ring through stacking and hydrogen bonding interactions. Structure probing shows minimal conformational changes upon ligand binding, indicating that the aptamers adopt a largely preorganized fold. These findings strengthen the central premise of scaffolded selection: riboswitch-derived "superfolder" architectures can bias in vitro selections towards aptamers that conserve global structure while supporting locally diverse binding pockets. This balance between structural stability and local plasticity expands the capacity of a single RNA fold to recognize chemically distinct ligands and positions scaffolded selection as a powerful platform for engineering robust RNA-based sensing and regulatory devices. TOC Abstract (graphical) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/729643v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1c92ee4org.highwire.dtl.DTLVardef@1258d16org.highwire.dtl.DTLVardef@51d755org.highwire.dtl.DTLVardef@1102c63_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Structural basis for ring-opening fluorescence by the RhoBAST RNA aptamer.

Tagging RNAs with fluorogenic aptamers has enabled imaging of transcripts in living cells, thereby revealing novel aspects of RNA metabolism and dynamics. While a diverse set of fluorogenic aptamers has been developed, a new generation of aptamers are beginning to exploit the ring-opening of spirocyclic rhodamine dyes to achieve robust performance in live mammalian cells. These fluorophores have two chemical states: a colorless, cell-permeable spirocyclic state and a fluorescent zwitterionic state. Recently, the developed dye SpyRho555 almost exclusively adopts the closed state in solution and becomes fluorescent in complex with the RhoBAST aptamer. To understand the basis for RhoBAST-SpyRho555 fluorogenicity, we have determined crystal structures of RhoBAST in complex with 5-carboxytetramethylrhodamine and a SpyRho555 analogue, MaP555. RhoBAST is organized by a perfect four-way junction that positions two loops to form the dye-binding pocket. The core of the ligand resides between a tri-adenine floor and a single guanine base, largely driven by {pi}-stacking interactions. Importantly, the unpaired guanine interacts with the 3-position group of MaP555 to stabilize the open conformation, supported by mutagenesis data, and may play an active role in promoting the open conformation of the dye. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/630784v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1daaee9org.highwire.dtl.DTLVardef@11773corg.highwire.dtl.DTLVardef@ab41b9org.highwire.dtl.DTLVardef@167fae0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Targeting riboswitches with beta-axial substituted cobalamins

RNA-targeting small molecule therapeutics an emerging field hindered by an incomplete understanding of the basic principles governing RNA-ligand interactions. One way to advance our knowledge in this area is to study model systems where these interactions are better understood, such as riboswitches. Riboswitches bind a wide array of small molecules with high affinity and selectivity, providing a wealth of information on how RNA recognizes ligands through diverse structures. The cobalamin-sensing riboswitch is a particularly useful model system as similar sequences show highly specialized binding preferences for different biological forms of cobalamin. This riboswitch is also widely dispersed across bacteria and therefore holds strong potential as an antibiotic target. Many synthetic cobalamin forms have been developed for various purposes including therapeutics, but their interaction with cobalamin riboswitches is yet to be explored. In this study, we characterize the interactions of eleven cobalamin derivatives with three representative cobalamin riboswitches using in vitro binding experiments (both chemical footprinting and a fluorescence-based assay) and a cell-based reporter assay. The derivatives show productive interactions with two of the three riboswitches, demonstrating simultaneously plasticity and selectivity within these RNAs. The observed plasticity is partially achieved through a novel structural rearrangement within the ligand binding pocket, providing insight into how similar RNA structures can be targeted in the future. As the derivatives also show in vivo functionality, they serve as several potential lead compounds for further drug development.

biochemistry↗