A Small-Molecule Approach Enables RNA Aptamers to Function as Sensors for Reactive Inorganic Targets

Abstract

Fluorescent light-up aptamer (FLAP) systems are promising (bio)sensing platforms that are genetically encodable. However, FLAP-mediated detection of each distinct target necessitates either in vitro selection or engineering of nucleic acid sequences. Furthermore, an aptamer that binds an inorganic target or a chemical species with a short lifetime is challenging to realize. Here, we describe a small-molecule approach that makes it possible for a single FLAP system to detect chemically unique, non-fluorogenic, and reactive inorganics. We developed functionalized pre-ligands of RNA aptamers that bind benzylidene imidazolinones (Baby Spinach, Broccolli, Squash). Reactive inorganics, hydrogen sulfide (H₂S/HS^-) and hydrogen peroxide (H₂O₂), can specifically convert these pre-ligands into native ligands that fluoresce with FLAPs. Adaptation of this platform to live cells opened an opportunity for constructing whole-cell sensors: Escherichia coli transformed with a Baby Spinach-encoding plasmid and incubated with pre-ligands generated fluorescence in response to exogenous H₂S/HS^- or H₂O₂. Leveraging the functional group reactivity of small molecules eliminates the requirement of in vitro selection of a new aptamer sequence or oligonucleotide scaffold engineering for distinct molecular targets. Our method allows for detecting inorganic, short-lived species, thereby advancing FLAP systems beyond their current capabilities.

Keywords: Aptamers; cell-based biosensor; redox processes; small-molecule ligand; synthetic biology.

Figure 1

Conceptual illustration of how a FLAP system can be repurposed to detect an inorganic molecular target without aptamer engineering. (A) Strong fluorescence induced by non‐covalent binding interactions between an aptamer and its native, intrinsically fluorogenic ligand. (B) Inorganic targets, like majority of biologically relevant molecules, may lack fluorogenicity, affinity for a structured nucleic acid, or both. (C) If exists, the chemical reactivity of the inorganic target can be utilized to form the native aptamer ligand in situ from a pre‐ligand that has a reduced fluorogenic binding. This would allow for the detection of inorganic targets by a single FLAP system, thereby expanding the application space of structured nucleic acids without in vitro selection or additional oligonucleotide scaffold engineering. (D) Interconversion between an aptamer ligand [e.g., 4‐hydroxybenzylidene imidazolinone (HBI) derivative] and pre‐ligand (e.g., 4‐O‐functionalized‐benzylidene imidazolinone).

Figure 2

Docked poses: binding mode of the selected 4‐O‐aryl‐modified derivatives of DFHBI. (A) Model of key hydrogen‐bonding interactions within the crystal structure of Spinach‐DFHBI (PDB:4TS2) near the binding site. (B) Binding mode of DFHBI docked using AutoDock Vina. (C–F) docked poses of the 4‐O‐aryl‐modified small molecule derivatives of DFHBI.

Figure 3

Baby Spinach pre‐ligand designs and native ligand formation. (A) Pre‐ligands synthesized from respective building blocks (yellow panels) for detecting (left) H₂S/HS⁻ or (right) H₂O₂. (B, top) Generation of DBrHBI from PyDSBz‐DBrHBI by Na₂S treatment. (Bottom) Overlay of extracted mass spectrum acquired following liquid chromatography. For PyDSBz‐DBrHBI, [M+H]⁺ requires 619.9131; found 619.9116; for DBrHBI, [M+H]⁺ requires 374.9162; found 374.9198. (C, top) Generation of MFHBI from MBBA‐MFHBI by H₂O₂ treatment. (Bottom) Spectrum acquired via direct injection. For MFHBI, [M+H]⁺ requires 235.0878; found 235.0922. No significant peak related to MBBA‐MFHBI was observed. (B and C, bottom) Crude reaction aliquot was taken after 1 hour of mixing pre‐ligand with the respective redox agent and analyzed in positive ionization mode. Peak heights are not an accurate representation of relative concentrations.

Figure 4

Fluorescence quenching with pre‐ligands. (A) Aptamer affinities of pre‐ligands were assessed via competitive ligand binding assay. Fluorescence intensities of (B) Baby Spinach/DBrHBI complex vs PyDSBz‐DBrHBI concentration, and (C) Baby Spinach/MFHBI complex vs MBBA‐MFHBI concentration. Error bars represent standard deviation, n=3.

Figure 5

Performances of PyDSBz‐DBrHBI and MBBA‐MFHBI with Baby Spinach in cell‐free conditions. (A, D) Time‐dependent fluorescence increase of pre‐ligands upon treatment with H₂S/HS⁻ (from Na₂S) or H₂O₂. (B, E) The fluorescence fold increase (F/F₀ ) measured 60 minutes after the addition of RSS (500 μM) and ROS (100 μM), respectively. Here, F and F₀ are the fluorescence intensities for samples with and without the redox species, respectively. (C, F) LoD measurements for Baby Spinach with PyDSBz‐DBrHBI or MBBA‐MFHBI incubated with H₂S/HS⁻ (from Na₂S) or H₂O₂ for 60 minutes. Conditions: DBrHBI, PyDSBz‐DBrHBI, MFHBI, and MBBA‐MFHBI (5 μM), Baby Spinach (1 μM), HEPES (pH 7.4, 50 mM), KCl (100 mM), MgCl₂ (10 mM), and Na₂S (500 μM) or H₂O₂ (100 μM). (A, B, and C) Data sets labeled as “H₂S” by convention. Single‐tailed Student's t test: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Error bars represent standard deviation based on triplicates.

Figure 6

Turning E. coli into redox sensors. (A) When transformed with pET‐21‐3xBaby Spinach and incubated with a Baby Spinach pre‐ligand, BL21 Star (DE3) E. coli can fluoresce in response to a redox cue. (B, D) Confocal microscopy images of live E. coli sensing (B) H₂S/HS⁻ or (D) H₂O₂. Top row panels represent positive and negative controls, including transfection with pUC19 plasmid, which contains no aptamer gene. Bottom row panels represent redox sensing at 0, 15, 30, 45, and 60 minutes. (C, E) Quantitative assessment of fluorescence outputs from cells over 60 minutes, along with the positive control measured at 60‐minute time point using either (C) DBrHBI or (E) MFHBI. Cells were incubated in M9 media containing 100 μM of the HBI derivative. Redox species introduced to the imaging media: (B, C) H₂O₂ (100 μM), (D, E) Na₂S (2 mM). For co‐staining, cells were treated with a FM 4‐64FX (red membrane dye, 1 μg/ml, 2 minutes). Channel/excitation (nm): DBrHBI/440, MFHBI/488, Fm4‐64FX/568. Scale bar: 5 μm. Error bars represent standard error of the mean.