A newly uncovered mode of nucleic-acid interaction allows two distinct strands of DNA to associate through a ligand-induced conformational rearrangement rather than conventional sequence complementarity. In this architecture, a structure-switching primary aptamer undergoes a defined spatial folding event upon binding a specific small molecule, exposing a previously inaccessible docking interface. An independently evolved secondary aptamer subsequently binds this newly presented docking interface primarily through noncanonical interactions, thereby reporting the ligand-bound state of the primary aptamer. Because Watson–Crick base-pairing typically governs nucleic-acid assemblies, this noncanonical mechanism expands the fundamental molecular features that synthetic oligonucleotides can be evolved to detect. The findings demonstrate that a ligand-induced structural state can itself act as the primary epitope for molecular recognition, shifting the focal point of nucleic-acid sensing away from direct chemical coordination of the target analyte and onto the physical conformation of the receptive DNA complex.
To establish the boundaries of this recognition mode, investigators developed a comprehensive selection workflow using four previously reported structure-switching primary aptamers that bind distinct small molecules: serotonin, dopamine, testosterone, and deoxycorticosterone 21-glucoside. The research team first examined the stability of these primary aptamer–ligand complexes using a fluorescence quenching assay involving a fluorescently labelled primary strand and a fully complementary anti-aptamer designed to hybridize and quench fluorophore emissions. These initial evaluations revealed pronounced operational differences across the four target systems. Serotonin produced clear, concentration-dependent stabilization at sub-micromolar thresholds, while deoxycorticosterone 21-glucoside exhibited comparable competitive behaviour at high micromolar concentrations. Dopamine required significantly higher concentrations of up to approximately twenty micromolar to achieve measurable separation from anti-aptamer hybridization, and testosterone demonstrated a much weaker concentration-dependent structural response under identical monitoring periods over five hours.
Recognizing that bulk fluorescence quenching assays capture only global conformational rearrangements that compete directly with duplex hybridization, the investigators reasoned that secondary aptamer recognition depends on localized docking surfaces that can emerge without large-scale structural remodeling. Consequently, all four candidate systems advanced into systematic systematic evolution of ligands by exponential enrichment, commonly termed SELEX, targeting the intact primary aptamer–ligand complex. Candidates were screened against immobilized biotinylated primary aptamers on streptavidin columns to filter out sequences prone to ligand-independent hybridization, followed by bead-based negative counter-selection against free ligand and unbound primary strands. Across thirteen iterative selection cycles, the seed-ligand concentration served as an experimental lever, regulating the equilibrium fraction of complexed versus unbound primary strands. This iterative strategy successfully enriched robust secondary aptamers for three of the four small molecules—serotonin, testosterone, and deoxycorticosterone 21-glucoside—while dopamine yielded no functional secondary binders under the tested ionic and stringency conditions.
The contrasting outcomes between serotonin and dopamine provided key structural insights into the rules governing secondary aptamer generation. Although both neurotransmitters belong to the monoamine class, serotonin possesses an indole core whereas dopamine contains a catechol scaffold, presenting distinct chemical surfaces for potential stacking interactions with nucleic-acid bases. The inability to isolate secondary aptamers against the dopamine complex, despite the known structure-switching ability of its primary aptamer, indicates that successful selection is governed not by the raw binding affinity of the primary aptamer for its target, but by how structurally distinct its ligand-bound conformation is from its unbound state. The degree of structural divergence between states dictates the distinctiveness of the exposed docking epitope and sets the baseline level of nonspecific background interactions, demonstrating that conformational contrast overrides conventional binding strength in enabling secondary aptamer selection.
Even among the systems that yielded functional secondary aptamers, nonspecific DNA–DNA interactions presented a significant challenge, prompting targeted sequence optimization in the testosterone system. Initial strand-displacement assays using the full forty-two-nucleotide testosterone primary aptamer displayed substantial background interaction with its cognate secondary aptamer in the absence of ligand. Hypothesizing that these spurious associations stemmed from partial hybridization to terminal stems that remained accessible across both folded states, researchers synthesized a series of systematically truncated variants comprising forty, thirty-eight, and thirty-six nucleotides. Ligand binding throughout this truncation series was verified independently through a thioflavin T displacement assay, in which dye intercalation within the DNA backbone decreases upon testosterone association. While thirty-six nucleotides abolished secondary aptamer recognition entirely, the thirty-eight-nucleotide construct preserved target binding, suppressed background hybridization, and produced the optimal signal-to-noise ratio in strand-displacement evaluations.
Solution-phase characterization using calibrated off-competition strand-displacement assays further delineated the binding kinetics and thermodynamic landscapes of the three successful pairs. In these assays, target-dependent assembly of the primary aptamer, ligand, and secondary aptamer displaces a Dabcyl-conjugated quenching strand from a 6-carboxyfluorescein-labelled secondary aptamer, yielding a measurable fluorescent signal. Serotonin and deoxycorticosterone 21-glucoside systems generated rapid fluorescence gains, whereas the testosterone complex required longer incubation times to reach equilibrium. In all three systems, fluorescent signal generation was strictly dependent on both the primary aptamer and the specific ligand. For the serotonin pair, fitting titration data to a standard competitive-binding isotherm under saturating ligand conditions indicated an apparent dissociation constant of approximately one micromolar. Analogous affinity values could not be extracted for testosterone or deoxycorticosterone 21-glucoside due to elevated ligand-independent background signals arising from coupled thermodynamic equilibria.
To track the structural mechanics underpinning complex formation, the researchers performed circular dichroism spectroscopy across all three functional dual-aptamer pairs. For the serotonin and deoxycorticosterone 21-glucoside assemblies, mixing the primary and secondary aptamers in the absence of ligand yielded negligible spectral changes, while subsequent addition of analyte triggered a redshift in circular dichroism absorbance for serotonin and a blueshift for deoxycorticosterone 21-glucoside. Unbound steroid ligands and secondary aptamers alone exhibited no intrinsic dichroic shifts under control conditions. These spectroscopic patterns confirmed that the primary aptamer undergoes the dominant conformational reorganization upon small-molecule binding, whereas the secondary aptamer engages the resulting complex without undergoing substantial secondary-structure restructuring. In the testosterone system, combining both aptamers caused a noticeable baseline displacement before ligand addition, consistent with persistent residual hybridization observed during fluorescence measurements.
High-resolution structural analyses focused on the lead serotonin system, combining proton nuclear magnetic resonance spectroscopy with molecular dynamics simulations to resolve interface formation at atomic detail. One-dimensional watergate proton nuclear magnetic resonance spectra revealed shifts in imino proton resonances upon serotonin addition, signifying localized hydrogen-bonding rearrangements that were completely absent when the primary aptamer was exposed to the structurally related metabolite 5-hydroxyindoleacetic acid. Explicit-solvent molecular dynamics simulations of two hundred nanoseconds, executed under physiological ionic conditions with the five-prime terminus of the primary aptamer tethered to model surface immobilization, corroborated these physical observations. The computational trajectories demonstrated that serotonin binding induces an extension of the primary aptamer backbone while preserving the overall geometry of the secondary aptamer, creating a stabilized docking cleft with minimal overall distortion of the participating secondary strand.
Transitioning the dual-aptamer serotonin architecture from free solution to solid-phase enzyme-linked immunosorbent assays revealed unexpected interfacial complications. When biotinylated secondary aptamers were immobilized onto streptavidin-coated microplates and exposed to primary aptamers conjugated to horseradish peroxidase, substantial background absorbance at 370 nanometres occurred even in the complete absence of serotonin. Sequence analysis pinpointed an internal five-nucleotide stretch, spanning bases twenty-one to twenty-five within the secondary aptamer, as a primary driver of nonspecific hybridization with the primary strand. Because this hybridization-prone sequence occupied an internal segment rather than a terminal stem, end-truncation was unfeasible. The investigators resolved this issue by designing an eleven-nucleotide linear inhibitory strand complementary to this central region. Pre-hybridizing this inhibitory strand to the immobilized secondary aptamers effectively suppressed nonspecific background interactions and restored robust, target-dependent colorimetric signals.
The dual-aptamer platform was subsequently translated into a rapid point-of-need lateral flow assay, providing an even more demanding test of conformational recognition. Citrate-stabilized gold nanoparticles of fifty-nanometre diameter were functionalized with thiolated primary aptamers through a freezing-assisted conjugation method, achieving a surface density of roughly five hundred oligonucleotides per nanoparticle with an estimated molecular footprint of sixteen square nanometres. When combined with secondary aptamers pre-incubated with the eleven-nucleotide inhibitory strand, the gold nanoparticles generated clear, visible test lines on streptavidin-printed nitrocellulose dipsticks exclusively in the presence of serotonin. The lateral flow strip assay displayed robust analytical selectivity, yielding statistically significant signal discrimination when challenged with buffer blanks and tenfold higher concentrations of structural analogues, including dopamine, tryptophan, tyrosine, 5-hydroxytryptophan, and 5-hydroxyindoleacetic acid.
Crucially, this conformational sandwich configuration bypasses the high-dose hook effect that frequently degrades traditional sandwich immunoassays and point-of-care lateral flow strips. Because the secondary aptamer specifically targets the ligand-induced spatial fold of the primary aptamer rather than binding a second epitope on the small molecule itself, escalating analyte concentrations cannot prematurely saturate both recognition reagents independently. Although the sandwich architecture does not surpass the absolute analytical sensitivity achieved by single-aptamer sensing formats, it decouples the initial molecular recognition event from downstream signal generation. By reading the physical state of a folded oligonucleotide rather than the chemical composition of the analyte, this dual-aptamer framework provides a versatile molecular logic gate for diagnostics, signal amplification networks, and modular nucleic-acid computing systems.
Subject of Research: Conformational DNA recognition using paired aptamer systems
Article Title: Aptamers enable a distinct mode of conformational DNA recognition
Article References: Stuber, A., Yang, K., Escobar, S. H., Cléry, A., Douaki, A., Moretó Bravo, N., Lamm, D., Jirasko, V., Allain, F. H. T., Garoli, D., & Nakatsuka, N. (2026). Aptamers enable a distinct mode of conformational DNA recognition. Nature Chemistry. https://doi.org/10.1038/s41557-026-02286-4
Image Credits: AI Generated
DOI: 10.1038/s41557-026-02286-4
Keywords: Aptamers, DNA Recognition, Structural Biology, Biosensors, Lateral Flow Assays, Molecular Dynamics, Biochemistry, Molecular Recognition, enable, distinct, mode, conformational
