Aug 2026· International Journal of Molecular Sciences· Vol 27· 0 citations· 61 references
Medicine
TL;DR
Results show that sequence changes outside the binding site can modulate ligand binding indirectly, and that the ligand interaction network is useful for evaluating edited aptamers.
Abstract
RNA aptamers are often used as ligand-recognition modules in engineered RNA systems, but integration into larger RNA constructs can influence stability and ligand binding. As a result, aptamer sequences may need to be adapted to new environments while preserving essential properties. Here, we examine this sequence editability problem for the theophylline RNA aptamer. Starting from the experimentally determined structure, we introduced targeted mutations in peripheral structural elements while leaving the recognition site unchanged. The native aptamer, mutated variants, a Mg2+-depleted system, and a caffeine-bound control were analyzed using three independent 1 μs molecular dynamics simulations. Binding energetics were estimated with multiple end-point as well as alchemical free energy approaches. Results were interpreted together with base pair stability, the conformational landscape of the binding pocket, and per-nucleotide energy contributions. This allows us to predict whether an edit is tolerated or disruptive. Some mutations retained structural and energetic profiles comparable to the native aptamer, whereas others reduced ligand affinity by propagating structural distortions into the binding pocket. These results show that sequence changes outside the binding site can modulate ligand binding indirectly, and that the ligand interaction network is useful for evaluating edited aptamers. The introduced workflow provides a novel combination of established computational strategies for efficient in silico screening of aptamer variants before experimental testing and can be integrated into the design of larger RNA structures. This works particularly well when an experimental structure is available and the tested mutations are small enough not to disrupt the folding pathway.
Aptamers are valuable recognition elements for biosensing and therapeutic applications; however, an accessible strategy to link structure with function during postselection engineering remains challenging. Here, we introduce the concept of a structure-function landscape for aptamers, constructed by systematic single-point mutagenesis that maps the contribution of each nucleotide to target binding. Using OTC2, an aptamer that recognizes multiple tetracycline antibiotics, as a model, we identified critical binding sites and variable regions within the landscape. Guided by this landscape, we engineered OTC2 through combinatorial mutagenesis of only five nucleotides, yielding an evolved aptamer (OTC2M1) with enhanced affinity and substantially improved binding kinetics. The improved performance translated directly into sensing applications: an aptamer switch based on OTC2M1 achieved a 40-fold improvement in detection sensitivity for tetracycline antibiotics compared to the original aptamer. This work establishes the structure-function landscape as an informative and accessible platform for rational aptamer engineering and highlights its potential to guide the development of high-performance biosensors.
Zhuoer Chen, Qi Sun, Xianlu Lei et al.· Analytical Chemistry· 0 citations
The effectiveness of the SaCas9 modular base editors, the robustness of the platform’s modularity, and its feasibility for convenient screening of target-specific base editors are demonstrated.
J. Collantes, Kellen Xu, Melany Ruiz-Urigüen et al.· The CRISPR Journal· 0 citations
The Cluster of Differentiation 47 (CD47)–signal regulatory protein alpha (SIRPα) immune checkpoint is a key regulator of tumor immune evasion and a promising target in cancer immunotherapy. To overcome the limitations of monoclonal antibodies, this study aimed to identify high-affinity nucleic acid aptamers targeting CD47. A systematic single-nucleotide mutagenesis workflow was performed on a known CD47-binding DNA aptamer to generate 270 single-point variants, enabling unbiased evaluation of each nucleotide position. The variants were first screened for structural stability, yielding 81 structurally stable candidates. These candidates were then subjected to molecular docking against CD47, and 45 variants showed improved docking scores compared with the native aptamer. The docking score improved from −226.07 for the native aptamer to −301.94 for the best-performing variant. Based on structural stability and docking performance, the ten top-ranked candidates were selected for molecular dynamics simulations. These variants exhibited improved conformational stability, as reflected by lower root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) values and increased hydrogen bonding, with Seq198 and Seq262 showing the most stable profiles. Binding free energy calculations confirmed improved affinity. The native aptamer exhibited a ΔTOTAL of −97.16 kcal/mol, whereas Seq198 (−173.13 kcal/mol), Seq244 (−152.35 kcal/mol), and Seq112 (−147.19 kcal/mol) showed markedly stronger binding. Seq198 emerged as the most promising candidate. These findings demonstrate that systematic computational mutagenesis is an effective strategy for optimizing aptamer performance and identifying high-affinity CD47-targeting candidates.
Sumeyye Altunok, Emre Can Buluz, Ehed Muhammed Aymaz· Journal of the Turkish Chemi...· 0 citations
The chemical richness of RNAs is greatly enhanced by post-transcriptional modifications with RNA methylation as the most prominent type. RNA modifications modulate the stability, folding and interaction pattern of RNA molecules. Furthermore, emerging data suggests RNA modifications also directly regulate the activity of catalytic RNA molecules, i.e., ribozymes. Here, we employ classical and hybrid quantum-classical (QM/MM) molecular dynamics (MD) simulations to investigate the reaction mechanism of an artificial methyltransferase ribozyme MTR1. Importantly, we pinpoint how 2’-O-methylations of active site nucleotides synergistically enhance ribozyme activity by reducing the conformational flexibility of the ribose rings and rigidifying the active site. Finally, the herein reported crystal structure of the modified MTR1, solved at 2.6 Å resolution, validates the results of our simulations. Taken together, our work supports the purported central role of modified RNA for early RNA catalysis and may guide rational design of more efficient ribozymes. Emerging data indicate RNA modifications are important regulators of ribozyme activity. Here, the authors show how 2’-O-methylation of active site nucleotides enhances catalytic activity by reducing ribose rings’ conformational flexibility.
Jana Aupič, Hsuan-Ai Chen, Carolin P. M. Scheitl et al.· Nature Communications· 0 citations
RNA molecules explore heterogeneous conformational ensembles that are essential for their biological function and molecular recognition, yet this intrinsic flexibility poses a major challenge for structure-based drug discovery. In particular, the absence of well-defined binding pockets in static structures limits the identification of ligandable sites. Here, we present an integrative ensemble-based approach that combines enhanced-sampling molecular dynamics simulations with Nuclear Magnetic Resonance data to characterize the conformational landscape of the HIV-1 TAR RNA at atomic resolution. Starting from extensive sampling, we refined the resulting conformational distribution through maximum-entropy reweighting to achieve quantitative agreement with experimental data. Analysis of the reweighted ensemble reveals a diverse set of conformational substates, including compact arrangements that exhibit pocket features compatible with ligand recognition and overlap with known ligand-bound structures. At the same time, highly ligandable conformations, which are only marginally populated, might nonetheless be critical for RNA recognition. Our results demonstrate that integrative ensemble modeling can reveal pharmacologically relevant RNA conformations that are not apparent from experimental static structures, providing a framework for ensemble-based strategies in RNA-targeted drug discovery.
Stefano Bosio, Vincent Schnapka, Mattia Bernetti et al.· bioRxiv· 0 citations
Kissing complexes involve interactions between complementary loops of two RNA hairpins. These complexes are continously found in RNA structures and are essential for several biological processes, including the dimerization of retroviral RNA genomes, regulation of plasmid copy number, and small RNA-based control of mRNA translation. We previously identified novel RNA-RNA and RNA-DNA kissing complexes through the in vitro selection of oligonucleotides from synthetic libraries. In order to expand the use of kissing complexes to aptamers and DNA nanotechnology, we describe the selection and characterization of DNA-DNA kissing complexes and their application in the development of DNA adenosine aptasensors. Four DNA/DNA kissing complexes were identified from SELEX and characterized by EMSA and fluorescence anisotropy. The couple dk25/dk26 was further modified to generate an adenosine-triggered aptaswitch, where the addition of adenosine selectively induces the dissociation of loop-loop interactions. This paves the way for a new class of nucleic acid modules, which could be useful for the design of controlled nanostructure assembly and disassembly.
É. Dausse, N. Tourasse, J. Toulmé et al.· bioRxiv· 0 citations
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