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Abstract Staphylococcus aureus(S. aureus), a common foodborne pathogen, poses significant public health challenges due to its association with various infectious diseases. A key player in its pathogenicity, which is the IsdA protein, is an essential virulence factor inS. aureusinfections. In this work, we present an integrated in‐silico and experimental approach using MD simulations and surface plasmon resonance (SPR)‐based aptasensing measurements to investigateS. aureusbiorecognition via IsdA surface protein binding. SPR, a powerful real‐time and label‐free technique, was utilized to characterize interaction dynamics between the aptamer and IsdA protein, and MD simulations was used to characterize the stable and dynamic binding regions. By characterizing and optimizing pivotal parameters such as aptamer concentration and buffer conditions, we determined the aptamer's binding performance. Under optimal conditions of pH 7.4 and 150 mM NaCl concentration, the kinetic parameters were determined;ka = 3.789 × 104/Ms,kd = 1.798 × 103/s, andKD = 4.745 × 10−8 M. The simulations revealed regions of interest in the IsdA‐aptamer complex. Region I, which includes interactions between amino acid residues H106 and R107 and nucleotide residues 9G, 10U, 11G and 12U of the aptamer, had the strongest interaction, based on ΔG and B‐factor values, and hence contributed the most to the stability of the interaction. Region II, which covers residue 37A reflects the dynamic nature of the interaction due to frequent contacts. The approach presents a rigorous characterization of aptamer‐IsdA binding behavior, supporting the potential application of the IsdA‐binding aptamer system forS. aureusbiosensing.
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Computational generation and characterization of IsdA‐binding aptamers with single‐molecule FRET analysis
Abstract Staphylococcus aureusis a major foodborne bacterial pathogen. Early detection ofS. aureusis crucial to prevent infections and ensure food quality. The iron‐regulated surface determinant protein A (IsdA) ofS. aureusis a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in‐silico approach to develop and validate high‐affinity binding aptamers for the IsdA protein detection using custom‐designed in‐silico tools and single‐molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in‐silico oligonucleotide screening methods and metadynamics‐based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA‐aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in‐silico approach to support aptamer discovery. This study showcases a computational SELEX method in combination with single‐molecule binding studies deciphering effective aptamers againstS. aureus IsdA, protein. The established approach demonstrates the ability to expedite aptamer discovery that has the potential to cut costs and predict binding efficacy. The application can be extended to designing aptamers for various protein targets, enhancing molecular recognition, and facilitating the development of high‐affinity aptamers for multiple uses.
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- PAR ID:
- 10459532
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology Journal
- Volume:
- 18
- Issue:
- 12
- ISSN:
- 1860-6768
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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