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Stoichiometric approach to quantitative analysis of biomolecules: the case of nucleic acids

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Abstract

Majority of protocols for quantitative analysis of biomarkers (including nucleic acids) require calibrations and target standards. In this work, we developed a principle for quantitative analysis that eliminates the need for a standard of a target molecule. The approach is based on stoichiometric reporting. While stoichiometry is a simple and robust analytical platform, its utility toward the analysis of biomolecules is very limited due to the lack of general methodologies for detecting the equivalence point. In this work, we engineer a new target/probe-binding model that enables detecting the equivalence point while maintaining an appropriate level of specificity. We establish the probe design principles through theoretical simulations and experimental confirmation. Further, we demonstrate the utility of the stoichiometric analysis via a proof-of-concept system based on oligonucleotide hybridization. Overall, the approach that requires neither standard nor calibration yields quantitative results with an adequate accuracy (> 90–110%) and a high specificity. The principles established in our work are very general and can extend beyond oligonucleotide targets toward quantitative analysis of many other biomolecules such as antibodies and proteins.

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Acknowledgements

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM135796.

Funding

The reported studies were supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM135796.

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  1. Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, 60115, USA

    Adeyinka Adegbenro, Seth Coleman & Irina V. Nesterova

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All of the authors participated in the methodology design, performed experiments, and analyzed and discussed the data. I.V. Nesterova produced an original idea of this work, designed the general experimental methodology, and supervised the project. All of the authors contributed to the discussion of this manuscript and its writing. All of the authors read and approved the manuscript.

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Correspondence to Irina V. Nesterova.

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Adegbenro, A., Coleman, S. & Nesterova, I.V. Stoichiometric approach to quantitative analysis of biomolecules: the case of nucleic acids. Anal Bioanal Chem 414, 1587–1594 (2022). https://doi.org/10.1007/s00216-021-03781-y

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