Impact of Chemical Dynamics of Commercial PURE Systems on Malachite Green Aptamer Fluorescence
Abstract
The malachite green aptamer (MGapt) is known for its utility in RNA measurement in vivo and in lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect RNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence, which leads to inaccurate RNA calculations. Specific to the commercial system, we posit that MGapt fluorescence is significantly affected by the system’s chemical properties, governed notably by the presence of dithiothreitol (DTT). We propose a model that, on average, accurately predicts MGapt measurement within a 10% margin, leveraging DTT concentration as a critical factor. This model sheds light on the complex dynamics of MGapt in cell-free systems and underscores the importance of considering environmental factors in RNA measurements using aptamers.
Copyright and License
© 2024 American Chemical Society
Acknowledgement
We thank Zachary Martinez for their encouragement during the start of this project. We sincerely appreciate Manisha Kapasiawala, Dr. Yan Zhang, and Dr. John Marken for their invaluable insights and thought-provoking discussions on the experiments and modeling framework throughout the project. We would also like to acknowledge the contributions of Dr. Ayush Pandey, who provided assistance and feedback on the proposed model’s modeling, analysis, and inference. Their involvement has been indispensable to its completion.
Funding
Research supported by the National Science Foundation award number 2152267. The computations presented here were conducted in the Resnick High Performance Computing Center, a facility supported by the Resnick Sustainability Institute at the California Institute of Technology, Pasadena, CA, USA.
Contributions
Z.J. conceived the project, developed the mathematical model, conducted all experiments and analyzed results, fit the model to the data collected, and wrote the manuscript. R.M.M. provided supervision and assisted in editing the manuscript.
Supplemental Material
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00211.
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Comparison of the chemical compositions between PURE systems; measured pH of the PURE system using SNARF-5F; MGapt calibration curves; measure MGapt concentration under different buffer conditions; non-normalized MGapt concentration under different experimental conditions presented in Figure 3; control experiments of MG at different DTT concentrations; list of initial conditions and parameter values used in models, absolute error plots; example of back-calculation of RNA using the model; and a list of primers used to make all constructs used in this study. (PDF)
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Additional details
- National Science Foundation
- 2152267
- Accepted
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2024-09-05Accepted
- Available
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2024-09-17Published online
- Publication Status
- Published