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 lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect mRNA concentration. Our study finds that MGapt fluorescence varies in commercial PURE systems, leading to inaccurate RNA calculations. MGapt fluorescence is not solely linked to waste products or buffering agent interactions. MGapt dynamics are also independent of DNA and RNA concentrations, suggesting an influence beyond general system dynamics. 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.

Optimizing protein production in the One-Pot Pure system: insights into reaction composition and expression efficiency

“…We sincerely thank the Murray Lab members, particularly Dr. Zoila Jurado and Miryong (Miki) Yun, who contributed to the early setup and discussions of the project. Dr. Jurado also prepared the P T7 -MGA-UTR1-deGFP plasmid 33 used in this work. We also thank Dr. Jurado, Manisha Kapasiawala, and Dr. John Marken for their review and feedback on this manuscript.…”