Engineering a Cell-free Uric Acid Detection System with Application to Congenital Heart Disease Screening in Rural India

Introduction: Approximately 9/1000 babies are born with congenital heart disease (CHD) annually in India, 20% of whom are anticipated to require medical intervention within the first year of life. Prenatal screening for congenital heart disease typically occurs at 20 gestational weeks via ultrasound or fetal echocardiogram. In rural areas, however, CHD is incredibly underdiagnosed and underreported due to the lack of appropriate diagnostic tools in community health centers. To address this problem, we initiated the design and testing of a mechanism that could be used to build a simple, cost-effective screening tool for CHD. A positive result, ideally interpreted within a couple of hours, would allow pregnant mothers to seek further testing at a high-resource tertiary care center, supported by recent expansions of India’s maternal and child health programs.

Cell-free synthetic gene networks facilitate fluorescent or colorimetric response to small-molecule biomarkers in an abiotic environment. This technology allows for freeze-drying cell-free extracts in a paper-based in vitro system, which is conducive for relatively cheap biosensors. Our goal was to develop and characterize a cell-free reaction that was responsive to changes in the concentration of uric acid, a clinically-found biomarker for CHD.

As a byproduct of the purine degradation cycle, uric acid binds to transcriptional regulator pucR (Fig-1). We built and optimized a cell-free reaction around the synthetic promoter-pucR uric acid binding system, and by placing a fluorescent protein under the control of the synthetic promoter, we elicited a fluorescent response to uric acid.

Materials and

Methods: The goal of the work was (i) to translate the synthetic promoter-uric acid binding system into a cell-free environment and (ii) to characterize its responsiveness to varying uric acid concentrations.

The pucR gene as well as two promoters (pucrpro, a previously designed synthetic promoter sequence, and pucpro_CFopt, pucrpro with cell free-optimized consensus sequences at the -35 and -10 positions) were obtained as gene blocks. The pucR block was cloned into a linear expression template via Gibson assembly. Each promoter was separately cloned into a standard plasmid backbone encoding an origin of replication, antibiotic resistance, and GFP (placed in-frame with the promoter sequences).

In multiple experiments, critical small molecules (PEP, ATP, etc.) and cellular extracts were combined with the linear pucR fragment and one of the promoters (suspended in water) (Fig-2). Aqueous uric acid suspensions ranging from 0 𝜇M-560 𝜇M (460 𝜇M is the concentration that is clinically correlated to CHD⁷ and concentrations above 560 𝜇M are not observed in human blood) were created by prolonged vortex mixing interspersed with 37-deg. C incubations (given low solubility). Immediately after vortexing, suspensions were added to separate cell-free reactions and samples were incubated at 30-deg. C for 14-hours on a GFP-reading plate reader. An identical experiment was performed with uric acid suspended in fetal bovine serum (FBS), so as to assess the ability of the cell-free system in detecting uric acid in a blood-like mixture, which is more similar to the clinical use case.

Results, Conclusions, and Discussions: When using the standard promoter (pucrpro), a steady rise in fluorescence was observed for ~7 hrs. (at which point the maximum fluorescence was reached) across all samples. The 0-𝜇M sample (negative control) displayed a similar fluorescence pattern to that of the non-zero uric acid samples, but maximum fluorescence was lower. The limit of detection for uric acid was determined to be ~4 hrs. Using the optimized consensus sequence-promoter (pucrpro_CFopt), overall fluorescence was higher compared to pucrpro, but the discrimination between zero and non-zero uric acid samples was greatly reduced. As such, the clinically relevant concentration of 460 𝜇M was not distinguishable from other samples within the 14 hr. incubation period (yielding a much higher limit of detection compared to pucrpro). Additionally, the use of FBS inhibited fluorescence completely–the system did not respond to changes in uric acid concentration at all.

Consensus sequences optimized for cell-free systems were found to be counterproductive for the successful detection of uric acid in a sample compared to non-native consensus sequences (E. coli’s, in this case); the significantly reduced discrimination between the negative control and non-zero uric acid samples suggests that cell-free consensus sequences enhanced the number of favorable interactions between polymerase and the promoter (and therefore, the rate of transcription) to a level where the effect of uric acid binding to pucR was rendered negligible as a rate control. From this, we uncovered a critical modulator that can tune the cell-free reaction’s uric acid detection capabilities in future work.

The elimination of fluorescence response with the introduction of FBS suggests blood proteins could physically interfere with critical binding steps necessary for GFP expression. This yields a need for isolated blood protein cell-free experiments so that the protein(s) blocking GFP expression can be controlled via a secondary binding/neutralization mechanism.

In this work, the foundations for a cell-free-based CHD screening tool were laid. A preliminary characterization of a uric acid-responsive cell-free system has uncovered the key next steps in developing a robust screening tool that can be deployed in low-resource settings.

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