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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster H19 - Building Protease Sensors using FAST and HaloTag

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Emily R. Pan

Undergraduate Student
University of California, San Diego
Avila Beach, California, United States

Co-Author(s)

RW

Ryan Woloschuk, PhD

Postdoctoral Fellow
Howard Hughes Medical Institute, Virginia, United States

Primary Investigator(s)

Alison Tebo, PhD

Janelia Group Leader
Howard Hughes Medical Institute, Virginia, United States

Introduction: Proteases are incredibly important in many physiological processes including wound healing, embryonic development, and cancer metastasis. However, detecting and measuring protease activity remains a challenge due to limitations of current protease biosensors which primarily rely on the use of fluorescent proteins. First, fluorescent proteins take roughly 30-80 minutes to undergo maturation which limits the measurement of fast occurring reactions [1]. The fluorescent proteins can also only undergo maturation in the presence of molecular oxygen which makes it difficult to study processes in oxygen-poor conditions such as cancer tumors [2]. Additionally, many current biosensors emit signals in the green and blue light regions, but these shorter wavelengths can cause phototoxicity in cells [3]. Therefore, new chemogenetic biosensors that can function in oxygen-poor conditions and have reduced phototoxicity are necessary to facilitate the study of protease activity. Our project aims to develop new protease biosensors using genetically encoded protein tags: FAST and HaloTag. FAST and HaloTag are self-labeling proteins that can rapidly bind fluorogenic dyes to become fluorescent. We aim to make dye-binding activity protease-dependent to study protease activity and address the limitations outlined above. FAST and HaloTag can function in oxygen-poor conditions, bind dye immediately, and be measured in the red and infrared regions as we are proposing to use a red fluorogenic dye, enabling the study of rapid protease activity in cancer metastasis and other oxygen-poor environments [4].

Materials and

Methods: We used autoinhibited coiled coils coupled with a split protein approach in our sensor design [5, 6]. In the presence of a protease, the cleavable linker is cut and the coiled coils are rearranged based on binding affinity to facilitate reconstitution of the split protein, thereby enabling dye-binding (Fig. 1A). To enable testing of our sensor, two controls were built alongside each sensor. A negative control was designed to measure the background affinity and fluorescence emitted by the split protein itself, while a positive control consisting of the split protein reconstituted through the interactions of complimentary coiled coils was used to measure the maximum fluorescence emitted by the sensor (Fig. 1B). Each sensor and control was designed as a plasmid, consisting of the split protein, sensor or control sequence, and an mTurquoise2 fluorescent protein expression marker to allow for visualization of the cells. To detect protease-mediated activity, TEV protease was used for testing. Sensors and controls were cloned, validated with Sanger and Whole Plasmid Sequencing, and transfected in human embryonic kidney cells (HEK293 WT). Each sensor was transfected in four different conditions at 500ng of DNA per well: negative control, positive control, sensor without protease, and sensor with protease. Immediately prior to imaging, 200uL of a red fluorogenic dye (HBR-3,5DOM for FAST and JF635 for HaloTag) was added to each well. Imaging of the cells was completed 24 and 48 hours after transfection with confocal microscopy. Analysis was conducted using Fiji and Excel.

Results, Conclusions, and Discussions: Since expression of the cells varied from sample to sample and we expect the expression of mTurquoise2 to be relatively stable, all data were normalized to mTurquoise2 by calculating the ratio of FAST or HaloTag expression to mTurquoise2 for each cell. Then, the average and standard deviation of each condition were calculated. Our results show the negative and positive control function as expected, with the negative control showing very little fluorescence and the positive control giving a large positive signal (Fig. 1C). However, the sensor with and without TEV protease showed no statistically significant difference in normalized fluorescence signal (Fig. 1C, 1D). The performance of the controls suggests this autoinhibited coiled coil and split protein approach is a promising design for developing biosensors without the use of fluorescent proteins. However, optimizing the coiled coil affinity, split protein orientation, and transfection conditions are necessary to improve the functionality of the biosensor itself.

Acknowledgements (Optional): Thank you to Janelia Postdoctoral Fellow, Dr. Ryan Woloschuk, for initiating this project and guiding and assisting the entire experimental process. Thank you to Janelia Group Leader, Dr. Alison Tebo, for supporting and guiding this project. Thank you to Dr. Erik Snapp, the Janelia-Meyerhoff Undergraduate Summer Program, and the UC San Diego PATHS Scholars Program for supporting this summer research initiative.

References: [1] Balleza E et al., (2018). Nat Methods, 15(1): 47-51. [2] Lidsky P et al., (2018). Development, 145(4):dev156257. [3] Jensen E. (2012). Anat Rec, 295: 2031-2036. [4] England C et al., (2015). Bioconjug Chem, 26(6): 975-986. [5] Shekhawat S et al., (2009). J Am Chem Soc, 131(42): 15284-15290. [6] Huppertz M et al., (2024). Science, 383,890-897.