Innovation and Partnerships Office

Genetically Encoded Sensors for Imaging Proteins and Their Complexes IB-3259

APPLICATIONS OF TECHNOLOGY:
Fluorescent protein labeling for

  • Medical diagnostics
  • Drug screening
  • Biology research

ADVANTAGES:

  • Smaller size than green fluorescent protein (GFP)
  • Long fluorescence lifetime
  • Extends convenient tagging and fluorescence anisotropy (FA) study for larger sized proteins
  • Can be used for FA-imaging of target proteins in living cells
  • Reduces false positive and negative rate in FA-based drug screening
  • Higher Förster resonance energy transfer (FRET) efficiency

ABSTRACT:

Gerard Marriott of Berkeley Lab has invented an example of a genetically encoded sensor for fluorescence anisotropy (FA) imaging in living systems. The invention provides a simple and robust means to create fusion proteins suitable for FA measurements in medical diagnostics, biomarker detection, and biology research.

The new genetically encoded FA sensors are based on small proteins that bind tightly to a fluorescent cofactor with all components being produced and provided by, and assembled within, the living cell. The combination of small protein size (as low as 10 kD) and long fluorescence lifetime (15 ns for one of the new sensors and 4.5 ns for another) allows the sensor to be used for FA measurement. The genetically encoded nature of the sensor enables convenient tagging FA sensor to any protein.

The invention enables FA-based drug screening for protein interaction inhibitor / enhancer using natural proteins or domains as substrates. Currently, fluorescent dye tagged peptides serve as protein substrate analogues during high throughput drug screening such as direct binding assay, competition binding assay or enzyme assay. FA sensor-protein substrate fusion with excellent dynamic range can be constructed for FA-based drug screening. The protein / domain is a more natural substrate than peptides and allows reduction in false positives and negative rates during drug screening.

The Berkeley Lab invention also provides a far better donor probe in Förster resonance energy transfer (FRET) compared to conventional techniques using cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) as FRET probes. For example, at 27 kD, the large size of CFP limits the effective range of FRET that can be measured in protein interactions in cells to usually less than 15%. On the other hand, the new class of FA sensor is 10kD, which will allow the Berkeley Lab donor probe to more closely approach the YFP acceptor probe, and thereby extend the mass range of FRET systems that can be studied. In addition, the longer lifetime vs. CFP will also help to extend the mass of the sensing unit in a genetically encoded FRET sensor.

FA imaging is presently carried out by using small molecule fluorophores, none of which are optimized for FA measurements in living cells. For example, conventional probes or sensors such as fluorescein and rhodamine have short lifetimes (2-3 nanoseconds), which do not provide enough time to adequately quantify the hydrodynamic properties of target proteins within a living cell. Even more problematic is that previous sensors’ synthetic composition prevents them from completely linking to the protein of interest in a cell, resulting in compromised FA imaging.

DEVELOPMENT STAGE:  Proven principle

FOR MORE INFORMATION:
Hoepker, A., Wang, A., LeMarouis, A., Suhling, K., Yan, Y., Marriott, G. (2015). Genetically encoded sensors of protein hydrodynamics and molecular proximity, PNAS, 1424021112v1-201424021.

STATUS: PCT Application US2014/038644 has been published (International Publication Number WO 2014/189854). Available for licensing or collaborative research.

REFERENCE NUMBER: IB-3259