A molecular imaging biosensor detects in vivo protein folding and misfolding.
Aberrant protein folding represents the molecular basis of many important human diseases. Although the discovery of new anti-misfolding drugs is a major priority in molecular therapeutics, there is currently no generalizable protein folding assay for use in cell-based high throughput screening (HTS) of chemical libraries, or for in vivo imaging. We molecularly engineered a bioluminescence-based biosensor composed of rationally split Firefly luciferase reporter fragments flanking a test protein, and used this in a protein-fragment complementation assay to quantitatively measure folding of the test protein. We comprehensively validated this biosensor in vitro, in cells, and by optically imaging protein folding and misfolding in living mice using several test proteins including enhanced green fluorescent protein, Renilla luciferase, Gaussia luciferase, and SIRT1. Applications of this novel biosensor are potentially far-reaching in both cell-based HTS approaches to discover new anti-misfolding drugs, and when using the same biosensor in validation studies of drug candidates in small animal models.



Novel anti-misfolding drugs are needed as molecular therapeutics for many diseases. We developed first in vivo imaging protein folding biosensor to aid drug discovery. Biosensor created by flanking a test protein with rationally split Firefly luciferase. Biosensor validated by detecting folding of test proteins EGFP, Rluc, Gluc, and SIRT1. Generalizable molecular biosensor for translational applications in drug screening.
Laboratory of Experimental and Molecular Neuroimaging, Molecular Imaging Program at Stanford (MIPS), and Bio-X Program, Stanford University School of Medicine, Stanford, CA, 94305-5427, USA.
Anjali V
AV
Sheahan
Laboratory of Experimental and Molecular Neuroimaging, Molecular Imaging Program at Stanford (MIPS), and Bio-X Program, Stanford University School of Medicine, Stanford, CA, 94305-5427, USA.
Thillai V
TV
Sekar
Laboratory of Experimental and Molecular Neuroimaging, Molecular Imaging Program at Stanford (MIPS), and Bio-X Program, Stanford University School of Medicine, Stanford, CA, 94305-5427, USA.
Kai
K
Chen
Laboratory of Experimental and Molecular Neuroimaging, Molecular Imaging Program at Stanford (MIPS), and Bio-X Program, Stanford University School of Medicine, Stanford, CA, 94305-5427, USA. paulmur8@stanford.edu.
Ramasamy
R
Paulmurugan
Laboratory of Experimental and Molecular Neuroimaging, Molecular Imaging Program at Stanford (MIPS), and Bio-X Program, Stanford University School of Medicine, Stanford, CA, 94305-5427, USA. tmassoud@stanford.edu.
Tarik F
TF
Massoud
D000072283:A549 Cells; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D055808:Drug Discovery; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D056564:Sirtuin 1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly; C447939:SIRT1 protein, human; D056564:Sirtuin 1
Bioluminescence imaging; Drug discovery; High throughput screening; Molecular imaging; Protein folding; Protein misfolding