Systems and methods for binding amyloid fibrils using fluorescent protein

#Amyloid fibrils are insoluble aggregates of enzymes, proteins, polypeptides, peptides, and hormones that form long, ordered fibers including β-sheet structures. In the human body, formation of amyloid fibril related species, including protofibrils, mature fibrils and oligomers has been associated with more than 20 human diseases, including neurodegenerative diseases, e.g., Huntington's disease, Alzheimer's disease, Parkinson's disease, fatal familial insomnia, familial amyloid polyneuropathy, atherosclerosis, cerebral amyloid angiopathy, transmissible spongiform encephalopathy; and diseases affecting other organs, e.g., the liver, pancreas, heart, thyroid, such as diabetes mellitus type 2, medullary carcinoma of the thyroid, cardiac arrhythmias, isolated atrial amyloidosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, heredity non-neuropathic systemic amyloidosis, dialysis related amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, systemic AL amyloidosis, sporadic inclusion body myositis, etc. In addition, amyloid fibrils present in the seminal fluid have been shown to enhance HIV infectivity. Currently there is no cure for these diseases and in many cases even diagnostics, i.e., identification of the presence of amyloid fibril related species, can be performed only on postmortem tissues.

#Some embodiments of the disclosed subject matter are directed to a method of detecting amyloid fibril related species. Some embodiments of the disclosed subject matter are directed to a method of monitoring for the presence or progression of amyloid diseases. In some embodiments, the method includes providing a sample from an individual to be monitored. In some embodiments, the method includes providing a sample including amyloid fibril related species. In some embodiments, the method includes treating the sample with a reagent including a fluorescent protein. In some embodiments, the method includes binding the fluorescent protein to amyloid fibril related species. In some embodiments, the method includes detecting a signal from fluorescent protein bound to the treated sample utilizing, e.g, confocal microscopy, fluorescent microscopy, fluorescent spectroscopy, absorption spectroscopy, ELISA, mass spectroscopy, radioactive detection, etc., or combinations thereof. In some embodiments, the method includes quantifying amyloid fibril related species in said treated sample via quantification of fluorescent protein.

#In some embodiments, the fluorescent protein is a cnidarian fluorescent beta-barrel protein. In some embodiments, the treated sample has a concentration of fluorescent protein above about 1 fM. In some embodiments, the treated sample has a concentration of fluorescent protein above about 1 nM. In some embodiments, the treated sample has a concentration of fluorescent protein above about 10 nM. In some embodiments, the amyloid fibril related species include Aβ, IAPP, amylin, PAPf39, SEMI, α-synuclein, tau, insulin, Huntingtin, PrPSc, Medin, Apolipoprotein AI, Atrial natriuretic factor, β-2 microglobulin, transthyretin, gelsolin, lysozyme, keratoepithelin, calcitonin, prolactin, serum amyloid A, immunoglobulin light chain AL, or combinations and/or variants thereof. In some embodiments, the sample includes blood, blood plasma, urine, seminal fluid, seminal plasma, cerebrospinal fluid, lymphatic fluid, intraocular fluid, synovial fluid, serous fluid, endolymph, perilymph, peritoneal fluid, pleural fluid, pancreatic tissue, brain tissue, liver tissue, heart tissue, thyroid tissue, corneal tissue, a biopharmaceutical, a therapeutic drug, or combinations thereof.

#The drawings show embodiments of the disclosed subject matter for the purpose of illustrating the invention. However, it should be understood that the present application is not limited to the precise arrangements and instrumentalities shown in the drawings, wherein:

#FIG. 1A is a chart of a method for monitoring a sample for the presence of amyloid fibril related species according to some embodiments of the disclosed subject matter;

#FIG. 1B is a chart of a method for detecting amyloid fibril related species in a sample according to some embodiments of the disclosed subject matter;

#Referring now to FIGS. 1A-1B, aspects of the disclosed subject matter include a method 100 of detecting amyloid fibril related species, e.g., amyloid protofibrils, mature amyloid fibrils, amyloid oligomers, or combinations thereof, in a sample. Referring specifically to FIG. 1A, at 102A, a sample is provided from an individual, e.g, a patient, to be monitored for the presence of amyloid fibril related species. Referring specifically to FIG. 1B, at 102B, a sample is provided that includes or is suspected to include amyloid fibril related species. In some embodiments, the sample is a bodily fluid or tissue provided by the individual. In some embodiments, the sample includes blood, blood plasma, urine, seminal fluid, seminal plasma, cerebrospinal fluid, lymphatic fluid, intraocular fluid, synovial fluid, serous fluid, endolymph, perilymph, peritoneal fluid, pleural fluid, pancreatic tissue, brain tissue, liver tissue, heart tissue, thyroid tissue, corneal tissue, a biopharmaceutical, a therapeutic drug, or combinations thereof. In some embodiments, the amyloid fibril related species to be detected include Aβ, IAPP, amylin, PAPf39, SEMI, α-synuclein, tau, insulin, Huntingtin, PrPSc, Medin, Apolipoprotein AI, Atrial natriuretic factor, β-2 microglobulin, transthyretin, gelsolin, lysozyme, keratoepithelin, calcitonin, prolactin, serum amyloid A, immunoglobulin light chain AL, or combinations and/or variants thereof.

#Still referring to FIGS. 1A and 1B, at 104, the sample is treated with a reagent including a fluorescent protein. In some embodiments, the fluorescent protein is a cnidarian fluorescent beta-barrel protein. In some embodiments, the fluorescent protein is green fluorescent protein (“GFP”), e.g., as isolated from Aequorea victoria; a variant green fluorescent protein, e.g., superfolder GFP, supercharged GFPs, yellow fluorescent protein, etc.; red fluorescent protein, e.g., as isolated from Discosoma sp.; a variant red fluorescent protein, e.g., mCherry; Dendra, e.g., as isolated from octocoral Dendronephthya sp.; variant Dendra, e.g., Dendra2; Dronpa, e.g., as isolated from coral Pectiniidae; variant Dronpa, e.g., Dronpa-2; mEosFP; variant mEosFP, e.g., mEos2; or combinations thereof. In some embodiments, treatment step 104 is performed in vitro. In some embodiments, treatment step 104 is performed in vivo.

#At 106, fluorescent protein from the reagent is bound to the amyloid fibril related species. Without wishing to be bound by any theory of how the fluorescent protein from the reagent is bound to the amyloid fibril related species, and as shown in FIG. 2, aromatic groups disposed on the surface of the β-barrel portion of the fluorescent protein, such as those provided by surface tyrosine and phenylalanine moieties, increase affinity of the fluorescent protein for the amyloid fibril related species. In some embodiments, binding step 106 is performed in vitro. In some embodiments, binding step 106 is performed in vivo. Referring back to FIGS. 1A-1B, at 108, a signal from the treated sample indicating fluorescent protein is bound to the treated sample is detected, indicating the presence of amyloid fibril related species in the sample. In some embodiments, detection 108 includes confocal microscopy, fluorescent microscopy, fluorescent spectroscopy, absorption spectroscopy, ELISA, mass spectroscopy, radioactive detection, etc., or combinations thereof. In some embodiments, detection 108 of the signal from the treated sample is performed in vitro. In some embodiments, detection 108 of the signal from the treated sample is performed in vivo. At 110, the amount of amyloid fibril related species in the sample is quantified via measurement of fluorescence intensity. The signal detection and intensity quantification is measured by any method known to one of skill in the art, including, but not limited to, confocal microscopy, fluorescent microscopy, fluorescent spectroscopy, absorption spectroscopy, ELISA, mass spectroscopy, radioactive detection, etc., or combinations thereof. In some embodiments, the treated sample has a concentration of fluorescent protein above about 1 fM. In some embodiments, the treated sample has a concentration of fluorescent protein above about 1 nM. In some embodiments, e.g., when detecting and/or quantifying native fluorescence, the treated sample has a concentration of fluorescent protein above about 10 nM.