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US-12624070-B2 - High contrast photoconvertible fluorescent proteins and methods of use

US12624070B2US 12624070 B2US12624070 B2US 12624070B2US-12624070-B2

Abstract

Disclosed herein, are photoconvertible fluorescent proteins or analogs thereof, and in particular, green-to-red photoconvertible fluorescent proteins or analogs thereof of the EosFP family; and compositions comprising the same and methods for analyzing a physiologically active substance in a cell wherein the fluorescent proteins are expressed in the cell.

Inventors

  • Brian J. Stoveken
  • James D. Lechleiter

Assignees

  • BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM

Dates

Publication Date
20260512
Application Date
20200619

Claims (13)

  1. 1 . A photoconvertible fluorescent protein comprising the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
  2. 2 . The photoconvertible fluorescent protein of claim 1 , wherein the amino acid sequence of SEQ ID NO: 2 is red at an excitation maximum at 571 mm.
  3. 3 . The photoconvertible fluorescent protein of claim 1 , wherein the amino acid sequence of SEQ ID NO: 2 is red at an emission maximum at 585 nm.
  4. 4 . The photoconvertible fluorescent protein of claim 1 , wherein the amino acid sequence of SEQ ID NO: 3 has an absorbance of UV/violet light around 385 nm.
  5. 5 . The photoconvertible fluorescent protein of claim 1 , wherein the protein is in a circularly-permutated form.
  6. 6 . The photoconvertible fluorescent protein of claim 5 , wherein the photoconvertible fluorescent protein is divided at residue 74 and 75, such that the N- and C-termini of the photoconvertible fluorescent protein are relocated.
  7. 7 . The photoconvertible fluorescent protein of claim 5 , wherein the circularly-permutated form is a photocleavable tag.
  8. 8 . A method for analyzing a physiologically active substance in a cell, comprising attaching the physiological substance to the photoconvertible fluorescent protein of claim 1 , expressing the attached photoconvertible fluorescent protein in the cell and observing the fluorescence of the protein.
  9. 9 . A method of performing live cell imaging, wherein the photoconvertible fluorescent protein claim 1 is expressed in the cell and the cell is then subjected to imaging.
  10. 10 . The method of claim 8 , wherein the physiologically active substance is a protein or a vector.
  11. 11 . The method of claim 8 , comprising analyzing localization or dynamic situation of a protein in the cell that is attached to photoconvertible fluorescent protein.
  12. 12 . A method of identifying and localizing an individual fluorescent molecule, wherein the fluorescent molecule is one or more of the photoconvertible fluorescent proteins of claim 1 and wherein said one or more photoconvertible fluorescent proteins is subjected to fluorescent imaging.
  13. 13 . The method of claim 12 , wherein the method comprises photo-activated localization microscopy or stochastic optical reconstruction microscopy.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2020/038749, filed Jun. 19, 2020 and claims the benefit of the filing date of U.S. Provisional Application No. 62/863,517, filed on Jun. 19, 2019. The content of these earlier filed applications is hereby incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING The Sequence Listing submitted herein as a text file named “21105_0072U2_SL.txt,” created on Dec. 14, 2021, and having a size of 28,672 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52 (e)(5). BACKGROUND Genetically-encoded photoconvertible fluorescent proteins are important tools for single molecule photoactivated localization microscopy, a technique that permits imaging below the diffraction limit with nanometer precision. Certain characteristics such as poor photoconversion contrast and high photoblinking of the bright, fixation-resistant, green-to-red photoconvertible fluorescent protein, mEos4b, limit its utility in quantitative applications. Alternative photoconvertible fluorescent proteins are desired that can enhance single molecule photoactivated localization microscopy and that can address the limitations of currently available photoconvertible fluorescent proteins. SUMMARY Disclosed herein are photoconvertible fluorescent proteins comprising one or more mutations or substitutions of the mEos4b protein coding sequence (SEQ ID NO: 1). Disclosed herein are photoconvertible fluorescent proteins, wherein the photoconvertible fluorescent proteins comprise the coding region of the mEos4b protein, wherein the coding region comprises at least one or more mutations or substitutions. Disclosed herein are photoconvertible fluorescent proteins, wherein the photoconvertible fluorescent proteins comprise the coding region of the mEos4b protein, wherein the coding region comprises a mutation or a substitution at residues 41 and 70, wherein the mutation or substitution at residue 41 is a methionine to an isoleucine residue mutation or substitution (Met4Ile); and the mutation or substitution at residue 70 is a valine to a threonine residue mutation or substitution (Val70Thr). Disclosed herein are photoconvertible fluorescent proteins, wherein the photoconvertible fluorescent proteins comprise the coding region of the mEos4b protein, wherein the coding region comprises a mutation or a substitution at residues 41, 70 and 197, wherein the mutation or substitution at residue 41 is a methionine to an isoleucine residue mutation or substitution (Met41Ile); and the mutation or substitution at residue 70 is valine to a threonine residue mutation or substitution (Val70Thr); and the mutation or substitution at residue 197 is an isoleucine to a methionine residue mutation or substitution (Ile197Met). BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-B shows the fluorescence spectra of mEos4b. FIG. 1A shows the excitation and emission spectra of green mEos4b. FIG. 1B shows the excitation and emission spectra of red mEos4b after 385 nm LED photoconversion. FIGS. 2A-C show the absorbance and photoconversion properties of mEos4b. FIG. 2A) shows native (green) and alkali-denatured absorbance spectra of equimolar mEos4b solutions. Note the decay in absorbance of the denatured chromophore over time. FIG. 2B shows maturation of mEos4b chromophore absorbance. Peak absorbance at 505 nm increased with incubation at 37 C and room temperature (RT). Peak absorbance is achieved by 96 hours at room temperature. FIG. 2C shows progressive photoconversion of mEos4b in vitro with 385 nm LED illumination. FIGS. 3A-B shows the chromophore acidities of green and red mEos4b. FIG. 3A shows the titration of the green mEos4b chromophore, and corresponding fit to the Henderson-Hasselbalch relationship. N=5. Mean±SD. Inset: Absorbance spectrum of mEos4b at each pH. Note sub-maximal absorbance since titrations were performed thawed solutions (see, FIG. 11). FIG. 3B shows the titration of the red mEos4b chromophore. N=3. Mean±SD. FIGS. 4A-C show widefield photoconversion of PC-FPs in cellulo. FIG. 4A shows the photoconversion contrast of mEos4b (N=9), mEos3.2 (N=4), and Dendra2 (N=4) in Hela cells. Mean field average of contrast in individual cells ±SD. ****=p<0.001 by two-way ANOVA with Tukey's post-hoc test for multiple comparisons. FIG. 4B shows green state decay of mEos4b, mEos3.2 and Dendra2 in the same experiment. FIG. 4C shows the representative wide-field images of green channel (FITC filter set) and red channel (TRITC filter set, magenta pseudocolor). Note the increased intensity of mEos4b and mEos3.2 after 30 seconds of photoconversion. FIGS. 5A-F show the estimated relative widefield photoconversion yields. FIG. 5A shows red Dendra2 fluorescence vs. initial green fluorescence. FIG. 5B shows a model of photoconversion pathways suggested by widefield results. FIG. 5C shows red mEos3.2 fluorescence vs. initial gr