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EP4581364A1 - Aptamère fret monomoléculaire pour identification de protéines et analyse structurelle - Google Patents

Aptamère fret monomoléculaire pour identification de protéines et analyse structurelle

Info

Publication number
EP4581364A1
EP4581364A1 EP23762572.8A EP23762572A EP4581364A1 EP 4581364 A1 EP4581364 A1 EP 4581364A1 EP 23762572 A EP23762572 A EP 23762572A EP 4581364 A1 EP4581364 A1 EP 4581364A1
Authority
EP
European Patent Office
Prior art keywords
protein
probe
donor
binding site
acceptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23762572.8A
Other languages
German (de)
English (en)
Inventor
Chirlmin JOO
Raman Gautam VAN WEE
Mike FILIUS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technische Universiteit Delft
Original Assignee
Technische Universiteit Delft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technische Universiteit Delft filed Critical Technische Universiteit Delft
Publication of EP4581364A1 publication Critical patent/EP4581364A1/fr
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the invention relates to a method for characterization of a structure of a protein using a first probe and a second probe.
  • the invention further relates to a system for characterization of a structure of a protein.
  • the invention further relates to a data carrier.
  • WO2018102759A1 relates to methods and systems for identifying a protein within a sample.
  • a panel of antibodies are acquired, none of which are specific for a single protein or family of proteins. Additionally, the binding properties of the antibodies in the panel are determined. Further, the protein is iteratively exposed to a panel of antibodies. Additionally, a set of antibodies which bind the protein are determined. The identity of the protein is determined using one or more deconvolution methods based on the known binding properties of the antibodies to match the set of antibodies to a sequence of a protein.
  • W02021049940A1 describes an analysis method for characterization of a tagged protein using FRET donor-acceptor pair chromophores, wherein the FRET donoracceptor pair chromophores comprise a first chromophore and a second chromophore, wherein the FRET donor-acceptor pair chromophores have a donor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range, wherein one of the FRET donor-acceptor pair chromophores is excitable by donor excitation radiation in the donor excitation radiation range, wherein the other of the FRET donor-acceptor pair chromophores is configured to provide acceptor emission radiation in the acceptor emission radiation range upon excitation with donor excitation radiation in the donor excitation radiation range of the one of the FRET donor-acceptor pair chromophores when the first chromophore and the second chromophore are configured within a predetermined distance, wherein the tagged protein comprises a first amino acid tagged with a first tag and
  • WO2022096677A1 describes a method for obtaining quantitative information on average donor-acceptor distance changes within a molecule or in between molecules using ensemble Forster resonance energy transfer (eFRET), and a measurement system comprising a controller adapted for performing the same, wherein the method comprises the steps of performing an eFRET measurement for at preferably a donor-, an acceptor-, and at least a donor-acceptor-labelled sample, wherein each sample comprises respective labelled molecule copies and wherein each eFRET measurement is performed using multiple respective labelled molecule copies, under a first and a second condition, correcting the obtained results for fluorophore-specific, condition-specific and inter-condition effects, determining conditionspecific eFRET efficiencies based on the corrected results, and determining quantitative information on donor-acceptor distance changes within the molecule between the first and the second condition based on the respective condition-specific eFRET efficiency.
  • eFRET ensemble Forster resonance energy transfer
  • WO20 16050813 Al describes a method for detecting a spatial proximity of a first and a second epitope f a protein or of a first and a second protein of a protein complex in a sample of a subject.
  • the method comprises binding a first binding member having a first oligonucleotide conjugated thereto to the first epitope, binding a second binding member having a second oligonucleotide conjugated thereto to the second epitope, and determining whether a Fluorescence Resonance Energy Transfer (FRET) effect is present between a donor fluorophore and an acceptor fluorophore, which are associated with the first oligonucleotide and the second oligonucleotide, wherein the presence of the FRET effect indicates a spatial proximity of the first and the second oligonucleotide and, thus, the spatial proximity of the first and the second epitope.
  • FRET Fluorescence Resonance Energy Transfer
  • Proteins are biochemical workhorses in all living cells. The many thousands of different proteins sustain the functions of the cell, from copying DNA and catalyzing basic metabolism to producing cellular motion and more. For understanding of biological processes and their (dys)regulation, including diseases, it may be critical to identify and monitor the protein composition of cells by sequencing (i.e. determination of the amino acid sequence of proteins) and/or structural analysis (i.e. determination of the three-dimensional structure of proteins).
  • sequencing i.e. determination of the amino acid sequence of proteins
  • structural analysis i.e. determination of the three-dimensional structure of proteins.
  • assigning the function to proteins remains one of the biggest challenges in fundamental and biomedical research. This is partly because it is not known how large the human proteome is. Currently there are reports suggesting that the human proteome can be as small as 20,000 proteins to as large as several millions.
  • a term “proteoform” has been defined. It refers to each individual molecular form of a protein that is derived from a single protein-encoding gene.
  • the number of protein encoding genes in the human genome may be estimated to be about 20,000. If all protein encoding genes result in a single protein, then the number of different proteins in the human proteome may also be about 20,000. However, a process known as alternative splicing may increase the number of transcripts to -80,000.
  • the complexity of the proteome further increases due to post-translational modification (PTMs).
  • PTMs post-translational modification
  • the PTMs may result in many hundreds of thousands of additional protein variants.
  • the large variety and number of PTMs may affect the protein function. Subtle differences in highly similar proteoforms may have profound effects on health.
  • the concentration range at which different proteoforms exist in the cell spans several orders of magnitude, with some of them being present with just a few copies per cell. To address these challenges, a method that can detect and discriminate them with single-molecule sensitivity may be needed.
  • protein sequencing and structural analysis remains a challenge, especially when only a small sample of the protein is available.
  • Modern protein analysis commonly utilizes mass spectrometry-based identification techniques — determining the precise mass of a protein. Such current methods may suffer from limitations. First, they can analyze only fragments of proteins. Information on those fragments is then used to reconstruct the full-length amino acid sequence, but this often fails due to the combinatorial complexity. Second, they often fail to recognize minor protein species among highly abundant protein species, since sequence prediction is made through analysis of complex spectral peaks. As many important cellular proteins such as signaling proteins exist in very low abundance, it can be difficult to obtain comprehensive proteomic information. Early detection of diseases may also rely on detection of low concentrations of protein biomarkers and thereby forms a demand for protein sequencing and structural analysis techniques capable of working at the single-molecule scale. In addition, it may be particularly challenging to analyze proteoforms of a protein using mass spectrometry approaches.
  • Single-molecule techniques are cutting-edge detection tools to study biological processes at the nanoscale and may be suited for samples containing target molecules (such as proteins) with low copy numbers.
  • target molecules such as proteins
  • Potential single-molecule protein sequencing approaches have recently been explored. For example, nanopores based on a-hemolysin have been used to distinguish between non-phosphorylated and phosphorylated proteins. Further, a comparable biological nanopore in combination with a motor protein complex has been used to control protein translocation through a nanopore. Edman degradation has been used for fluorescence detection of single peptides and single-molecule fluorescence fingerprinting of peptides and cellular proteins has been demonstrated using a motor protein complex.
  • proteins may typically comprise up to 20 different amino acid building blocks. Independent of the readout method of choice, full protein sequencing may require the detection of 20 distinguishable signals, which has so far not been demonstrated.
  • Prior art methods for protein characterization and identification may require linearization and fragmentation of the protein, hence losing the folded conformation and structure of the whole protein. Further, such methods may require the use of enzymes for translocation, linearization, and fragmentation of the protein, all of which may alter the protein structure via enzymatic activity.
  • Prior art methods may require (covalently) attaching linkers to specific amino acid residues of a protein. Thereby, such methods may be limited to analyses only of those amino acid residues for which there is linker chemistry available. Further, the attaching of such linkers to amino acid residues may be labor-intensive, wasteful with regards to reagents (and thus costly), and may result in incomplete or off-target labelling. In addition, the attachment of such linkers may complicate the procedure, may hamper flexibly analyzing the protein, and may result in changes to the structure of the protein, such as to the secondary, tertiary and/or quaternary structure of the protein.
  • the present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
  • the invention provides a method for characterization of a structure of a protein using a first probe and a second probe.
  • the method may comprise an exposure stage.
  • the exposure stage may comprise exposing the protein to the second probe.
  • the exposure stage may further comprise providing excitation radiation (or “radiation”) to the protein.
  • the radiation (or: “light”) may especially have a wavelength selected from a donor excitation radiation range.
  • the exposure stage may additionally measure emission to provide an emission signal.
  • the emission may especially be measured in a donor emission radiation range and an acceptor emission radiation range.
  • the exposure stage may be protein degradation-free.
  • the protein may comprise a first binding site and a second binding site.
  • the first probe may in embodiments be covalently bound to the protein at the first binding site.
  • the first probe may in other embodiments be configured to transiently bind the protein at the first binding site.
  • the first probe may especially comprise a first chromophore.
  • the second probe may be configured to transiently bind the protein at a second binding site.
  • the second probe may comprise a second chromophore.
  • the second probe may comprise a second affinity-based probe, especially a second affinity-based probe selected from the group comprising an aptamer, an antibody, a nanobody, and a small-molecule moiety.
  • the invention provides a method for characterization of a structure of a protein using a first probe and a second probe, wherein the method comprises: an exposure stage comprising: (i) exposing the protein to the second probe, (ii) providing radiation to the protein, wherein the radiation has a wavelength selected from a donor excitation radiation range, and (iii) measuring emission in a donor emission radiation range and an acceptor emission radiation range to provide an emission signal; wherein the exposure stage is protein degradation-free; and wherein: the protein comprises a first binding site and a second binding site; the first probe is: (i) covalently bound to the protein at the first binding site; or (ii) configured to transiently bind the protein at the first binding site, wherein the first probe comprises a first chromophore; the second probe is configured to transiently bind the protein at the second binding site with an off-rate selected from the range of 0.01 - 10 s’ 1 , wherein the second probe comprises a second chromophore
  • the protein fingerprinting method employs a first probe and a second probe.
  • the first probe and second probe may interact with a localized protein substructure, such as a protein domain, protein sequence, or amino acid, optionally in a highly specific manner. Thus their binding may be a reliable indicator for the presence of a particular localized protein substructure. Examining the binding of a different first probe and second probe to a protein or protein substrate in relation to each other may provide information about the presence and locations of the localized protein substructure that may be present in the protein. This information may facilitate characterizing, such as identifying, the protein.
  • the protein may especially be immobilized at the first binding site.
  • the first probe may be (covalently) bound to the protein at the first binding site, and may be immobilized on a structure, such as via a binding to an analytical surface.
  • the binding stage may comprise binding the protein to the first probe during the binding stage, wherein the first probe is immobilized (on a structure), or wherein the binding stage further comprises immobilizing the first probe on a structure.
  • the protein may be immobilized at a different binding site, such as a localized protein substructure, than the first binding site.
  • the protein may be immobilized (at either the first binding site or at a different binding site) prior to the first probe binding to the first binding site.
  • the second probe may comprise an aptamer.
  • the aptamer may comprise 2-80 aptamer monomers.
  • the aptamer may comprise 5-80 aptamer monomers, such as 5-70 aptamer monomers.
  • the aptamer may in embodiments comprise 10-70 aptamer monomers, especially 10-60 aptamer monomers,
  • the aptamer may comprise 20-60 aptamer monomers, such as 20-50 aptamer monomers.
  • the aptamer may especially comprise a DNA aptamer.
  • the DNA aptamer may comprise 10-80 aptamer monomers, i.e., 10-80 nucleotides.
  • the DNA aptamer may comprise 12-80 aptamer monomers, such as 12-70 aptamer monomers.
  • the DNA aptamer may in embodiments comprise 15-70 aptamer monomers, especially 15-60 aptamer monomers, Moreover, the DNA aptamer may comprise 20-60 aptamer monomers, such as 20-50 aptamer monomers.
  • the second probe may comprise an antibody (or: “immunoglobulin”).
  • Antibodies are glycoproteins that may be naturally produced by the immune system of vertebrate animals and may be configured to selectively bind specific molecules or molecule domains, such as protein domains.
  • the target molecule or molecule domain of an antibody is called an epitope.
  • the immune system of vertebrate animals may be able to produce many varieties of antibodies with different epitopes, thereby facilitating the ability of the vertebrate animal immune system to recognize and neutralize foreign compounds.
  • Antibodies with specific epitopes may be generated for use in biotechnological applications. Hence, antibodies may be generated for use as a second probe in the current invention.
  • the invention may provide a method to investigate the epitope of an antibody on a protein.
  • the second probe may comprise a nanobody.
  • Nanobodies are the recombinant variable domains isolated from antibodies, retaining the specific binding abilities for an epitope but losing other parts of the antibody glycoprotein. Nanobodies may hence have a smaller molecular weight and better solubility than antibodies. Hence, nanobodies may be generated for use as a second probe in the current invention.
  • the second binding site may be a localized protein substructure with a known localization within the protein structure, especially in such embodiments employing the method with the goal of identifying the presence or absence of a known protein in a (biological) sample.
  • the second binding site may also be a localized protein substructure with an unknown localization within the protein structure, especially in such embodiments employing the (fingerprinting) method with the goal of investigating the structure of a known or unknown protein in a (biological) sample.
  • the second probe may be configured to transiently bind the protein at the second binding site.
  • a transient binding may be described using binding kinetics that comprise (i) an on-rate (also: “association rate constant”, “association rate coefficient”, K on ), being the second-order rate constant for the binding of the second probe to the second binding site, and (ii) an off-rate (also: “dissociation rate constant”, “dissociation rate coefficient”, K O ff), being the first-order rate constant for the disassociation of the second probe from the second binding site.
  • an on-rate also: “association rate constant”, “association rate coefficient”, K on
  • an off-rate also: “dissociation rate constant”, “dissociation rate coefficient”, K O ff
  • probes may have different on-rates and off-rates for the protein, especially for binding sites in the protein.
  • probes may be designed, for instance via computational modeling and/or via binding assays, with specific binding affinities for one or more specific binding sites.
  • the second probe may bind the protein at the second binding site with an on-rate selected from the range of 0.05 * 10 6 - 5 * 10 6 M’ 1 , such as 0.05 * 10 6 -
  • the second probe may bind the protein at the second binding site with an on-rate selected from the range of 0.1 * 10 6 - 2 * 10 6 M’ 1 , such as 0.1 * 10 6 - 1 * 10 6 M' 1 .
  • the second probe may bind the protein at the second binding site with an on-rate selected from the range of 0.2 * 10 6 - 1 10 6 * M’ 1 , such as 0.2 * 10 6 - 0.8 * 10 6 M' 1 .
  • the second probe may bind the protein at the second binding site with an on-rate selected from the range of 0.4 * 10 6 - 0.8 * 10 6 M’ 1 , such as 0.4 * 10 6 - 0.6 * 10 6 M'
  • the second probe may bind the protein at the second binding site with an off- rate selected from the range of 0.01 - 10 s' 1 . Further, the second probe may bind the protein at the second binding site with an off-rate selected from the range of 0.02 - 10 s' 1 , such as 0.02 - 5 s' 1 . In embodiments, the second probe may bind the protein at the second binding site with an off-rate selected from the range of 0.05 - 5 s' 1 , such as 0.05 - 2 s' 1 . Moreover, the second probe may bind the protein at the second binding site with an off-rate selected from the range of 0.1 - 2 s' 1 , such as 0.1 - 1 s' 1 .
  • the second probe may bind the protein at the second binding site with an off-rate selected from the range of 0.2 - 1 s' 1 , such as 0.2 - 0.5 s' 1 .
  • the binding kinetics of the second probe may be controlled. This may be done by f.e. altering the ion strength of the solution which may affect aptamer folding. Through adjusting specific ion concentrations, the binding kinetics of aptamers may be sped up (which may result in for a faster read-out) or slowed down (which may result in a higher signal-to- noise ratio).
  • the binding kinetics of the second probe may be controlled by altering the labeling (or “binding”) of the second chromophore to a different position on the second probe.
  • the second probe comprises an aptamer
  • the second chromophore may be labeled to a different position in the aptamer sequence, which may affect the binding kinetics of the second probe.
  • the method, especially the exposure stage may comprise controlling the off-rate (or the on-rate) of the second probe at the second binding site in the range of 0.02 - 5 s' 1 (or in the range of 0.01 * 10 6 - 10 * 10 6 M ⁇ s' 1 ), and especially in the other ranges mentioned hereabove.
  • the first probe may bind the protein at the first binding site with an on-rate selected from the range of 0.2* 10 6 - 1 * 10 6 M’ 1 , such as 0.2* 10 6 - 0.8 * 10 6 M’ 1 .
  • the first probe may bind the protein at the first binding site with an on-rate selected from the range of 0.4* 10 6 - 0.8 * 10 6 M'V, such as 0.4* 10 6 - 0.6 * 10 6 M'V.
  • the first probe may be configured to transiently bind the protein at the first binding site with an off-rate selected from the range of 0.01 - 10 s' 1 .
  • the second probe may comprise the acceptor chromophore
  • the molar ratio between the second probe and the first probe may be selected from the range of 3: 1 - 50:1, such as from the range of 3:1 - 30: 1.
  • the molar ratio between the second probe and the first probe may be selected from the range of 2: 1 - 30: 1, especially from the range of 2: 1 - 20: 1, such as from the range of 3:2 - 20: 1, especially from the range of 3:2 - 10: 1.
  • the molar ratio between the second probe and the first probe may be selected from the range of 1 : 1 - 10: 1, such as 1 : 1 - 5: 1.
  • Aptamers with complementary sequences may be able to transiently bind each other instead of their respective first binding site or second binding site, which may result in the donor and acceptor being within FRET distance and thus providing acceptor emission radiation.
  • the use of aptamers with complementary sequences may interfere with the FRET method due to providing an emission signal based on probe-to-probe binding.
  • the first probe may bind (covalently or transiently) to the first binding site, optionally with a known localization within the protein structure, whereas the second probe may transiently bind to the second binding site with a (known or unknown) localization within the protein structure.
  • the second probe provides information about the second binding site in the protein structure in relation to the (predetermined or known) localization of the first binding site as a reference point.
  • the first chromophore and the second chromophore may be selected from FRET donor-acceptor pair chromophores.
  • FRET is a method based on the transfer of the energy of a donor fluorophore to an acceptor fluorophore. This energy transfer may only occur when the two fluorophores are placed within several nanometers. As the transfer efficiency is sensitive to sub-nanometer distance change, the transmission efficiency of the radiation that is being emitted by the acceptor fluorophore after excitement by the donor fluorophore depends on this sub-nanometer distance change. FRET may hence be used as a spectroscopic ruler for probing biological systems.
  • the method may especially relate to the use of FRET donor-acceptor pair chromophores.
  • FRET Form Resonance Energy Transfer
  • Fluorescence Resonance Energy Transfer may herein refer to the transfer of the energy of a donor chromophore to an acceptor chromophore, which may occur when the donor-acceptor pair chromophores are within a FRET distance, such as within several nanometers.
  • the FRET donor-acceptor pair chromophores may comprise a first chromophore and a second chromophore, wherein the FRET donor-acceptor pair chromophores have a donor excitation radiation range, an acceptor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range, wherein one of the FRET donor-acceptor pair chromophores is excitable by donor excitation radiation in the donor excitation radiation range, wherein the other of the FRET donor-acceptor pair chromophores is configured to provide acceptor emission in the FRET acceptor emission radiation range upon excitation with donor excitation radiation in the donor excitation radiation range of the one of the FRET donor-acceptor pair chromophores when the first chromophore and the second chromophore are configured within a FRET distance.
  • the donor chromophore and the acceptor chromophore are arranged within a FRET distance, which may vary for different FRET donor-acceptor pairs, the donor chromophore may upon excitation with donor excitation radiation transfer energy to the acceptor chromophore, whereupon the acceptor chromophore may emit acceptor emission radiation.
  • This energy transfer may occur with a specific FRET efficiency depending on the (exact) distance between the donor chromophore and the acceptor chromophore.
  • FRET transfer
  • the FRET transfer efficiency may be sensitive to sub-nanometer distance changes, which may make FRET an outstanding spectroscopic ruler for probing, for example, biological systems.
  • the FRET efficiency (E) may be defined as: wherein I a is the intensity of the acceptor emission, and wherein la is the intensity of the donor emission.
  • the distance between the donor chromophore and the acceptor chromophore may then be estimated by comparing the measured value of E (equation above) to an estimated value of the FRET Efficiency E e as a function of the distance r: wherein R is the Forster radius, which may be specific for the donor-acceptor pair.
  • the distance between the first binding site and the second binding site may then be estimated based on the estimated distance between the donor chromophore and the acceptor chromophore, as well as, for example, based on the (length of) the probes.
  • the FRET donor-acceptor pair chromophores may have an excitation radiation range and an emission radiation range for both the donor and the acceptor.
  • the donor of the FRET donor-acceptor pair chromophores may be excitable by exposure to donor excitation radiation with a wavelength range in the donor excitation radiation range. When the donor has thus been excited, it may re-emit donor emission radiation with a wavelength range in the donor emission radiation range.
  • the acceptor of the FRET donor-acceptor pair chromophores may be excitable by exposure to acceptor excitation radiation with a wavelength range in the acceptor excitation radiation range. When the acceptor has thus been excited, it may re-emit acceptor emission radiation with a wavelength range in the acceptor emission radiation range.
  • the FRET donor-acceptor pair chromophores may be selected such that the donor emission radiation range substantially overlaps with the acceptor excitation radiation range.
  • the donor when the donor becomes excited upon exposure to donor excitation radiation, it may emit donor emission radiation that falls within the acceptor excitation radiation range. If the donor and acceptor are in sufficient proximity for FRET coupling and hence exposure of the acceptor to donor emission radiation, the acceptor may then become excited and emit acceptor emission radiation.
  • the FRET donor-acceptor pair chromophores may herein be configured to provide acceptor emission radiation in the acceptor emission radiation range upon excitation with donor excitation radiation of the donor.
  • the acceptor excitation radiation range may comprise a (sub)range selected from the range of 200 - 1500 nm, especially from the range of 400 - 800 nm.
  • the acceptor emission radiation range may comprise a (sub)range selected from the range of 200 - 1500 nm, especially from the range of 400 - 800 nm.
  • the FRET excitation and emission ranges will in general depend on the used FRET pairs.
  • the donor-acceptor chromophore pair may comprise Cy3 and Cy5, which may respectively be maximally excited at 552 nm and 650 nm, and may provide emission radiation at about 568 nm and 666 nm.
  • the donor-acceptor chromophore pair may comprise Cy3 and Cy7, which may respectively be maximally excited at 488 nm and 750 nm, and may provide emission radiation at about 568 nm and 788 nm.
  • the donor-acceptor chromophore pair may comprise Cy5 and Cy7, which may respectively be maximally excited at 650 nm and 750 nm, and may provide emission radiation at about 666 nm and 788 nm.
  • the FRET donor-acceptor pair chromophores may especially comprise a chromophore pair selected from the group comprising Atto488/Cy3, Atto488/Cy3b, Atto488/Cy5, Atto488/Atto647n, Cy3/Cy5, Cy3b/Cy5, Cy3/Cy7, Cy3b/Cy7, and Cy5/Cy7.
  • the term “predetermined distance range” and similar terms may herein especially refer to a distance range wherein FRET energy transfer can occur for the FRET donor-acceptor pair chromophores, which may vary for different sets of FRET donor-acceptor pair chromophores.
  • FRET pairs Different combinations may be used for probing different regions of the protein.
  • the most commonly used FRET pair Cy3-Cy5
  • Cy3-Cy7 may be used that may be most sensitive at a distance of ⁇ 3 nm.
  • Cy2-Cy3 may be used that may be most sensitive at a distance of ⁇ 7-nm.
  • the end- to-end distance of a FRET pair may also be altered by placing a chromophore at a different position in the probe, such as at a different part of the sequence of an aptamer.
  • the FRET donor-acceptor pair may be optimized by altering the chromophore pair.
  • the FRET donoracceptor pairing may be (essentially) unaffected by being bound to the first probe or second probe and may remain valid if switched, i.e. Cy3 may be bound to the first probe and Cy5 may be bound to the second probe and vice versa.
  • the FRET pairing may occur when the first chromophore and the second chromophore are configured within a FRET distance.
  • the FRET distance may be selected from the range of 0.05 - 20 nm. In further embodiments, the FRET distance may yet be selected from the range of 0.1 - 10 nm. In further embodiments, the FRET distance may be selected from the range of 1 - 9 nm, such as from the range of 2 - 8 nm, especially from the range of 3 - 7 nm.
  • radiation may then be provided having a wavelength selected from the donor excitation radiation range to the protein. This may excite the donor chromophore and may consequently result in the donor providing donor emission radiation. If the donor chromophore and acceptor chromophores separation is within the FRET distance, FRET from the donor to the acceptor may take place. Hence, the acceptor may become excited and consequently provide acceptor emission radiation. The efficiency of FRET may depend on the precise distance between the donor and acceptor up to a sub-nanometer distance difference. If the donor and acceptor chromophores separation is not within the FRET distance, FRET may (essentially) not occur. Hence, the presence or absence of acceptor emission radiation, and the efficiency of the FRET of such acceptor emission radiation, may provide information about the distance between the donor and acceptor.
  • the method may further comprise a distance estimation stage comprising estimating a distance (di) between the first binding site and the second binding site based on the emission signal, especially based on a FRET efficiency.
  • the emission signal may further comprise the ratio between the emission from the donor emission radiation range and the emission from the acceptor emission radiation range. This ratio comprises information on the transfer efficiency of the FRET that has occurred between the acceptor and the donor.
  • the emission signal may allow for a sub-nanometer distance di estimation between the first binding site and the second binding site.
  • the current invention provides a method to determine the structure of (essentially) the whole protein.
  • the method of the invention may facilitate determining at least part of the structure of the whole protein, i.e., of an unfragmented and non-degraded protein.
  • currently applied methods may commonly analyze protein degradation products rather than the whole protein.
  • the structure of protein degradation products may be different from the structure of the whole protein. The current invention therefore may provide an approach allowing the characterization and identification of the structure of the whole protein.
  • detection of alternatively spliced proteins may be of high relevance in disease detection and/or monitoring as several diseases, such as cystic fibrosis, cancer and Parkinson disease have been associated with mutations in their spliceoforms that lead to alternative splicing and abnormal protein production.
  • current methods that employ protein degradation to characterize and identify proteins may not be able to differentiate between different proteoforms with largely overlapping amino acid sequences. Through protein fingerprinting of the whole protein, the method of the invention may be able to differentiate between such proteoforms with high accuracy.
  • the method may be a non-medical method. In further embodiments, the method may be a non-diagnostic method.
  • the method may, prior to the exposure stage, comprise providing the protein from a protein source comprising the protein.
  • the protein source may be a biological sample, such as from a bacterial sample, or such as from an archaeal sample, or such as from a protozoan sample, or such as from a fungal sample, or such as from a mammalian sample, or such as from a plant sample.
  • the protein source may be a clinical sample obtained from a patient, or a research sample obtained from a cell culture, or a research sample obtained in situ.
  • the exposure stage may comprise exposing the protein to different second probes.
  • the exposure stage may comprise sequentially exposing the protein to different second probes.
  • at least two of the different second probes may be configured to transiently bind to the protein at different respective second binding sites.
  • the transient binding of the second probe may allow probing two or more second binding sites. Further, the transient binding of the second probes may ensure that the probing of different second binding sites on the protein may be (temporally or spatially) separated.
  • the temporal separation may be achieved via successive exposure of the protein to the at least two different second probes.
  • the exposure stage may comprise a washing step.
  • the washing step may follow after exposure of the protein to one second probe, and may comprise removing the one second probe (such that the protein is no longer exposed to the one second probe).
  • the washing step may then be followed by exposure of the protein to another second probe.
  • the protein may be exposed to the at least two second probes separately, i.e., not simultaneously.
  • the emission signal may be measured throughout the successive exposure of the protein to the at least two different second probes.
  • the exposure stage may comprise flushing the protein (with first probe bound thereto) over a well-plate with different second probes arranged in different wells.
  • the exposure stage may comprise concurrently exposing the protein to different second probes within the same container.
  • at least two of the different second probes may be configured to transiently bind to the protein at respective second binding sites.
  • the different second probes may especially have different second chromophores.
  • the different second chromophores may be selected such that (i) they may each form a FRET donor-acceptor pair with the first chromophore, and (ii) they may provide a distinguishable emission signal upon FRET with the first chromophore.
  • the first probe may comprise a donor chromophore and the different second chromophores may comprise different acceptor chromophores, and the different second chromophores may be distinguished based on different acceptor emission radiation upon excitation by donor emission radiation.
  • the first probe may comprise an acceptor chromophore and the different second chromophores may comprise different donor chromophores, and the different second chromophores may be distinguished based on different donor emission radiation upon binding to the second binding site.
  • the different second probes may further be distinguished based on different binding kinetics.
  • the protein may be exposed to at least two or more different second probes concurrently and within the same container, but through at least two or more distinguishable emission signals this configuration may allow for the accurate probing of their different second binding sites.
  • the exposure stage may comprise exposing the protein to different second probes with different second binding sites.
  • the method may comprise exposing the protein to the different second probes sequentially, one second probe before a different second probe.
  • the method may comprise exposing the protein to the different second probes in different containers, each providing an individual emission signal.
  • the method may comprise exposing the protein to the different second probes simultaneously within the same container, one second probe comprising a different chromophore than the chromophore comprised by a different second probe.
  • the transient binding of the second probe may further circumvent photobleaching issues.
  • a probe comprising a photobleached chromophore may dissociate from the protein, and a (same) probe comprising a fresh chromophore may again associate to the protein at the same binding site.
  • the protein may be probed multiple times using the transiently binding second probes.
  • the transient interactions may be monitored in real time using a fluorescence microscope, such as especially a single-molecule fluorescence microscope. This repetitive probing may allow for the FRET efficiency to be determined with less than 0.1% error, which may provide a smaller than 0.1 nm resolution.
  • the emission signals from the FRET pairings may be repetitively recorded for the protein until a FRET “fingerprint” sufficient for profiling of the structure of the protein is obtained.
  • a FRET histogram may be provided from recording the emission signal per second probe.
  • the FRET histogram may comprise mean FRET efficiency for each observed FRET event.
  • the FRET histogram may be fit using a Gaussian function.
  • the center of the peak may be determined with an accuracy (standard error) of up to a maximum of 2%, such as up to a maximum of 1%, like up to a maximum of 0.5%, especially up to a maximum of 0.1%, moreover up to a maximum of 0.05%, when at least 100 binding events are recorded.
  • the distribution of the center of the peaks from all measurements may be called a ‘FRET fingerprint’ that is unique for the structure of the protein.
  • the term “protein fingerprint” may herein refer to a protein-specific (unique) signal, especially wherein the protein fingerprint is suitable for identification of the protein.
  • the protein fingerprint may especially refer to one or more of an array of FRET efficiency values; an array of estimated distances; and/or raw data, especially one or more emission signals, obtained according to the method of the invention.
  • the invention may provide a system for characterization of a structure of a protein.
  • the system may comprise (i) an analytical space, (ii) a probe supply, (iii) a radiation source, (iv) a fluorescence microscope, especially a single-molecule fluorescence microscope, and (v) a control system.
  • the analytical space may especially comprise an analytical surface and may be configured to host the protein.
  • the probe supply may be configured to provide probes to the analytical space.
  • the radiation source may be configured to provide radiation, especially donor excitation radiation, to the analytical space.
  • the fluorescence microscope may especially comprise a single-molecule fluorescence microscope and be configured to measure emission radiation having a wavelength in a donor emission radiation range and in an acceptor emission radiation range in the analytical space.
  • the fluorescence microscope may be configured to (then) provide an emission signal to the control system.
  • the control system may in some embodiments comprise an analysis system, and in other embodiments, the control system may be functionally coupled to an analysis system.
  • the system In an operational mode (of the system), the system, especially the control system, may be configured to execute the method of the invention.
  • a system may be provided that performs the method as described above for characterization of a structure of a protein.
  • the control system may be configured to have the system execute the various stages of the method as described above.
  • controlling and similar terms especially refer at least to determining the behavior or supervising the running of an element.
  • controlling and similar terms may e.g. refer to imposing behavior to the element (determining the behavior or supervising the running of an element), etc., such as e.g. measuring, displaying, actuating, opening, shifting, changing temperature, etc..
  • controlling and similar terms may additionally include monitoring.
  • controlling and similar terms may include imposing behavior on an element and also imposing behavior on an element and monitoring the element.
  • the controlling of the element can be done with a control system, which may also be indicated as “controller”.
  • the control system and the element may thus at least temporarily, or permanently, functionally be coupled.
  • the element may comprise the control system.
  • the control system and element may not be physically coupled. Control can be done via wired and/or wireless control.
  • the term “control system” may also refer to a plurality of different control systems, which especially are functionally coupled, and of which e.g. one control system may be a master control system and one or more others may be slave control systems.
  • a control system may comprise or may be functionally coupled to a user interface.
  • the control system may be configured to estimate a distance di between the first binding site and the second binding site based on the emission signal.
  • the control system, especially the analysis system may further be configured to predict a structure of the protein based on the estimated distance di.
  • the control system may in embodiments be configured to provide an estimated distance di and to predict a structure of the protein.
  • Such embodiments may be especially suitable for the characterization of a protein that has not been characterized previously, e.g. the investigation of a protein being developed as a novel drug candidate.
  • control system may be configured to predict a protein structure of the protein (using a computational process), especially based on the estimated distance, or especially based on the FRET efficiency.
  • the computational process may especially comprise a computational algorithm.
  • control system may be configured to execute in a controlling mode the analysis method according to the invention.
  • the control system may especially receive program instructions from a data carrier such that the control system executes the method according to the invention.
  • the invention may provide a data carrier having stored thereon program instructions. Such program instructions when executed by the system described above may cause the system to execute the method described above.
  • the data carrier may facilitate the execution of pre-programmed operational modes of the system. This may increase user convenience and adherence to standard use of the system.
  • the data carrier may comprise the reference data that may be used in the protein identification stage.
  • stage and similar terms used herein may refer to a (time) period (also “phase”) of a method and/or an operational mode.
  • the different stages may (partially) overlap (in time).
  • the exposure stage may, in general, be initiated prior to the fingerprint provision stage, but may partially overlap in time therewith.
  • the binding stage may typically be completed prior to the exposure stage. It will be clear to the person skilled in the art how the stages may be beneficially arranged in time.
  • Fig. 1A-B schematically depict embodiments of the method for characterization of a structure of a protein using a first probe and a second probe.
  • Fig. 2 schematically depicts embodiments of the system for characterization of a structure of a protein.
  • Fig. 3 A-B depict further aspects of embodiments of the invention.
  • Fig. 4 schematically depicts results of fingerprinting experiments.
  • Fig. 5A-C schematically depict results of further fingerprinting experiments.
  • Fig. 6A-B schematically depict results from binding kinetics experiments.
  • Fig. 7A-C schematically depict results from further binding kinetics experiments.
  • the schematic drawings are not necessarily on scale.
  • Fig. 1 A schematically depict embodiments of the method for characterization of a structure 13 of a protein 10 using a first probe 31 and a second probe 32.
  • the method comprises an exposure stage comprising (i) exposing the protein 10 to the second probe 32, (ii) providing excitation radiation 50 having a wavelength selected from a donor excitation radiation range to the protein 10, and (iii) measuring emission radiation 60 in a donor emission radiation range and an acceptor emission radiation range to provide an emission signal.
  • the exposure stage is protein degradation-free.
  • the protein 10 comprises a first binding site 11 and a second binding site 12.
  • the FRET donor-acceptor pair chromophores 23,24 have a donor excitation radiation range, the donor emission radiation range 63 and the acceptor emission radiation range 64.
  • a donor chromophore 23 of the FRET donor-acceptor pair chromophores 20 is excitable by donor excitation radiation 53 in the donor excitation radiation 53 range.
  • an acceptor chromophore 24 of the FRET donor-acceptor pair chromophores 23,24 is configured to provide acceptor emission radiation 64 (also see Fig.
  • the system further comprises a probe outlet 225 configured for the removal of probes from the analytical surface 210.
  • the single-molecule fluorescence microscope 240 comprises or is functionally coupled to a plurality of optical elements configured to separate the radiation emitted from the analytical space 210 (by the donor chromophore 23 and/or the acceptor chromophore 24) into the donor emission radiation 53 and acceptor emission radiation 64.
  • the single-molecule fluorescence microscope 240 may comprise an EMCCD camera 241 to measure the donor emission radiation 53 and the acceptor emission radiation 64. It will be clear to the person skilled in the art, that many variations of the single-molecule fluorescence microscope 240 and/or the optical elements may be possible without deviating from the scope of the invention as described herein.
  • Fig. 2 further schematically depicts a data carrier 400 having stored thereon program instructions, which when executed by the system 200 according to the invention, especially by the control system 300, causes the system 200 to execute the method 100 according to the invention.
  • the control system 300 may comprise the data carrier 400.
  • Fig. 3A-B depict further embodiments for the immobilization of a protein 10 that may be used to investigate a structure 13 of the protein 10.
  • Fig. 3 A depicts the immobilization of a protein 10 using an antibody 38.
  • a first probe 31 may comprise an affinitybased probe 35 comprising an acceptor chromophore 24 targeting the first binding site 11.
  • a second probe 32 may comprise an aptamer 36 and a donor chromophore 23 targeting the second binding site 12.
  • Fig. 3B depicts the immobilization of a protein 10 with a covalently bound aptamer 36. This may provide for the probing of a structure 13 of the protein 10 by an affinitybased probe 35 comprising an aptamer 36 and a donor chromophore 23.
  • Imaging buffer the experiments were performed in imaging buffer comprising 100 mM NaCl and 10 mM Na2HPO 4 /NaH 2 PO4 pH 7.4 or 100 mM KC1 and 10 mM K 2 HPO 4 /KH 2 PO4.
  • the ions in this buffer may promote aptamer 36 folding, in particular the stabilization of the G-quadruplex and duplex, which are structures that some DNA/RNA aptamers 36 may adopt.
  • the aptamers 36 have off-rates of 0.05 - 0.2 s' 1 .
  • the binding affinity of an aptamer 36 can be fine-tuned by altering the position of the fluorescent dye, which resulted in up to 20-fold difference in binding affinity.
  • the emission 60 was collected using an ssFRET TIRF microscope 240. TIR excitation radiation 50 and FRET pair 20 emission 60 detection using prism type TIRF. Immobilized molecules were exposed to donor excitation radiation 53 by TIR using a green and/or red laser. Fluorescence emission 60 was collected by an objective and the slit created images of half the size of the EM-CCD camera. The fluorescence emission 60 signal was split into donor and acceptor signal by a dichroic mirror and was imaged side by side on the EM- CCD. Collected movies were processed and analyzed using custom written software.
  • aptamers were synthesized with a short Thymine stretch at their 5' to increase the distance from the aptamer-protein binding site and the chromophore (see SEQ ID NO: 1-4).

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Abstract

L'invention concerne un procédé de caractérisation d'une structure d'une protéine à l'aide d'une première sonde et d'une seconde sonde, le procédé comprenant : une étape d'exposition consistant à (i) exposer la protéine à la seconde sonde, (ii) fournir un rayonnement à la protéine, le rayonnement ayant une longueur d'onde choisie dans une plage de rayonnement d'excitation de donneur, et (iii) mesurer l'émission dans une plage de rayonnement d'émission de donneur et une plage de rayonnement d'émission d'accepteur pour fournir un signal d'émission, l'étape d'exposition se faisant sans dégradation de la protéine, et la protéine comprenant un premier site de liaison et un second site de liaison; la première sonde étant : (i) liée de manière covalente à la protéine au premier site de liaison; ou (ii) configurée pour lier de façon transitoire la protéine au premier site de liaison, la première sonde comprenant un premier chromophore;la seconde sonde étant configurée pour se lier de façon transitoire à la protéine au second site de liaison avec un taux de décroissance choisi dans une plage de 0. 01 à 10 s-1, la seconde sonde comprenant un second chromophore, la seconde sonde comprenant une sonde par affinité choisie dans le groupe comprenant un aptamère, un anticorps, un nanocorps et une fraction de petite molécule; le premier chromophore et le second chromophore étant choisis parmi les chromophores de la paire donneur-accepteur FRET, les chromophores de la paire donneur-accepteur FRET présentant la plage de rayonnement d'excitation du donneur, la plage de rayonnement d'émission du donneur et la plage de rayonnement d'émission de l'accepteur, un donneur des chromophores de la paire donneur-accepteur FRET étant excitable par le rayonnement d'excitation du donneur dans la plage de rayonnement d'excitation du donneur, un accepteur des chromophores de la paire donneur-accepteur FRET étant configuré pour fournir un rayonnement d'émission d'accepteur dans la plage de rayonnement d'émission d'accepteur lors de l'excitation par le rayonnement d'excitation du donneur lorsque le premier chromophore et le second chromophore sont configurés à une distance FRET choisie dans la plage de 0. 1 - 10 nm.
EP23762572.8A 2022-08-31 2023-08-28 Aptamère fret monomoléculaire pour identification de protéines et analyse structurelle Pending EP4581364A1 (fr)

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EP1692486B1 (fr) * 2003-12-12 2015-10-28 Saint Louis University Biocapteurs permettant de detecter des macromolecules et d'autres analytes
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WO2018102759A1 (fr) 2016-12-01 2018-06-07 Ignite Biosciences, Inc. Procédés d'analyse de protéines
NL2023825B1 (en) * 2019-09-12 2021-05-17 Univ Delft Tech Single-molecule FRET for protein characterization
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