EP4278180A1 - Procédé et systèmes d'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique - Google Patents

Procédé et systèmes d'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique

Info

Publication number
EP4278180A1
EP4278180A1 EP22702887.5A EP22702887A EP4278180A1 EP 4278180 A1 EP4278180 A1 EP 4278180A1 EP 22702887 A EP22702887 A EP 22702887A EP 4278180 A1 EP4278180 A1 EP 4278180A1
Authority
EP
European Patent Office
Prior art keywords
heteropolymer
sequence
residual current
nanopore
monomer building
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
EP22702887.5A
Other languages
German (de)
English (en)
Inventor
Jan Behrends
Tobias ENSSLEN
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.)
Albert Ludwigs Universitaet Freiburg
Original Assignee
Albert Ludwigs Universitaet Freiburg
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 Albert Ludwigs Universitaet Freiburg filed Critical Albert Ludwigs Universitaet Freiburg
Publication of EP4278180A1 publication Critical patent/EP4278180A1/fr
Pending legal-status Critical Current

Links

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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C07K1/128—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

Definitions

  • the present invention relates to a method for identifying a sequence of monomer building blocks of a biological or synthetic heteropolymer.
  • the invention also relates to the use of a nanopore to identify a sequence of monomer building blocks of a biological or synthetic heteropolymer.
  • the invention also relates to a computer-implemented method, a computer program code and a data processing system for identifying a sequence of monomer building blocks of a biological or synthetic heteropolymer.
  • the identification of proteins in complex mixtures currently relies on mass spectrometry of ionized molecules in the gas phase, a powerful but expensive technology that requires large equipment.
  • the present invention consists in a novel approach that involves a highly controlled and automated, preferably enzymatic, fragmentation using both sequence-specific endopeptidases and exopeptidases with a newly developed principle of "peptide spectrometry through nanopores" for the purpose of label-free characterization of protein mixtures, including identification , discrimination and finally protein sequencing combined.
  • Nanopore size spectroscopy was first demonstrated for synthetic polymers, but recently it was shown to be applicable to peptides and enable their highly sensitive label-free discrimination (Piguet et al. 2018; Ouldali et al. 2020). Importantly, this technique is able to detect differences in individual amino acid residues and - in contrast to mass spectrometry - distinguish between peptides of the same mass, e.g. peptides containing either the stereoisomers leucine or isoleucine (Ouldali et al. 2020), or are characterized by sequence isomerism.
  • the current standard method for identifying proteins from mixtures involves a series of separation steps, such as liquid chromatography or (2D) gel electrophoresis, followed by tryptic digestion to peptide fragments and mass spectrometry, eg electrospray ionization (ESI), or matrix-assisted laser desorption/ionization ( MALDI), followed by a separation according to the transit time (TOF), or in a quadrupole (Q)/multipole field and subsequent correlation with known proteins in databases.
  • mass spectrometry while a powerful technique, requires expensive and bulky equipment and suffers from significant deficiencies in terms of detection limits and dynamic sensitivity range.
  • a more fundamental disadvantage is that peptides of the same mass but different composition (eg containing leucine or isoleucine) cannot be differentiated without derivatization.
  • novel solutions are required to identify, discriminate, and ultimately sequence proteins with single-molecule sensitivity.
  • nanopore-mediated single-molecule DNA sequencing in which only 4 nucleobases with the same charge have to be distinguished, the problem of protein structure elucidation is much more complex because of the 20 proteinogenic amino acids (aa). To date, this area is still in its infancy, but some progress has already been made, which is summarized below.
  • Single molecule detection through nanopores is based on the analysis of the reduction in electrical conductivity that occurs when an analyte, e.g. a DNA strand or a peptide, diffuses into a molecularly dimensioned water-filled channel located in an insulator, i.e. into a nanopore, or migrated.
  • the principle of electrical detection of the transport of molecules through a nanopore which is a protein channel or an artificial channel, e.g. a nanoscale aperture in a solid-state membrane or a nanotube (nanotube) or a DNA origami structure embedded in a lipid membrane or a introduced into a solid membrane introduced nanoscale hole can act is known.
  • the membrane is subjected to a potential difference that induces an ionic current across the nanopore in the presence of an electrolyte solution or other ionically conductive medium (e.g., an ionic liquid).
  • an electrolyte solution or other ionically conductive medium e.g., an ionic liquid.
  • the interaction of a molecule with the channel of a nanopore in particular the entry of the molecule into the channel, the presence of the molecule in the channel or the passage of the molecule through the channel, induces a measurable reduction in current if the conductive medium in the channel is a has higher electrical conductivity than the analyte and vice versa.
  • Biological (protein) nanopores which form such channels through insulating lipid bilayers, were the first nanopores to demonstrate the ability to detect single molecules, and they enable current nanopore-based DNA sequencing techniques.
  • nanoscopic pores can be produced in solid materials such as thin SiN membranes by various drilling or etching processes.
  • solid-state nanopores are promising, although the production of solid-state nanopores that are as identical as possible is a technical challenge.
  • are pore-forming Proteins are constructed with atomic precision and have evolved over millions of years to allow solute transport across membranes.
  • Fig. 1 a sketch of the principle of single molecule detection by nanopores is shown.
  • a constant potential difference AE across an insulator drives an ionic current through the pore.
  • a single analyte molecule in the pore partially blocks the current (resistance pulse). Both the depth of the blockage or the residual current and the duration and temporal variations of this current signal carry information about the analyte.
  • the reduction in conductivity is measured as a change in ionic current induced by a constant voltage across the insulator in which the pore forms the only (or the dominant) electrically conductive link.
  • These signals correspond to individual analyte molecules entering the pore and interacting with the inner wall of the pore - possibly, but not necessarily, traversing the pore, i.e. translocating through the pore from one side of the insulator to the other .
  • the analyte is a polymer (eg a peptide, polynucleotide or a synthetic polymer such as poly(ethylene glycol))
  • two regimes must be distinguished, as shown in Figure 2: in the threading regime is the polymer stretched and few of its monomers contribute to the change in resistance. In this regime, the current signal is sensitive to the identity of the monomers in the narrowest part of the pore and can therefore be used for sequencing if the polymer is threaded through the pore in a regular manner, i.e. with a speed that is as uniform as possible.
  • the collapsed regime was used for the nanopore-mediated determination of the molecular size distribution of neutral synthetic polymers (Baaken et al. 2015). It is assumed that in this regime there is a non-specific binding of the collapsed polymer to the pore wall (binding regime; Talarimoghari, M., G. Baaken, R. Hanselmann, and JC Behrends. 2018. Size-dependent interaction of a 3-arm star polyethylene glycol) with two biological nanopores . Eur. Phys. JE 41:6288-8.
  • the two regimes of the polymer-nanopore interaction are shown in FIG.
  • the threading/translocation regime is favored when long polyelectrolyte chains in relation to the pore length in low to medium salt concentration (0.1 to 0.3 M KCl) interact with the pore, with relatively high electrical voltages (>50 to >100 mV) to the Used to move the polymer through the pore in an electric field.
  • the collapsed/binding regime also: trapping regime, since the pore acts as a molecular trap here
  • the collapsed/binding regime can only be used for polymers that are short enough or and/or sufficiently collapsed to fully fit within the pore. Binding and trapping of a polymer in the pore is also possible for charged polymers and also for polymers in the non-collapsed or not fully collapsed state, as long as they are not too long for the pore.
  • the investigations on which this invention is based showed that carrying out the current measurement method (step b) in claim 1) in the collapse regime (also: collapsed, binding or trapping regime) is particularly advantageous.
  • the method according to the invention serves to identify a sequence of monomer building blocks of a biological or synthetic heteropolymer and has the following steps: a) carrying out a fragmentation method in which the heteropolymer is fragmented in particular enzymatically, chemically and/or physically, and a fragment mixture is thereby obtained , whose fragments are molecules with different sequence sections of the heteropolymer; b) performing a current measurement method in which current signals of a current through the channel of a single nanopore, or a current that occurs in parallel through a plurality or plurality of channels of a plurality or plurality of nanopores, are detected, each current signal on the interaction of a Fragments with the channel of the nanopore is based, the current signals being characteristic for the different fragments, it being possible to determine a representative set of characteristic current signals which represents the fragment mixture; c) Carrying out an evaluation method in which a sequence of monomer building blocks of the heteropolymer is determined from the representative set of the characteristic current signals.
  • the fragments of the fragment mixture are obtained by successive degradation of the heteropolymer.
  • n-2, n-1 , n so that the length fragments have a total length of n-(n-1), n-(n-2)...to n-(nn) monomer building blocks) to obtain a heteropolymer consisting of n monomer building blocks, each length fragment having the identical sequence to the heteropolymer Having monomer building blocks starting from position 1 (start of chain) to position n-(ni).
  • a fragment mixture is also referred to here as a “ladder” or as a heteropolymer ladder, ie a “peptide ladder” if the heteropolymer is/has a peptide.
  • the sequence of monomer building blocks of the heteropolymer determined in step c) can be part of the total sequence (partial sequence) of monomer building blocks of the heteropolymer, or, preferably, be the total sequence of monomer building blocks of the heteropolymer.
  • the heteropolymer is a peptide.
  • the fragmentation method is or includes Edman degradation.
  • the fragmentation procedure can be designed to involve the cleavage of the protein by endopeptidases into peptides, and in particular the treatment of the peptides by exopeptidases to obtain the peptide ladder.
  • the method according to the invention preferably has the following steps: in particular preferably in each case in step b):
  • a characteristic residual current value designates the measurement results of the current value measurement resulting from the interaction of a specific fragment, which is characterized by the characteristic residual current value, with the nanopore.
  • the characteristic residual current value contains in particular the residual current value that can be assigned to the corresponding current signal.
  • the characteristic residual current value can also be a vector-valued variable which, in addition to the residual current value, has other Includes components, the number of which determines the dimension of the vector value. Such components can be a duration of the current signal or another variable describing the time course of this current signal, or can be parameters that describe an interpolation curve that is used to describe the current signal.
  • a characteristic residual current value in each case describes a fragment type, in particular fragment size, of the number n of fragment types of a fragment mixture formed from the heteropolymer.
  • a fragment mixture formed as a peptide ladder contains a total of n fragment types starting from a peptide with n amino acids as monomer building blocks.
  • the peptide solution containing the fragment mixture usually contains a large number of fragments of each fragment type (peptide type).
  • a mixture of fragments obtained by 100% efficient fragmentation of a starting quantity M of the peptide to be sequenced also contains a number M of fragments for each of the n fragment types of the peptide. If the term “fragment” is spoken of in this application, the fragment type in particular can be meant, depending on the context.
  • the method according to the invention is preferably defined as an extended method which serves to determine a sequence of a protein, comprising the steps i) cleaving the protein, in particular by enzymatic and/or chemical and/or physical cleavage, in order to obtain peptides as cleavage products of the protein; optional: obtaining the peptides by chromatographic or electrophoretic separation of a peptide mixture obtained by the cleavage; ii) application of the method according to the invention for determining the sequence of amino acids (monomer building blocks) of at least one, in particular each, of the peptides (heteropolymer); iii) carrying out a recognition method for recognizing the sequence of the protein, in which the sequence of the protein is determined from the sequence of amino acids of the at least one peptide.
  • the method according to the invention or the above-mentioned embodiment of the method according to the invention can advantageously be used to elucidate the, in particular complete, primary structure of a macromolecule, in particular a biological macromolecule, in particular a protein, the biological macromolecule containing different heteropolymers, in particular formed from different heteropolymers bonded to one another is:
  • the method according to the invention is preferably defined as an extended method which is used to determine the primary structure of a macromolecule, in particular a protein, comprising the steps i) cleavage of the macromolecule, in particular protein, in particular by enzymatic and/or chemical and/or physical cleavage in order to to obtain heteropolymers, in particular peptides, as cleavage products of the macromolecule; optional: Obtaining heteropolymers, in particular the peptides, by separation, in particular chromatographic or electrophoretic separation, of a heteropolymer mixture obtained by the cleavage, in particular a peptide mixture; ii) application of the method according to the invention for determining a sequence of monomer building blocks, in particular amino acids, at least one, in particular each, of the heteropolymers, in particular peptides; iii) Carrying out a macromolecule recognition method, in particular protein recognition method, in which the primary structure of the macromolecule, in particular protein, is determined from the sequence of the at least one
  • the method according to the invention can be designed to determine part of the complete sequence of monomer building blocks from which the heteropolymer is composed. If only part of the complete sequence of monomer building blocks of a heteropolymer is determined, the method according to the invention can be used in particular to implement a determination method in which the partial sequence of monomer building blocks of a heteropolymer determined using the method according to the invention is used to determine which previously known heteropolymer from a set T (1 to T) of previously known different (namely different in terms of their sequence) heteropolymers was determined.
  • “Prior art” here means that the nearly complete, or full sequence of monomer building blocks of any previously known heteropolymer is known.
  • the partial sequence determined using the method according to the invention represents a “fingerprint” of the heteropolymer to be determined from the previously known quantity of heteropolymers, i.e. a feature that makes the heteropolymer sought clearly identifiable from the other heteropolymers in the quantity 1 to T.
  • the steps of such a detection method can be described as follows: i) providing the information about the previously known sequence of each heteropolymer of a set of 1 to T different heteropolymers; ii) use of a heteropolymer to be determined which is identical to exactly one heteropolymer of this quantity of 1 to T different heteropolymers, it not being known in particular which heteropolymer of this quantity the heteropolymer to be determined is identical to; iii) carrying out the method according to the invention for determining a partial sequence of the heteropolymer to be determined; iv) comparing the partial sequence determined in iii) with the previously known sequences of all heteropolymers in the amount from 1 to T different Heteropolymers and determining the desired heteropolymer from the set of previously known heteropolymers using the partial sequence that makes the desired heteropolymer compared to the other heteropolymers of the set 1 to T clearly identifiable.
  • the determination method mentioned allows the determination of the complete sequence of a desired heteropolymer, without the complete sequence of the desired heteropolymer having to be elucidated by means of the method according to the invention, if the desired heteropolymer comes from a set T of previously known heteropolymers, each with a previously known sequence, with a partial sequence -after Fingerprint type - uniquely identifies the sought-after heteropolymer from the remaining heteropolymers of that lot.
  • the detection method is the more efficient way to determine the complete sequence of the searched heteropolymer compared to the alternative of using the method according to the invention to elucidate the complete sequence of the searched heteropolymer instead of the partial sequence of the searched heteropolymer.
  • the nanopore is preferably a biological nanopore, ie a pore-forming toxin or a porin.
  • the nanopore is preferably a solid nanopore or a hybrid of a solid and biological and/or chemical components.
  • a solid body in particular a substrate, can have or be formed from at least one of the following materials: SiNx, SiO2, HfO2, MOS2, CNT, graphene, nanopipettes.
  • Biological or chemical components can, each preferably, contain or consist of at least one of the following: pore-forming toxins, porins, ⁇ -barrel proteins, alpha-helical membrane proteins, DNA origami structures. Hybrids, i.e. combinations of all the components mentioned above, are possible.
  • the heteropolymer is preferably fragmented by enzymes.
  • these are endo/exo-peptidases for proteins/peptides and common ones for DNA Restriction enzymes (nucleases).
  • nucleases DNA Restriction enzymes
  • Possible peptidases are mentioned, for example, in: https://www.ebi.ac.uk/merops/ Possible nucleases are mentioned, for example, in: https://wikivisually.com/wiki/List_of_restriction_enzyme_cutting_sites%3A_Bst%E2%80%93Bv#Whole_list_navigation
  • the heteropolymer is fragmented chemically and non-enzymatically.
  • proteins/peptides one can use Schlack-Kumpf and Edman degradation.
  • DNA enzymes are usually used for this.
  • the heteropolymer is preferably fragmented physically, for example by exposure to heat, cold, sound waves, electromagnetic radiation, in particular infrared, ultraviolet or X-ray radiation, microwaves or visible light. Examples of this are documented in https://doi.Org/10.1073/pnas.0901422106 or https://doi.Org/10.1007/s13361-017-1794-9 and https://doi.Org/10.1002/mas.20214 .
  • the nanopore is preferably selected from the group of preferred nanopore proteins containing aerolysin, alpha-hemolysin, MspA, CsgG, VDAC or another protein from the beta-barrel protein family, as well as genetically optimized variants of these pore proteins.
  • the pore proteins and the other measurement conditions are preferably optimized for an interaction of the analyte (of the fragment) with the pore, which results in an interaction between the analyte and the pore that is optimally long-lasting for the respective analyte.
  • a preferred configuration of the nanopore is as follows: the nanopore is preferably an aerolysin pore, in particular a variant of the aerolysin pore.
  • the single-molecule trap of the aerolysin pore can be adapted and optimized to the analyte by single-point mutation in the dimension and depth of the potential well.
  • Aerolysin variants R220S/A/C/K/H/E/D/Q/N, R288S/A/C/K/H/E/D/Q/N, R282S/A/C/K /H/E/D/Q/N, D222S/A/C/F/R/K/H/E/Q/N, D276S/A/C/F/R/K/H/E/Q/N, D209S/A/C/F/R/ K/H/E/Q/N,
  • K238S/A/C/F/R/D/H/E/Q/N K242S/A/C/F/R/D/H/E/Q/N, K244S/A/C/F/R/ D/H/E/Q/N,
  • E254S/A/C/F/R/D/H/K/Q/N E252S/A/C/F/R/D/H/K/Q/N and any combination thereof.
  • the aerolysin pore in its natural form (wild type) or as a variant thereof is particularly preferred for use as a nanopore within the scope of the invention.
  • the variant can be designed to differentiate and characterize fragments of heteropolymers that differ only by positional isomerism, for example.
  • positional isomerism derived from acetylation was distinguished (“Resolving isomeric posttranslational modifications using a nanopore”, Tobias Ensslen, Kumar Sarthak, Aleksei Aksimentiev, Jan C. Behrends, bioRxiv 2021.11.28.470241; doi: https://doi.org/10.1101/2021.11.28.470241).
  • a translocation or passage of the analyte through the pore is not necessary, although it is permitted in principle. Rather, it is particularly advantageous if the same analyte visits its binding site in the pore for as long as possible, or visits it again several times and binds there after it has left the molecular trap again in the direction of the entry opening. Accordingly, “interaction” of the fragment (analyte, molecule) with the channel of the nanopore preferably means that the fragment enters the channel but does not pass through the channel, which ultimately results in a non-destructive multiple determination of the same molecule.
  • step b) carrying out the current measurement method (step b) in claim 1) in the collapse regime (also: collapsed, binding or trapping regime) is particularly advantageous.
  • the current measurement method carried out in step b) is preferably carried out in such a way that the fragment mixture is present in an electrolyte solution which, in particular, has dissolved salts of the form AX, A 2 X and AX 2 etc., with substance A (e.g. selected from the alkali and alkaline earth metals Na, K, Cs, Rb, Li) provides the cation and substance X (e.g. selected from the halogens F, Cl, Br) provides the anion.
  • substance A e.g. selected from the alkali and alkaline earth metals Na, K, Cs, Rb, Li
  • substance X e.g. selected from the halogens F, Cl, Br
  • the substance groups A and X can include other components in terms of inorganic or organic derivatives of such salts (where, for example, substance A is a quaternary ammonium, imidazolium, phosphonium, pyridinium and pyrrolidinium ion such as tetramethylammonium and substance X is a nitrate, a sulfate, phosphate , an amino acid such as glutamate, a carboxylic acid such as gluconate, citrate, a (bi)carbonate, or a simple hydroxide).
  • the electrolyte solution can preferably also contain mixtures of different combinations of different salts.
  • the total salt concentration of the electrolyte solution in which the fragment mixture is present during the implementation of the current measurement method is between 0.5 M and 20 M, preferably between 2 M and 10 M and particularly preferably between 3 M and 5 M.
  • the fragment mixture can alternatively to an electrolyte solution in one ionic liquid present.
  • Such configurations of the electrolyte ensure that conditions such as charge shielding and solubility of the analyte in the electrolyte solution are optimally adjusted for the collapsed/binding regime and the analyte remains in the molecule trap of the pore for as long as possible, and at the same time the highest possible signal-to-noise ratio is achieved. ratio of the current measurement is achieved.
  • the invention also relates to the use of a nanopore for carrying out the method according to the invention for identifying a sequence of monomer building blocks of a biological or synthetic heteropolymer.
  • the invention also relates to a computer-implemented method for determining a sequence of monomer building blocks of a heteropolymer (heteropolymer sequence) from the measurement data of a current measurement method that contains information about current signals that are determined during the interaction of different fragments formed from the heteropolymer with a nanopore, having the steps :
  • the invention also relates to a computer program code that is stored on a data carrier and that determines a sequence of monomer building blocks of a heteropolymer (heteropolymer sequence) from the measurement data of a current measurement method when it is executed by the central processor of a computer, the measurement data containing information about current signals that are the interaction of different fragments formed from the heteropolymer with a nanopore can be determined, having the steps implemented in each case by the program code: A) determination of residual current values (the current signals) from the measurement data, with a residual current describing the interaction of one of the different fragments of the heteropolymer with a nanopore;
  • the invention also relates to a data processing system for determining a sequence of monomer building blocks of a heteropolymer (heteropolymer sequence) from the measurement data of a current measurement method that contains information about current signals that are determined during the interaction of different fragments formed from the heteropolymer with a nanopore, having a computer with a central processor, and a program code, in particular the program code according to the invention, the computer being programmed to carry out the following computer-implemented steps:
  • the evaluation method in which the sequence of the monomer building blocks of the heteropolymer is determined from the representative set of the characteristic current signals, preferably provides the following computer-implemented steps:
  • a prediction algorithm can be used to indicate a probability or an evaluation factor for evaluating the reliability of a primary structure of the heteropolymer determined by estimation from the incomplete data, in particular from an incomplete representative set of characteristic residual current values.
  • the prediction algorithm can have been determined by machine learning using, in particular, labeled training data.
  • the labeled data may contain variations of incomplete representative sets of the residual current characteristic values of previously known heteropolymers.
  • the prediction algorithm can contain an artificial neural network, in particular a convolutional neural network (CNN), which can be trained using the labeled training data.
  • CNN convolutional neural network
  • 1 shows a sketch of the principle of single molecule detection by nanopores, which can be used in the method 100 according to the invention.
  • 2 shows the two possible regimes of a polymer-nanopore interaction.
  • FIG 3 shows the detection of the twenty proteinogenic amino acids (aa) using the aerolysin nanopore, in particular according to the prior art.
  • 5a, 5b and 5c each show exemplary embodiments of the method according to the invention and its components.
  • Figure 6a shows, in relation to an embodiment of the invention: sequences of the six heterodeca peptides constituting the start peptide of the ladder.
  • 6b shows in relation to an embodiment of the invention: a schematic representation of the experimental setup.
  • 6c shows in relation to an embodiment of the invention: a control measurement curve in 4 M KCl
  • 6d shows in relation to an embodiment of the invention: an exemplary measurement curve after addition of the peptide ladder L1 with all peptides in an equimolar concentration.
  • Figure 6e shows in relation to an embodiment of the invention: a schematic mean level histogram over the main level for a peptide ladder sequencing experiment.
  • FIG. 7 shows in relation to an embodiment of the invention: residence time scatter diagrams over the residual pore flow l/lo (red) with superimposed level histograms (black) averaged over the main level for all six peptide ladders.
  • FIG. 8 shows: data correlation plots for all six peptide ladders.
  • FIG. 9a shows in relation to an embodiment of the invention: Reproducibility of l/lo of the homo-arginine peptides R3, R4, R5, R7 (blue) in comparison to R3-R7 from Piguet et al. 2018 (red), and conductors L1 (green, solid line, circle), L3 (green, dashed, pointing triangle), L4 (green, dotted, pointing triangle), L2 (pink, solid line, circle), L5 (pink , dashed, pointing triangle), L6 (pink, dotted, pointing triangle).
  • 9b shows in relation to an embodiment of the invention: A I/Io boxplot for each cleaved amino acid type with median (blue) and mean (white).
  • Figure 9c shows, in relation to an embodiment of the invention: A I/Io values for arginine cleavage classified by the nearest neighbor aa of arginine as C-terminal aa (alanine blue, arginine red, serine green, tyrosine yellow) of homo- (dots) and hetero-peptides (circles); Data for homo-peptides were taken from Piguet et al. 2018
  • Fig. 9d shows in relation to an embodiment of the invention: residence time scatter diagrams over the residual pore flow l/lo with superimposed main level averaged level histograms for the deca peptides of Porteri (red), Porter2 (blue), Porters (green), Porter4 (yellow) ,
  • IDEs pink
  • auerß black
  • FIG. 10 shows in relation to an exemplary embodiment of the invention: residence time scatter diagrams over the residual pore flow l/lo (red) with superimposed level-averaged histograms (black) sample A (left) and B (right). Below each graphic, the suggested sequences (prop) and the correct sequences (corr) are shown using the first reading aid. The green box indicates the correct reading frame.
  • Fig. 11 shows in relation to an embodiment of the invention: Data table for double-blind study.
  • Figure 1a shows an illustration of the principle of single molecule capture by nanopores which can be used to implement the invention.
  • a constant voltage AU across an insulator draws ionic current through the nanopore.
  • a single analyte particle, e.g. a fragment, in the nanopore partially blocks the current (resistance pulse or current signal, or residual current value). Both the depth of blockage and the duration carry information about the analyte.
  • FIG. 2 shows the two possible regimes of a polymer-nanopore interaction.
  • the threading/translocation regime is favored when long polyelectrolyte chains in low to medium salt concentration (0.1 to 1.0 M KCl) interact with the pore.
  • the binding trapping, or collapsed, regime typically occurs under high salt conditions (e.g., 4M KCl) and does not require loading of the analyte.
  • the collapsed regime is preferably used in the invention.
  • an electrolyte-filled first compartment 11 is electrically insulated from an electrolyte-filled second compartment 12 by a membrane formed in particular by means of a lipid double layer 2; a current flow is essentially only possible through the nanopore 3 built into the lipid bilayer, which electrically connects the compartments 11 and 12 .
  • the lipid bilayer can be stretched over the micro-aperture or over a micro-cavity of a microstructure device (not shown in Figure 2), such as is described in document WO 2013/083270.
  • the threading/translocation regime the analyte 4a is elongated, in the collapsed/binding regime, the analyte 4b is collapsed and compact.
  • FIG. 3 shows the detection of the twenty proteinogenic amino acids (aa) using the aerolysin nanopore.
  • peptides or other heteropolymers
  • peptides which can initially be produced preferably by enzymatic or chemical or physical cleavage of proteins, preferably be separated using known chromatographic or electrophoretic methods, or in which peptides or other heteropolymers are already isolated, and, preferably in a second step, either the action of exopeptidases, which cleave individual N- or C-terminal amino acids from a peptide, or chemical Methods such as the Edman reaction are subjected to produce a mixture of peptides or heteropolymers, ie a fragment mixture, in which several species or characteristic types of fragments are present in a representative set, which preferably represent all or most of the possible fragments that by removing the amino acids (or Mon omer building blocks) are generated one after the other, so that for a peptide (or a heteropolymer) of degree of polymerization (d.p.)
  • the measurement evidence shows the ability of the invention to correlate, for example, short, known peptide sequences in this way with the data of the nanopores (see FIG. 4).
  • Figure 4 shows:
  • A, B Scatterplots with event histogram obtained from the interaction of aerolysin with two peptide ladders containing a triarginine handle.
  • the removal of aa leads to a species-specific shift in the residual current that is characteristic of a monomer building block type (here aa).
  • C,D Plot of change in peptide volume and relative residual current for the two ladders shown above. A clear correlation between the two parameters as well as the sequence dependency is evident.
  • Fig. 5a shows an exemplary method 100 according to the invention for identifying a sequence of monomer building blocks of a biological or synthetic heteropolymer, comprising the steps: a) carrying out a fragmentation method in which the heteropolymer is fragmented in particular enzymatically, chemically and/or physically, and thereby a fragment mixture is obtained, the fragments of which are molecules with different sequence sections of the heteropolymer; (101) b) performing a current measurement method in which current signals of a current through a nanopore are detected, each current signal being based on the interaction of a fragment with the nanopore, the current signals being characteristic for the different fragments, so that a representative set of characteristic current signals can be determined which represents the mixture of fragments; (102) c) Carrying out an evaluation method in which the sequence of the monomer building blocks of the heteropolymer is determined from the representative set of the characteristic current signals. (103)
  • the method 100 can be used in particular in a method (200) for determining the primary structure of a protein, comprising the steps (see FIG. 5b) i) cleavage of the protein, in particular by enzymatic and/or chemical and/or physical cleavage to form peptides to recover as cleavage products of the protein; optional: obtaining the peptides by chromatographic or electrophoretic separation of a peptide mixture obtained by the cleavage; (201) ii) application of the method according to the invention for determining the sequence of amino acids (monomer building blocks) of at least one, in particular each, of the peptides (heteropolymer); (202 or 100) iii) carrying out a protein recognition method in which the primary structure of the protein is determined from the sequence of the at least one peptide. (203) In particular, method 100 can be carried out for all peptides obtained by cleavage of the protein.
  • the evaluation method (103 or 300), in which the sequence of the monomer building blocks of the heteropolymer is determined from the representative set of the characteristic current signals, can have the following steps in particular (see FIG. 5c):
  • the method according to the invention is described as a “method for peptide sequence recognition with regard to peptide sequencing in a derivatization-free single-molecule experiment using the wt-aerolysin (wt-AeL) nanopore by a bottom-up peptide ladder strategy”.
  • wt-AeL wt-aerolysin
  • wt-AeL wt-aerolysin
  • six peptide ladder-type sample pools were designed. Each pool consisted of the same deca-peptide but with a scrambled sequence and the respective ladder down to the tri-arginine polycationic carrier.
  • the exemplary embodiment uses the wt AeL nanopore.
  • a deca-peptide was designed consisting of a polycationic C-terminal carrier, R 3 , preceded by a heterogeneous stretch of seven aa recruited from the five different aa SRAKY (eg SRASKYR).
  • the sequence of the aa part was scrambled to obtain six different hetero-deca peptides that have exactly the same mass of 1335.65 Da (Fig. 6a).
  • peptide ladders mixture of fragments
  • Stepwise degradation of a peptide in a ladder generation process was simulated by successively adding the peptides of a ladder to the measurement chamber with the nanopore (e.g. Edmann degradation). The step thus corresponds to step a) of the method according to the invention.
  • Step b) of the method according to the invention, or steps A) and B), was carried out as follows:
  • a single wt-AeL channel was inserted into a DPhPC lipid bilayer containing a single 50 ⁇ m opening of the microelectrode used -Cavity arrays (MECA16) spanned.
  • a trans-negative bias of 40 mV was used to drive an ionic current (Io) through the protein channel connecting two reservoirs filled with electrolyte solution (4 M KCl) otherwise electrically isolated from each other by the lipid bilayer.
  • Individual peptides that enter the channel defined by the protein and thereby change the ion current (I) are detected via the resulting resistance pulses, FIG. 6b.
  • FIG. 6e schematically shows a result of a nanopore-based peptide ladder experiment.
  • the peptide ladder of an aa 7 R3 peptide would consist of eight peptides, each leading to a single peak in the histogram of event-averaged residual current values.
  • the sequence of maxima of the residual current histogram represents the sorting of the measured current signal values I as fractions of the current through the unblocked pore Io (also referred to as relative residual current values (l/lo) or relative residual conductivities with possible values between 0 and 1) into a sequence of characteristic residual current values ( Step C)). It thus defines a representative set of 8 different, characteristic residual current values with a likewise characteristic scatter, each of which represents a fragment of the peptide ladder. It is expected that the longest peptide, aa 7 R 3 , would result in the deepest blockade, while the shortest peptide, R 3 , would be represented with the highest l/lo.
  • the sequence of the maxima can also be clearly assigned to the steps of the ladder and the difference in l/lo of two neighboring maxima corresponds to the difference that the splitting off of a single aa would produce in the ladder generation process (used in step D).
  • the size of the distance A l/lo is sensitive to the identity of the cleaved aa, which makes it easier to identify the sequence of the peptide.
  • An evaluation method in which the sequence of the monomer building blocks (here: aa) of the heteropolymer (here: peptide) is determined from the representative set of the characteristic current signals results from using the differences A l/Io of the residual current values of neighboring maxima in the representative set of characteristic residual current values .
  • Step D the determination of the above aa, is carried out by assigning the residual current value differences A l / Io to aa of the peptide using previously known correlation data containing information about which aa is represented by which current value difference amount A l / Io in order to determine the sequence from aa (determining the sequence of aa of the peptide).
  • FIG. 6c and d show exemplary raw data (current traces) for the measurement of conductor L1. After addition of peptides (d), resistance pulses of different depth and duration were detected. It was seen that individual resistance pulses were heavily modulated, but to avoid falsifying the I/Io values, these modulations were excluded and only the main level of a pulse was considered in the data analysis. Such modulations are induced by the movement of the polymer itself within the AeL nanopore.
  • Figure 6a Sequences of the six heterodeca peptides, each representing the start peptide of a ladder. Black dashed boxes symbolize shifts of aa cassettes, black (and gray) lines symbolize inversion, while colored lines symbolize identity of aa in the different sequences; b: Schematic representation of the experimental setup. An external trans-negative voltage is applied to drive an ionic current Io through the open nanopore.
  • the longest Peptide (aa 7 R 3 ) generates the deepest, the shortest peptide (aaiR 3 ) the shallowest block.
  • the differences in I/Io values can be correlated with the identity of the lost aa.
  • the final aa can be determined against the polycationic C-terminal carrier peptide, R 3 (black).
  • Figure 7 Residence time scatter diagrams versus the residual pore current l/lo (red) with superimposed histograms of the relative residual current values (black) averaged over the main current level of the resistive pulses for all six peptide ladders.
  • the peptides were added sequentially, starting with the smallest peptide aaiR 3 and ending with the largest peptide aa 7 R 3 . All measurements of a ladder were made using the same AeL nanopore.
  • the green line indicates the location of the separately determined polycationic C-terminal carrier peptide, R 3 .
  • Figure 8 Data correlation plots for all six peptide ladders. Dwell time scatter plots and level histograms averaged over the main level were analyzed with regard to their differences in dwell time (red), residual current (blue) and number of modulations (black, dotted). The corresponding peptide volumes (green) and hydrophobicity (black, dashed) were also plotted. All values were normalized twice to enable direct comparability.
  • Figure 10 Residence time scatter diagrams over the residual pore flow l/lo (red) with superimposed level-averaged histograms (black) for samples A (left) and B (right). Below each graphic, the suggested sequences (prop) and the correct sequences (corr) are shown using the first reading aid. The green box indicates the correct reading frame.
  • the exemplary embodiment shows the method according to the invention for peptide identification by means of ladder fingerprinting, which can serve as the primary platform for a further development in the direction of peptide sequencing, in particular when using the highly sensitive wt-AeL nanopore.
  • Reliable detection of hetero-peptides consisting of a c-terminal polycationic R 3 carrier and up to seven n-terminal alternating heterogeneous aa was achieved.
  • peptide ladder-like sample pools ranging from aaiR 3 to aa 7 R 3 range
  • the position-sensitive contribution of a specific aa species to the overall block depth of a peptide was investigated and based on these findings a sequencing and fingerprinting reading aid was postulated. With their help, the robustness and reliability of this strategy was proven in a double-blind study by demonstrating the sequencing of a randomly selected peptide and the identification of a second peptide by fingerprinting.
  • peptides were used which were synthesized on demand. This is a model case that can easily be adapted for the case of unknown protein or peptide samples.
  • the more comprehensive analysis of larger heteropolymers is achieved by an initial step of splitting the heteropolymer into further fragmentable sub-components using fragmentation methods, from which ladders can then be formed
  • Sample preparation process are made available.
  • an endo-peptidase can be used to break down proteins into smaller peptides.
  • an exo-peptidase can be used to dynamically generate ladders from these peptides. Individual peptides produced by the protease could be presented sequentially to the nanopore and analyzed in a dynamic exopeptidase-coupled experiment.
  • the method according to the invention is of great value with regard to everyday laboratory applications.
  • Wild-type proaerolysin (pAeL) was produced in-house via standard protocols from E. coli BL21 (DE3) pLysS competent cells using the pET22b (+) vector.
  • pAeL was purified from cell lysates via His-tag chromatography. Stocks of pAeL were prepared at 1 pg pL' 1 , nitrogen frozen and stored at -80°C. Thawed pAeL was activated with trypsin (Promega GmbH, Walldorf, Germany) and used in a final pAeL concentration of 20 pmol L' 1 (or 3 pmol L' 1 AeL).
  • the preprotein construct was chosen in such a way that the affinity tag used for purification is separated from the protein during trypsin activation and native protein is obtained.
  • All membranes were made from 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) from octane.
  • DPhPC was dissolved in chloroform by Avanti Polar Lipids Inc. (Alabaster, AL, USA). The lipids were aliquoted, dried under argon and stored as a dry film at -20°C until used at a concentration of 1 mg mL' 1 .
  • MECA16 cavity arrays from lonera GmbH (Freiburg, Germany) with cavities of 50 pm diameter were used. Further digital filtering (25 kHz Bessel) and event detection was done with self-written LabView (National Instruments)-based software; the subsequent evaluation with Igor Pro 8 (Wavemetrics, Lake Oswego, OR, USA).
  • Suppl. 7 (Supplement 7): determined values for l/lo and residence time of homo-arginine
  • Ensslen et al. Denotes the embodiment of the invention.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique. L'invention concerne en outre l'utilisation d'un nanopore pour l'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique. L'invention concerne en outre un procédé mis en oeuvre par ordinateur, un code de programme informatique et un système de traitement de données pour l'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique.
EP22702887.5A 2021-01-18 2022-01-18 Procédé et systèmes d'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique Pending EP4278180A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102021200425.3A DE102021200425A1 (de) 2021-01-18 2021-01-18 Verfahren und Systeme zur Identifikation einer Sequenz von Monomerbausteinen eines biologischen oder synthetischen Heteropolymers
PCT/EP2022/050990 WO2022152933A1 (fr) 2021-01-18 2022-01-18 Procédé et systèmes d'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique

Publications (1)

Publication Number Publication Date
EP4278180A1 true EP4278180A1 (fr) 2023-11-22

Family

ID=80222084

Family Applications (1)

Application Number Title Priority Date Filing Date
EP22702887.5A Pending EP4278180A1 (fr) 2021-01-18 2022-01-18 Procédé et systèmes d'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique

Country Status (5)

Country Link
US (1) US20240077491A1 (fr)
EP (1) EP4278180A1 (fr)
CA (1) CA3207733A1 (fr)
DE (1) DE102021200425A1 (fr)
WO (1) WO2022152933A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4362028A1 (fr) * 2022-10-31 2024-05-01 Ecole Polytechnique Federale De Lausanne (Epfl) Aérolysine mutante et ses utilisations

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011120394B4 (de) 2011-12-06 2015-06-25 Universitätsklinikum Freiburg Verfahren und Mikrostrukturvorrichtung zur elektrischen Kontaktierung biologischer Zellen
US10139417B2 (en) 2012-02-01 2018-11-27 Arizona Board Of Regents On Behalf Of Arizona State University Systems, apparatuses and methods for reading an amino acid sequence
WO2017173392A1 (fr) 2016-03-31 2017-10-05 Two Pore Guys, Inc. Différenciation, à travers des nanopores, de polynucléotides cibles d'un arrière-plan d'échantillon par fragmentation et liaison de charge utile
FR3053119A1 (fr) 2016-06-24 2017-12-29 Excilone Procede de detection electrique de peptides, proteines et autres macromolecules
WO2020131103A1 (fr) * 2018-12-21 2020-06-25 Sri International Appareils et méthodes impliquant l'exploration de protéines par la protéolyse et la translocation de nanopores

Also Published As

Publication number Publication date
DE102021200425A1 (de) 2022-07-21
US20240077491A1 (en) 2024-03-07
CA3207733A1 (fr) 2022-07-21
WO2022152933A1 (fr) 2022-07-21

Similar Documents

Publication Publication Date Title
Cao et al. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore
JP3372862B2 (ja) 生体液の質量分析装置
DE3513168C2 (fr)
EP1287348B1 (fr) Procede et dispositif de determination qualitative et/ou quantitative d'un modele proteique et/ou peptidique d'un echantillon de liquide, preleve d'un corps humain ou animal
DE69514109T2 (de) Geruchssensor
EP1574837A1 (fr) Méthode et dispositif pour le séquencage de polymères par détection de la variation d'une conductance tunnel
Wang et al. The aerolysin nanopore: from peptidomic to genomic applications
DE112011101618T5 (de) Verwendung von Nanoporen-Arrays zur Multiplex-Sequenzierung von Nukleinsäuren
DE4438833A1 (de) Verfahren zur analytischen Trennung von Viren
Wang et al. Probing molecular pathways for DNA orientational trapping, unzipping and translocation in nanopores by using a tunable overhang sensor
EP4278180A1 (fr) Procédé et systèmes d'identification d'une séquence d'éléments monomères d'un hétéropolymère biologique ou synthétique
Yuan et al. The analysis of single cysteine molecules with an aerolysin nanopore
DE602004009824T2 (de) Analyse von massenspektraldaten in den ruhigen gebieten
Barry et al. Analysis of modified oligonucleotides by capillary electrophoresis in a polyvinylpyrrolidone matrix coupled with electrospray mass spectrometry
Okuno et al. A gold nano-electrode for single ion channel recordings
Machado et al. Effects of alkali and ammonium ions in the detection of poly (ethyleneglycol) by alpha-hemolysin nanopore sensor
DE10358366B4 (de) Massenspektrometrische Substanzidentifizierung
DE2926172A1 (de) Verfahren zur messung von aktivitaeten mit festen ionenleitern
Konovalov et al. Forming the nanosized molecular assemblies (nanoassociates) is a key to understand the properties of highly diluted aqueous solutions
Aoki et al. Single channel properties of lysenin measured in artificial lipid bilayers and their applications to biomolecule detection
EP1377818B1 (fr) Procede et dispositif de detection de la concentration d'un compose contenant de l'oxygene dans un gaz de mesure
Hellwig et al. LILBID-MS: using lasers to shed light on biomolecular architectures
DE10006491A1 (de) Verfahren und Probenträgersystem zur Trennung und Anreicherung von Stoffen in situ
US20230194499A1 (en) Nanopore preparation and detection method and detection apparatus thereof
RU2694131C1 (ru) Способ оценки константы диссоциации органических соединений методом капиллярного электрофореза

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20230629

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)