WO2012016306A1 - Procédé électrophorétique en une seule étape dans un tube capillaire fermé destiné à séparer précisément des oligonucléotides liés à des protéines - Google Patents

Procédé électrophorétique en une seule étape dans un tube capillaire fermé destiné à séparer précisément des oligonucléotides liés à des protéines Download PDF

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Publication number
WO2012016306A1
WO2012016306A1 PCT/BR2010/000264 BR2010000264W WO2012016306A1 WO 2012016306 A1 WO2012016306 A1 WO 2012016306A1 BR 2010000264 W BR2010000264 W BR 2010000264W WO 2012016306 A1 WO2012016306 A1 WO 2012016306A1
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Prior art keywords
capillary
protein
oligonucleotides
covered
bound
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PCT/BR2010/000264
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English (en)
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Luiz Augusto Pinto
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Luiz Augusto Pinto
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Priority to PCT/BR2010/000264 priority Critical patent/WO2012016306A1/fr
Publication of WO2012016306A1 publication Critical patent/WO2012016306A1/fr

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    • 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
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • oligonucleotides were named "aptamers" by the worldwide scientific community.
  • the oligonucleotide bound to such protein acts as a specific antibody for it as it was bound to it throughout the electrophoretic migration and it can be separated from it and reproduced in large industrial scales by several known methods, such as the Polymerase Chain Reaction (PCR) and the cloning in bacteria through cloning vectors.
  • PCR Polymerase Chain Reaction
  • cloning in bacteria through cloning vectors.
  • the attainment of the compound formed by the protein sample strongly bound to its specific oligonucleotide, its aptamer, in a high purity degree is obtained through the following eight steps, of which the seven first ones are mandatory and the eighth one is optional, in case of wishing to amplify the aptamer obtained in high purity degree:
  • the order of these two first steps may be any and both can be carried out simultaneously under identical conditions and apparatus.
  • 3 rd step - the capillary electrophoretic migration of the proteic sample is carried out after it has reacted to the library of oligonucleotides sample and it has been linked to one or more than one oligonucleotides; although the ideal condition to be attained at the end of this third electrophoretic migration is the clear physical separation in a narrow zone along the capillary of a compound formed by at least one oligonucleotide specifically and strongly bound to the sample protein, in practice, by the current State of the Art, this does not take place and the physical separation of such compound along the capillary tube appears as occupying a wide zone of the capillary, called fraction collection window.
  • the compounds of the sample protein, formed by its bound to one or more than one oligonucleotides that are located in the fraction collection window area, are isolated and amplified by the PCR (Polymerase Chain Reaction).
  • 5 th step - the oligonucleotides obtained by the amplification through the PCR technique, described in the 4 th step above, are subjected to the fourth electrophoretic migration under the same previous conditions and in the presence of the sample protein so that a purer compound is attained in the capillary tube region which is called fraction collection window by the current State of the Art.
  • 6 step - the compounds of the sample protein formed by its links with one or more than one oligonucleotides lying in the region of the fraction collection window that were attained at the end of the 5 th step are isolated and amplified by the PCR (Polymerase Chain Reaction).
  • the oligonucleotides obtained by the amplification through the PCR technique in the 6 th step are subjected to the fifth electrophoretic migration, under the same conditions and in the presence of the sample protein in order to obtain a purer compound in a clearer and narrower position inside the capillary tube, peak-like, which has the purpose to isolate the fraction in which the specific bound of the oligonucleotide to the sample protein subjected to the electrophoretic migration is stronger, that is, to obtain the aptamers with a high purity degree.
  • aptamers identified in the 7 th step described above, shown as peaks in the fifth electrophoretic migration, are isolated and subjected to the amplification for their attainment in large industrial scales by means of the several known methods, such as the Polymerase Chain Reaction (PCR) and the cloning in bacteria through the cloning vectors.
  • PCR Polymerase Chain Reaction
  • the electroosmotic flux is an inseparable consequence of the electrophoresis carried out in the fused silica capillaries.
  • capillary internal surface area/volume of the liquid inside it is very large - the capillaries have between 10 and 100 micron of internal diameter - and this makes the phenomena of interaction of the fused silica surface of the capillaries with the buffer solution to be significant.
  • Such ionization leads to the formation of: A - a fixed layer of positive ions overlapping the internal surface of the capillary of the electrophoretic solution compounds in order to keep the electroneutrality,
  • This phenomenon is called electroosmotic flux or electroosmosis.
  • the electrostatic field applied to the electrophoresis is of around 15 kV and it is applied from a distance of around 50cm, this means that an electrical field of about 300 V/cm is strong enough for making the migration of a water current towards the cathode to take place, such current that flows to the opposite direction of the flow of the molecules formed by the fusion of the oligonucleotides and proteins that start to migrate through the electrophoretic migration towards the anode of the apparatus.
  • the migration speed of the "protein - oligonucleotide" complex towards the anode is lower than the speed it would go if there were not the electroosmotic flux, that is, the water flow of the "mobile phase” towards the cathode, due to the negative polarization of the fused silica surface of the capillary.
  • the electroosmotic flux is something chaotic since it is constrained by the narrow diameter of the fused silica capillary and it delays the migration of the "protein - oligonucleotide" complex towards the anode, in such a way that during the time of migration, several of those complexes - formed by the protein bound to different kinds of oligonucleotides - which have very similar molecular charges and masses, they get very little away from each other, forming mixture of a wide spectrum called fraction collection window, that is, an extensive region of similar compounds - and, therefore, they are not pure - which is obtained with the conventional electrophoretic methods, when non-covered fused silica capillaries are used.
  • First innovation - The creation of a new electrophoretic process in a capillary tube that is internally covered with neutral hydrophilic substances, according to the proper description in this Report, process in which a compound formed by the sample protein bound to one or more than one oligonucleotides that have high specificity and high affinity with the proteic sample is collected with a high degree of purity, being that this compound appears in the electropherogram, in the form of a clear peak of separation and localization inside the capillary, reducing six of the mandatory seven steps and one optional step of the current State of the Art process into a single one with only one electrophoretic migration;
  • Second innovation - elimination of the fraction collection window by eliminating the electroosmotic effect due to the use of a capillary internally covered by neutral hydrophilic substances and, consequently, elimination of the need of carrying out consecutive electrophoretic migrations and amplifications through the PCR which constitute the first, second, fourth, fifth, sixth and seventh steps described in the State of the Art;
  • Third innovation - unlike the description of the State of the Art, in which the material of interest of the electrophoretic migration is collected in a large region of the capillary, called fraction collection window, in this new process, only a chosen and central part is collected from the highest point of the electrophoretic separation peak of the compound formed by the sample protein bound to one or more than one oligonucleotides with high specificity and high affinity with the proteic sample, thus being such collection technique an efficient purification technique as it will be properly illustrated by Figure 2, which is partly responsible for the reduction of the seven mandatory steps of the process described by the State of the Art into one single step.
  • the Figure 1 of this Report is the exact reproduction of the " Figure 15, Sheet 15 of 16 of the U.S. Patent US 7,672,786 granted on March 2, 2010" and it shows the typical electropherograms of the State of the Art and it corresponds to the results obtained by the 2 nd , 3 rd , 5 th and 7 th steps of the seven mandatory steps of the State of the Art, as it was previously cited in this paper.
  • the amount of the compound formed by the sample protein bound to the oligonucleotides of the library of oligonucleotides, obtained by this State of the Art is diffuse in a wide area of the capillary, forming the fraction collection window, which determines that through this technique, the oligonucleotides collected there must be subjected to the 4 th mandatory step of the State of the Art, which is the amplification by the PCR.
  • the chart (B) illustrates the 5 th mandatory step of the State of the Art, in which the oligonucleotides amplified by the PCR during the 4 th step are placed together with the sample protein in a new migration for a new collection in the fraction collection window, in the period between 4 minutes and 8 minutes.
  • the collected material is subjected to the 6 th step for amplification by the PCR.
  • the product of this amplification is subjected to the 7 th mandatory step, described in the State of the Art, which is represented by the chart ( C ) of the Figure 1 , where high affinity oligonucleotides, aptamers bound to the sample protein, can be identified, appearing as four peaks (Complexes of Aptamers with PFTase).
  • the aptamers identified in the 7 th step are isolated and they can be amplified by the 8 th step, which is optional, of the State of the Art.
  • Figure 2 is a typical electropherograms obtained in an electrophoretic migration of one single step and with a high discrimination power, according to the descriptions of the innovations of the object of this Patent; in it, we can see the Peak (P1 ) that corresponds to the oligonucleotides of the library of oligonucleotides that have not been bound to the sample protein and which, due to the electroosmotic effect elimination caused by the use of fused silica capillary covered internally with neutral hydrophilic substances, object of this Patent, migrated during the first 3,25 minutes of the electrophoretic migration; we can also see in the same Figure 2, the Peak (P2) that corresponds to the sample protein strongly linked to one or more than one oligonucleotides, being that the space (2) between Peak (1 ) and Peak (2) corresponds to several oligonucleotides with low and medium affinities with the sample protein and they are separated from it between 3,25 and 4,25 minutes and they will be discarded together with the oligonucleotides of the Peak
  • This collection of the compound fraction lying inside the Peak (P2) has its level of capacity of purification as a function of the "time-space" dimension of the segment of the variable extension for collection (S3) and it is determined by the analyst according to the level of purification he wants to impose to the process; the bigger the extension of the variable extension segment for collection (S3), the bigger the chance to have more than one high affinity and high specificity oligonucleotides bound to the sample protein; reciprocally, the smaller the extension determined for the variable extension segment for the smaller such probability is.
  • the formation of the fraction collection window is necessary for collecting the material in the third and fifth steps and at the end of the migration takes around 900 seconds whereas with the innovations to the State of the Art introduced by the object of this Patent, the total electrophoretic migration time is of 360 seconds and the collection period takes until 30 seconds.
  • the Ferritin Protein obtained from the human blood plasma, was used as sample protein to characterize and exemplify a typical result of the process, object of this Patent.
  • a library constituted of 10 14 oligonucleotides made of single strand DNA bought from IDT Technologies (USA - http://www.idtdna.com); this library is constituted of sequences of DNA marked with fluorescein in the end 5 " (line five) containing two preserved regions (19-mer and 22-mer) and a random region (39-mer), constituted of a same likelihood of occurrence of the four nucleotides A, T, C and G, being the sequence as the following: - 5 ' -FAM - CTT CTG CCC GCC TCC TTC CNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NGG NGG AGA CGA GAT AGG CGG ACA CT -3 ⁇ where N represents a random nucleotide.
  • object of this Patent exemplified in Figure 2 For performing the capillary electrophoretic process, object of this Patent exemplified in Figure 2, a commercial apparatus PACE MDQ model (Beckman-Coulter, EUA) equipped with a fluorescence induction laser at the frequency of 480 nm and a fluorescence detector at 520 nm was used.
  • the internally covered capillaries made of fused silica, object of this Patent, used in the electrophoretic process, have their development described below in this Report.
  • the internally covered fused silica capillary has the following specifications: total length of 60cm; detection point of the laser-induced fluorescence at 45cm from the initial end of the capillary; internal diameter of 75 pm and external diameter of 375 ⁇ .
  • Figure 2 shows the electropherogram of the electrophoretic migration carried out with the library oligonucleotides dissolved in a buffer (Tris-acetate 50 mM with pH 8.2, NaCI 100 mM and MgCI 2 5 mM) at the final concentration of 100 ⁇ and a final volume of 5 ⁇ , to which a sample of 1 ⁇ of the Ferritin protein dissolved in the concentration of 2,4 mg/ml was added to the same buffer of the library of oligonucleotides.
  • a buffer Tris-acetate 50 mM with pH 8.2, NaCI 100 mM and MgCI 2 5 mM
  • the sample of the Ferritin protein mixed with the library of oligonucleotides was kept in a temperature of 23°C for 15 minutes in order to be then subjected to the electrophoretic migration, in a way that this sample was injected into the capillary by means of a pressure pulse of 0,5 psi.
  • the capillary was washed for 5 minutes with a buffer solution Tris-acetate 50 mM with pH8.2, which was the same buffer used during the electrophoretic migration carried out in an electrical field of 18kV (300 V/cm).
  • the electropherogram obtained after 6 minutes of the electrophoretic migration in the internally covered capillary, object of this Patent is shown in Figure 2.
  • the fourth innovation the fused silica capillaries internal surfaces being covered with neutral hydrophilic molecules
  • object of this Patent - is purposed to nullify the polarizing effects of the fused silica surfaces of the conventional capillaries and to eliminate the electroosmotic flux.
  • Molecules that have the property of getting bound to the Silanol groups by surface adsortion are applied in the internally covered capillaries with hydrophilic substances after a specific physical-chemical treatment of the internal surface of the fused silica capillaries and, therefore, nullify the electrostatic forces that the ionized silanol groups have on the water molecules, thus eliminating the eletroosmotic flux that occurs during the electrophoretic migrations carried out in conventional fused silica capillaries that are not internally covered.
  • the neutral hydrophilic molecules used for covering the internal surfaces of the capillaries, object of this Patent can be octadecylsilyls, polyvynilsiloxanes, cyclodextrins, cellulose, polymetacrilate, amino acids, proteins, peptides, polyamines, other organic acids or any other ones that produce the same phenomenon, for having the same or similar properties.
  • the process for covering the internal surfaces of the fused silica capillaries, object of this Patent takes place in three stages:
  • the capillary internal surfaces are bound to the neutral hydrophilic molecules chosen to cover the internal surfaces and it is consisted of the following steps:
  • the choice of the substance that will be used for the covering can be: - octadecylsilyls, polyvynilsiloxanes, cyclodextrins, cellulose, polymetacrilate, amino acids, proteins, peptides, polyamines , other organic acids or any other ones that produce the same phenomenon, for having the same or similar properties;
  • the compounds cited above can be identified by fluorescence, laser-induced fluorescence, light absorbance, physical-chemical properties, charge and specific mass.
  • the following electrophoretic parameters can be optimized: temperature, voltage, buffer composition, addition of electrophoretic separation mediators to the buffer, buffer pH, capillary dimensions (length, internal diameter, external diameter), chemical composition of the capillary, chemical substance chosen for covering the 30 capillary internally.
  • oligonucleotides obtained through the object of this Patent "SINGLE- STAGE ELECTROPHORETIC PROCESS IN A COVERED CAPILLARY TUBE FOR ACCURATELY SEPARATING THE PROTEIN-BOUND OLIGONUCLEOTIDES" - can be used for the:

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Abstract

La présente invention, qui résout les problèmes de l'état de la technique, concerne : premièrement, un nouveau procédé électrophorétique destiné à être mis en œuvre dans un capillaire de silice fondue dont l'intérieur a été préalablement enduit avec des substances hydrophiles neutres réduisant ainsi les sept étapes obligatoires du procédé électrophorétique selon l'état de l'art à une seule étape de migration électrophorétique dans le nouveau procédé, dotée d'un pouvoir de séparation élevé du composé « protéine-oligonucléotide », permettant d'obtenir des résultats dans lesquels la protéine échantillon, ainsi que d'autres substances, sont physiquement bien séparées de complexes similaires, permettant ainsi d'obtenir une zone étroite de composés dans le tube capillaire dont l'intérieur est enduit, constituée de la protéine échantillon liée à, au moins, un oligonucléotide par le biais d'une liaison forte et spécifique, éliminant ainsi la fenêtre de collecte des fractions qui est un phénomène typique décrit dans l'état de l'art. Deuxièmement, l'invention concerne le développement d'un capillaire de silice fondue dont l'intérieur est enduit avec des substances hydrophiles neutres qui annulent les effets polarisants des surfaces internes de silice fondue des capillaires conventionnels, éliminant le flux électro-osmotique, servant de base à la création du nouveau procédé décrit ci-dessus, de telles substances étant des octadécylsilyles, des polyvinylsiloxanes, des cyclodextrines, de la cellulose, un polyméthacrylate, des acides aminés, des protéines, des peptides, des polyamines, d'autres acides organiques ou d'autres substances produisant le même phénomène, car ayant les mêmes propriétés ou des propriétés similaires.
PCT/BR2010/000264 2010-08-02 2010-08-02 Procédé électrophorétique en une seule étape dans un tube capillaire fermé destiné à séparer précisément des oligonucléotides liés à des protéines WO2012016306A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4376641A (en) * 1981-12-14 1983-03-15 The Dow Chemical Company Coated capillary chromatographic column
US5840388A (en) * 1995-01-27 1998-11-24 Northeastern University Polyvinyl alcohol (PVA) based covalently bonded stable hydrophilic coating for capillary electrophoresis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4376641A (en) * 1981-12-14 1983-03-15 The Dow Chemical Company Coated capillary chromatographic column
US5840388A (en) * 1995-01-27 1998-11-24 Northeastern University Polyvinyl alcohol (PVA) based covalently bonded stable hydrophilic coating for capillary electrophoresis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BEREZOVSKI, M.: "Kinetic capillary electrophoresis and its applications", PHD THESIS, GRADUATED PROGRAM IN HIGHER EDUCATION, July 2005 (2005-07-01), TORONTO, ONTARIO, CA, pages 109 - 111 *
HORVATH, J. ET AL.: "Polymer wall coatings for capillary electrophoresis", ELECTROPHORESIS, vol. 22, 2001, pages 644 - 655 *
MOSING, R. K. ET AL.: "Isolating Aptamers using Capillary Electrophoresis - SELEX (CE- SELEX)", NUCLEIC ACID AND PEPTIDE APTAMERS: METHODS AND PROTOCOLS, vol. 535, 2009, pages 33 - 43 *
PESEK, J. J. ET AL.: "Open Tubular Approaches to Capillary Electrochromatography", JOURNAL OF CHROMATOGRAPHY LIBRARY, vol. 62, 2001, pages 241 - 270 *

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