WO2011008173A1 - Criblage amélioré de biopolymères - Google Patents

Criblage amélioré de biopolymères Download PDF

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Publication number
WO2011008173A1
WO2011008173A1 PCT/SG2010/000266 SG2010000266W WO2011008173A1 WO 2011008173 A1 WO2011008173 A1 WO 2011008173A1 SG 2010000266 W SG2010000266 W SG 2010000266W WO 2011008173 A1 WO2011008173 A1 WO 2011008173A1
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WO
WIPO (PCT)
Prior art keywords
biopolymer
cleavable linker
biopolymers
composition
amino acid
Prior art date
Application number
PCT/SG2010/000266
Other languages
English (en)
Inventor
James R. Heath
Su Seong Lee
Jaehong Lim
Junhoe Cha
Sylvia Tan
Shi Yun Yeo
Yi Li Ang
Yiran Zhang
Original Assignee
Agency For Science, Technology And Research
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
Priority claimed from PCT/SG2009/000258 external-priority patent/WO2011010964A1/fr
Application filed by Agency For Science, Technology And Research filed Critical Agency For Science, Technology And Research
Priority to US13/384,510 priority Critical patent/US20120122711A1/en
Priority to EP10800125A priority patent/EP2454269A4/fr
Priority to CN2010800411371A priority patent/CN102498123A/zh
Priority to SG2012003547A priority patent/SG177697A1/en
Publication of WO2011008173A1 publication Critical patent/WO2011008173A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • OBOC libraries are referred to as one-bead-one-compound (OBOC) libraries.
  • OBOC libraries A common use of OBOC libraries is to identify molecules from the libraries that perform some function of interest.
  • an OBOC library may be used to identify a molecule (i.e., a peptide) that binds to a particular protein by screening the library for beads that are associated with the protein ("hit" beads). The hit beads can be separated from the rest of the library, and the identity of the peptide on a particular hit bead can be determined using a peptide sequencing strategy.
  • the second biopolymer has the same number of amino acids as the first biopolymer.
  • the biopolymers to the at least one surface such that only a portion of the biopolymers attached to the surface contain a cleavable linker.
  • the cleavable linker precursor comprises at least one amino acid.
  • the ratio of the amino acid precursor to the cleavable linker precursor is greater than 1:1.
  • the composition further comprises a library of unique biopolymers, wherein each of the biopolymers is attached to a separate particle.
  • FIG. 10 shows (A) overall flow from the initial anchor hexamer-2 to the three N- terminus elongated decameric peptides; and (B) dot blot experiments of the three decameric peptides in comparison with the hexamer-2 and commercially available polyclonal antibody for CAII, according to an embodiment;
  • embodiments allow a portion of a biopolymer species to be cleaved.
  • a cleavable linker may affect the binding strength of a biopolymer species for a target species. It may thus be desirable to have at least some of the biopolymer species be cleavable such that a sample of the biopolymer species may be collected and have the remaining biopolymer species be essentially free of the cleavable linker so as not to affect
  • the surface may be functionalized with a reactive group to which a monomer or biopolymer may be coupled.
  • the reactive group may be directly attached to the surface.
  • the reactive group may be indirectly attached to the surface using a linker (e.g., PEG).
  • linker e.g., PEG
  • Non-limiting examples of reactive groups include carboxyls, alcohols, amines, and thiols.
  • a portion 160 of the second biopolymer species may be cleaved from the particle, e.g., via cleavable linker 130, as described in more detail below, to form a mixture that can be subsequently analyzed by mass spectrometry (or other techniques) to determine the sequence of variable sequence 140.
  • the first biopolymer species and the second biopolymer species may have the same number of subunits. In some cases, the second biopolymer species may have more subunits than the first biopolymer species. In some instances, the second biopolymer species may have fewer subunits that the first biopolymer species. For example, in some embodiments, the second biopolymer species may have at least one more subunit, in certain embodiments at least two more subunits, in certain embodiments at least three more subunits, and in certain embodiments at least four more subunits. In some cases, the first biopolymer species and the second polymer species may be identical except at one or more locations where the second biopolymer species is modified.
  • the second biopolymer species may contain a cleavable linker, whereas the first biopolymer species may not contain a cleavable linker.
  • the first biopolymer species and a second biopolymer species may have identical sequences except that the cleavable linker may be inserted between two subunits of the second biopolymer species, thereby increasing the length of the second biopolymer species by one subunit relative to the first biopolymer species.
  • the first biopolymer species and the second biopolymer species may have identical sequences and identical lengths except at one or more locations where a subunit of the second biopolymer species is replaced with a cleavable linker.
  • a mixture of biopolymer species may not be used, and instead only a single biopolymer species may be used. In some embodiments, all of the single biopolymer species may be cleavable.
  • the binding region and the cleavable linker may be separated by at least one biopolymer subunit, in certain embodiments by at least two biopolymer subunits, in certain embodiments by at least three biopolymer subunits, in certain embodiments by at least four biopolymer subunits, in certain embodiments by at least five biopolymer subunits, in certain embodiments by at least six biopolymer subunits, in certain embodiments by at least seven biopolymer subunits, in certain embodiments by at least eight biopolymer subunits, in certain embodiments by at least nine biopolymer subunits, and in certain embodiments by at least ten biopolymer subunits.
  • a library may comprise at least 100 unique biopolymers, in certain embodiments at least 500 unique biopolymers, in certain embodiments at least 1000 unique biopolymers, in certain embodiments at least 5000 unique biopolymers, and in certain embodiments at least 10000 unique biopolymers.
  • each member of the library of biopolymers may comprise a fixed sequence region (e.g., an anchor sequence) and a variable sequence region.
  • the anchor sequence may comprise a sequence having at least some binding affinity for a target species.
  • cleavable linker precursor it may be necessary to use a larger amount of cleavable linker precursor to achieve essentially the same result, hi some embodiments, less than 10 equivalents of the cleavable linker precursor are mixed with the biopolymer chains, in certain embodiments less than 5 equivalents of the cleavable linker precursor are mixed with the biopolymer chains, in certain embodiments less than 1 equivalent of the cleavable linker precursor is mixed with the biopolymer chains, in certain embodiments less than 0.5 equivalents of the cleavable linker precursor are mixed with the biopolymer chains, and in certain embodiments less than 0.1 equivalents of the cleavable linker precursor are mixed with the biopolymer chains.
  • additional monomers may be added to the biopolymer chains.
  • the first method (21 ) represents a modification of the coupling step in the standard peptide coupling for constructing OBOC peptide libraries.
  • the beads (11) that are used for OBOC libraries are typically pre-equipped with molecular functionalities for further chemical modifications.
  • a standard example would be a polyethylene glycol oligomer that is terminated with an amine (-NH 2 ) chemical group, as shown in FIG. 2.
  • amide coupling chemistry which is used to couple amino acids serially to form peptides, can be employed on-bead.
  • amino acids are coupled onto the bead, they are typically protected from subsequent reactions through the use of finoc (fluoren-9-ylmethoxycarbonyl) group.
  • the second method (22) employs an activated ester form of fmoc-methionine, which undergoes amide formation in the presence of N,N'-diisopropylethylamine (DIPEA).
  • DIPEA N,N'-diisopropylethylamine
  • the incorporation of fmoc-protected methionine by using the activated ester form is controlled so that only a fraction of the exposed NH 2 groups are reacted.
  • This is similar to method (21), although the slowly reacting activated ester facilitates more control over the extent of partial methionine coupling to the bead-bound NH2 groups.
  • the resulting beads undergo fmoc deprotection and the subsequent coupling of an fmoc-protected amino acid (fmoc-AA-OH).
  • FIG. 3 illustrates two other methods for attaching amino acid sequences on beads by using two pre-mixed amino acid reagents.
  • the third method (31) involves the use of dimeric peptides having methionine appended to a fmoc-protected second amino acid. For example, a fraction of finoc-Leu-Met-OH (leucine-methionine) is pre-mixed with finoc- Leu-OH and used for coupling to amino resin if the amino acid at C-terminus is leucine.
  • Benzotriazole-1- yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N,N- diisopropylethylamine (DIEA) were purchased from GL Biochem (Shanghai) Ltd.
  • the libraries were incubated against 50 nM of bovine carbonic anhydrase (bCAII), conjugated with Alexa Fluor® 647 for fluorescence detection, at 25 °C for 20 h.
  • Hit beads were sorted into a 96 well plate in an automatic fashion by COPAS Plus and the appended peptides were released by treatment with CNBr, which were delivered to MALDI-MS station for characterization.
  • the position-dependent histograms in FIG. 7 illustrate clear difference between the two libraries, which implies the significant interference can be attributed to the linker portion, thereby to perturb the screening results.
  • FIG. 8 Another comparison was performed with two tetrameric peptide libraries, in which a cleavable linker was placed in the middle of the anchor peptide LHRYWF (SEQ ID NO. 7) (FIG. 8).
  • the libraries were synthesized in a similar way with 15% and 100% methionine incorporated between H and R, respectively.
  • the libraries were incubated against 10 nM of bovine carbonic anhydrase (bCAII), conjugated with Alexa Fluor 647 for fluorescence detection, at 25 °C for 20 h.
  • the sequencing results were depicted as the position-dependent histograms in FIG. 8, which illustrate distinct outcomes between the two libraries. These results support the interference of the linker portion to the screening process.
  • the Alexa Fluor® 647 labeled bCAII (bCAII-A647) was purified from the mixture by size exclusion purification resin in the kit.
  • the purified bCAII-A647 was characterized by NanoDrop (Thermo Scientific) and gel documentation (Typhoon) after SDS-PAGE. 200 mg of dried library resin was transferred into an 8 ml Alltech vessel and pre- incubated in blocking solution, 0.05% NaN 3 , 0.1% Tween 20 and 0.1% BSA in PBS buffer (pH 7.4) for 1 hr on 360-degree shaker at ambient temperature.
  • the buffer solution was drained and then 5 ml of 10 or 50 nM bCAII-A647 diluted in blocking solution was added to the swelled resin. The resulting mixture was incubated for 20 h at 360-degree rotating thermostat shaker. The liquid was drained and non-specifically bound proteins were eliminated by washing 3 times with blocking solution, 7 times with 0.1% Tween 20 in PBS, sequentially. After stringent washing, 200 mg of the assayed library resin was transferred into sample vessel of COPAS Plus (Union Biometrica) and diluted with 200 ml of 0.1% Tween 20 in PBS buffer.
  • COPAS Plus Union Biometrica
  • the final three decameric peptides were reconstructed with biotin labeled at C-terminus for validation by dot blot to exhibit significantly increased affinity compared to the precursor hexamer-2 (ifvykr) (SEQ ID NO. 9). It is noteworthy that their affinities appeared comparable to those of elongated hits from N-terminus to display the developed spots clearly up to 20 ng. Among the 3 elongated hits, the decamer-4 (ifvykr- wryp) (SEQ ID NO. 11) appeared most prominent for further investigation.
  • Biotin-NHS (1.5 equiv) and DIEA (5 equiv) were treated to beads in NMP (1.5 ml) with vortexing for 15 min, the resulting beads were thoroughly washed with NMP (1.5 ml ' x 3). The resulting beads were thoroughly washed by NMP (3 ml x 4). Next, 20 % piperidine in NMP (5 ml, v/v) was added and the RV was vortexed for 5 min. The liquid was drained and a fresh solution of 20 % piperidine in NMP (3 ml, v/v) was added and the RV was vortexed for another 15 min.
  • the resulting beads were thoroughly washed by NMP (3 ml x 4) and DCM (3 ml x 4).
  • the necessary amino acids or PEG group was sequentially introduced as described above using 18 non-natural Fmoc-protected amino acids, excluding cysteine and methionine, and Fmoc-PEG-COOH.
  • acetyl group was introduced at N-terminus by treatment of the beads with acetic anhydride (1 ml, 0.3 M solution in NMP) in the presence of DIEA (1 ml, 0.5 M solution in NMP) for 15 min.
  • the beads were transferred to an 8 ml reactor equipped with a filter, and incubated in trifluoroacetic acid (TFA)/water/TIS (2 ml, 94/3/3, /v/v/v) at room temperature for 2 h.
  • TFA trifluoroacetic acid
  • TIS trifluoroacetic acid
  • TIS water/TIS
  • the cleavage solution was collected and concentrated in a nitrogen stream.
  • the final purification was carried out by using a preparative HPLC to produce the desired peptide with carboxamide group at C-terminus (typical quantity 1-5 mg, purity >95 %) in a white solid.
  • Alexa Fluor 647-labeled bCAII was purified from the mixture using the size exclusion purification resin in the labeling kit.
  • a solution of bCAII was prepared as 10 mg/mL stocks in PBS buffer (pH 7.4). A serial dilution of the mother solution was applied to a nitrocellulose membrane, typically ranging from 2 ⁇ g to 5 ng per spot. The membrane was blocked at room temperature for 2 h in 5 % nonfat milk/TBS-T. The membrane was then washed with TBS-T.

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Abstract

L’invention concerne des compositions et des procédés ayant trait à l’échantillonnage de biopolymères, et, en particulier, à l’échantillonnage fractionnel de biopolymères. Dans un aspect, les formes de réalisation concernent généralement des espèces biopolymères uniques, une fraction de chaque espèce biopolymère contenant un lieur clivable. Dans certaines formes de réalisation, l’espèce biopolymère peut être fixée sur une surface. Par exemple, l’espèce biopolymère peut être fixée sur des billes. Dans certaines formes de réalisation, une partie d’une espèce biopolymère unique peut être échantillonnée par le clivage du lieur clivable. Dans certains cas, l’échantillon peut être analysé afin de déterminer la séquence du biopolymère.
PCT/SG2010/000266 2009-07-15 2010-07-15 Criblage amélioré de biopolymères WO2011008173A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US13/384,510 US20120122711A1 (en) 2009-07-15 2010-07-15 Screening of biopolymers
EP10800125A EP2454269A4 (fr) 2009-07-15 2010-07-15 Criblage amélioré de biopolymères
CN2010800411371A CN102498123A (zh) 2009-07-15 2010-07-15 改进的生物聚合物筛选
SG2012003547A SG177697A1 (en) 2009-07-15 2010-07-15 Improved screening of biopolymers

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US22588109P 2009-07-15 2009-07-15
US61/225,881 2009-07-15
PCT/SG2009/000258 WO2011010964A1 (fr) 2009-07-22 2009-07-22 Différentiation d’aminoacides isobariques et autres espèces
SGPCT/SG2009/000258 2009-07-22

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WO2011008173A1 true WO2011008173A1 (fr) 2011-01-20

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PCT/SG2010/000266 WO2011008173A1 (fr) 2009-07-15 2010-07-15 Criblage amélioré de biopolymères

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EP (1) EP2454269A4 (fr)
KR (1) KR20120093151A (fr)
CN (1) CN102498123A (fr)
WO (1) WO2011008173A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9863956B2 (en) 2009-07-22 2018-01-09 Agency For Science, Technology And Research Differentiation of isobaric amino acids and other species

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Publication number Priority date Publication date Assignee Title
CN111748089B (zh) * 2019-03-28 2023-07-18 成都先导药物开发股份有限公司 一种生物素标记化合物以及确定化合物结合靶标蛋白的方法

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US20020197653A1 (en) * 2001-03-05 2002-12-26 Matthew Shair System for detecting reporter gene expression
WO2005116656A1 (fr) * 2004-05-25 2005-12-08 2Curex Identification de composes modifiant une reponse cellulaire
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9863956B2 (en) 2009-07-22 2018-01-09 Agency For Science, Technology And Research Differentiation of isobaric amino acids and other species

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KR20120093151A (ko) 2012-08-22
EP2454269A1 (fr) 2012-05-23
CN102498123A (zh) 2012-06-13

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