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WO2025176862A1 - Procédé et dispositif pour déterminer des sites de liaison de protéines à des acides nucléiques, plus particulièrement de facteurs de transcription à l'adn - Google Patents

Procédé et dispositif pour déterminer des sites de liaison de protéines à des acides nucléiques, plus particulièrement de facteurs de transcription à l'adn

Info

Publication number
WO2025176862A1
WO2025176862A1 PCT/EP2025/054757 EP2025054757W WO2025176862A1 WO 2025176862 A1 WO2025176862 A1 WO 2025176862A1 EP 2025054757 W EP2025054757 W EP 2025054757W WO 2025176862 A1 WO2025176862 A1 WO 2025176862A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acids
proteins
dna
bonds
binding
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
PCT/EP2025/054757
Other languages
German (de)
English (en)
Inventor
Nina VIESSMANN
Christoph Russmann
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.)
Hochschule fuer Angewandte Wissenschaft und Kunst Hildesheim Holzminden Gottingen
Original Assignee
Hochschule fuer Angewandte Wissenschaft und Kunst Hildesheim Holzminden Gottingen
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 Hochschule fuer Angewandte Wissenschaft und Kunst Hildesheim Holzminden Gottingen filed Critical Hochschule fuer Angewandte Wissenschaft und Kunst Hildesheim Holzminden Gottingen
Publication of WO2025176862A1 publication Critical patent/WO2025176862A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • bonds of the proteins to the nucleic acids are fixed by short laser pulses with a pulse duration of not more than 100 ns, and the nucleic acids with the fixed bonds are sequenced by passing them through nanopores.
  • the nucleic acids to which the method according to the invention relates are, in particular, DNA, with the proteins in question being, in particular, transcription factors.
  • the DNA to which the proteins bind is usually double-stranded DNA.
  • the nucleic acids can be single-stranded DNA or RNA, although appropriate adaptation of sample preparation prior to the application of nanopore technology may be necessary.
  • the double-stranded DNA is split into two single strands, one of which is then randomly selected and sequenced. It is generally advantageous to sequence both single strands of the double-stranded DNA. If only one of the two strands is sequenced, information about fixed bonds may be lost. Using so-called duplex basecalling, both strands can be sequenced directly one after the other, providing complementary information.
  • the initial binding of the proteins to the nucleic acids can already occur before the method according to the invention is carried out.
  • the nucleic acids can also be contacted with one or more proteins only at the beginning of the method according to the invention in order to bind these proteins to the nucleic acids.
  • Fig. 2 is a flowchart of analysis steps following the steps according to Fig. 1 of an embodiment of the method according to the invention.
  • Fig. 3A and B show quality scores of analyzed DNA libraries as they occur in the method according to the invention, Fig. 3B, right, and in comparison experiments, Fig. 3A and Fig. 3B, left.
  • Fig. 5A and B show sequence information of a proof-of-concept study of the method according to the invention.
  • Fig. 6A, B, C and D show preliminary sequencing results of cross-linked interactions of TBP with double-stranded DNA and corresponding controls in the proof-of-concept study.
  • the method 1 according to the invention serves to determine binding sites of transcription factors 3 to DNA 4.
  • the method 1 according to the invention can also be carried out to determine the binding sites 3 of other proteins 5 to other nucleic acids 6.
  • a binding reactor 7 of a device 22 for carrying out the method 1 the respective protein 5 is contacted with the nucleic acid 6 in order to enable a binding reaction 8.
  • hydrogen bonds typically form between the protein 5 and the nucleic acid 6, which are not sufficiently stable for the determination of the binding sites 2. Therefore, the bonds are fixed in a fixing device 9.
  • short laser pulses 11 with a pulse duration of less than 1 ps and a wavelength in the UV range are directed onto the nucleic acids 6 with the bound proteins 5 using a laser 10.
  • the laser pulses 11 have a pulse duration in the femtosecond range, so that the laser 10 is also referred to as a femtosecond laser.
  • the laser pulses 11 cause cross-links 12 between the proteins 5 and the nucleic acids 6.
  • the cross-links are based on the conversion of hydrogen bonds into covalent bonds and have a bond length of zero, meaning they bind the respective protein 5 directly to the respective nucleic acid 6.
  • Quality control 27 ensures that only high-confidence reads 26, which are typically filtered based on Phred quality scores and read lengths, are retained for subsequent squiggle analysis 28.
  • Squiggle analysis 28 for example, uses Remora, another open-source program from Oxford Nanopore Technologies, to identify variations that could correspond to altered bases or cross-linking events.
  • Basecalling training 29 involves iterative refinement of the basecalling model used in basecalling 25, incorporating known modifications or experimental artifacts to improve the detection of DNA changes caused by cross-linking.
  • the identified cross-linked DNA bases are then used in downstream analyses not separately shown here to infer specific peptide residues bound to these sites.
  • the results of analysis 20 are the binding site of interest, 2 of protein 5 to nucleic acid 6, and the peptide residue 21 bound there.
  • the analysis 20 of the squiggle signal 18 must be modified compared to these commercial products so that the binding sites 2 of interest of the bound amino acids 21 are determined.
  • the analysis 20 or the basecalling model used can be trained using known combinations of binding sites and bound amino acids, as well as potential other DNA changes resulting from the laser pulses 11.
  • the laser pulses 11 can cause damage to the nucleic acids 5 in the fixation device 9, such as strand breaks or the formation of pyrimidine dimers. However, this does not impede the sequencing 16 in the nanopore sequencing device 17. Radiation-induced molecular modifications of the DNA, such as pyrimidine dimers and hydrates, can be detected by base calling after the signal has been learned and assigned to the original bases.
  • Fig. 3 shows quality scores of analyzed DNA libraries output by a commercial nanopore technology product.
  • Fig. 3A shows the quality score for 200 bp double-stranded DNA fragments without UV irradiation (“(-)UVR”) as a comparison value on the left, and the corresponding quality score after UV irradiation (“(+)UVR”) on the right.
  • Fig. 2B shows the quality scores of 200 bp double-stranded DNA fragments and transcription factors without cross-linking (“(-)DPC”) on the left, and with cross-linking (“(+)DPC”) on the right.
  • the protein hydrolysis and purification steps of DNA fragments following UV irradiation and cross-linking, respectively, were performed in parallel for both sample types.
  • the quality score of Fig. 3B, right, with an average of 9.8, is not significantly worse than the comparison value of Fig. 2A, left, with an average of 11.1.
  • Fig. 4 shows quality control metrics for Nanopore sequencing data from additional analyzed DNA libraries consisting of two biological replicates, Buffer A and Buffer B, as well as three technical replicates each.
  • Fig. 4A shows the distribution of read lengths of the sequenced DNA libraries from control samples (dsDNA only) and two independently cross-linked samples (dsDNA+TBP+FLIX in Buffer A and dsDNA+TBP+FLIX in Buffer B).
  • the raw sequencing data were converted to sequence information using a canonical basecalling model, and only reads with a quality score (q-score) of 10 were included. or higher were selected for analysis.
  • Figure 4B shows the Phred quality score distribution of the analyzed DNA libraries. The table below provides a statistical summary of the number of reads generated by Phred quality assessments and the average quality scores.
  • Control 9,301 Control 404,249 — Control 839,238
  • BufferA 12.4 BufferA 12.7 — BufferA 12.8
  • Fig. 5 shows sequence information for a proof-of-concept study of the method according to the invention, in which the binding behavior of the TATA-box binding protein (TBP) to corresponding double-stranded target sequences was investigated.
  • Fig. 5A shows i) the consensus sequence of the TATA-box motif (Seq_1) in reverse complementary orientation. Listed below are ii) the reference information for mapped cross-linking points (five-pointed empty stars) of TBP on short, double-stranded oligonucleotides (Seq_2) using MS (Reim et al.
  • Fig. 5B shows the 200 bp dsDNA sequence (Seq_4) used for in vitro binding studies in combination with TBP. Two reference regions, Region #1 and Region #2, are indicated, which were used to analyze the squiggle signals outside the TATA box.
  • Fig. 6 shows preliminary sequencing results of cross-linked interactions of TBP with double-stranded DNA and corresponding controls in the proof-of-concept study. The sequencing data were generated and analyzed using methods and equipment described in the previous figures.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Afin de déterminer les sites de liaison (2) de protéines (5) à des acides nucléiques (6), les liaisons de protéines (5) à des acides nucléiques (6) sont fixées à l'aide d'impulsions laser courtes (11) d'une durée d'impulsion ne dépassant pas 100 ns. Afin de déterminer les sites de liaison (2) de protéines (5) à des acides nucléiques (6), les liaisons de protéines (5) à des acides nucléiques (6) sont fixées à l'aide d'impulsions laser courtes (11) d'une durée d'impulsion ne dépassant pas 100 ns.
PCT/EP2025/054757 2024-02-22 2025-02-21 Procédé et dispositif pour déterminer des sites de liaison de protéines à des acides nucléiques, plus particulièrement de facteurs de transcription à l'adn Pending WO2025176862A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102024104997.9 2024-02-22
DE102024104997 2024-02-22

Publications (1)

Publication Number Publication Date
WO2025176862A1 true WO2025176862A1 (fr) 2025-08-28

Family

ID=94824324

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/054757 Pending WO2025176862A1 (fr) 2024-02-22 2025-02-21 Procédé et dispositif pour déterminer des sites de liaison de protéines à des acides nucléiques, plus particulièrement de facteurs de transcription à l'adn

Country Status (1)

Country Link
WO (1) WO2025176862A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014052433A2 (fr) * 2012-09-26 2014-04-03 Nabsys, Inc. Procédé et système d'analyse d'une protéine et d'autres modifications sur l'adn et l'arn
WO2015138405A2 (fr) * 2014-03-10 2015-09-17 The Board Of Trustees Of The University Of Illinois Détection et quantification de la méthylation de l'adn
WO2017104398A1 (fr) * 2015-12-17 2017-06-22 株式会社日立ハイテクノロジーズ Appareil de mesure de biomolécules
EP3404113A1 (fr) * 2017-05-19 2018-11-21 Universidad del Pais Vasco Procédé de détection d'interaction protéine-adn
DE102010020194B4 (de) 2010-05-07 2022-09-08 Carl Zeiss Meditec Ag Vorrichtung zur Stabilisierung der Augenhornhaut

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102010020194B4 (de) 2010-05-07 2022-09-08 Carl Zeiss Meditec Ag Vorrichtung zur Stabilisierung der Augenhornhaut
WO2014052433A2 (fr) * 2012-09-26 2014-04-03 Nabsys, Inc. Procédé et système d'analyse d'une protéine et d'autres modifications sur l'adn et l'arn
WO2015138405A2 (fr) * 2014-03-10 2015-09-17 The Board Of Trustees Of The University Of Illinois Détection et quantification de la méthylation de l'adn
WO2017104398A1 (fr) * 2015-12-17 2017-06-22 株式会社日立ハイテクノロジーズ Appareil de mesure de biomolécules
EP3404113A1 (fr) * 2017-05-19 2018-11-21 Universidad del Pais Vasco Procédé de détection d'interaction protéine-adn

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"Methods in Enzymology", vol. 582, 1 January 2017, ELSEVIER, ACADEMIC PRESS, NL, ISBN: 978-0-12-805382-9, article A.H. SQUIRES ET AL: "Single-Molecule Characterization of DNA-Protein Interactions Using Nanopore Biosensors", pages: 353 - 385, XP055394114, DOI: 10.1016/bs.mie.2016.08.010 *
GEORGIEVA, D. ET AL.: "Detection of base analogs incorporated during DNA replication by nanopore sequencing", NUCLEIC ACIDS RESEARCH, vol. 48, 4 September 2020 (2020-09-04), pages e88, XP055861275, DOI: 10.1093/nar/gkaa517
JIWOOK SHIM ET AL: "Detection and Quantification of Methylation in DNA using Solid-State Nanopores", SCIENTIFIC REPORTS, vol. 3, 11 March 2013 (2013-03-11), XP055101649, DOI: 10.1038/srep01389 *
MOSS T ET AL: "UV-Laser Crosslinking of Proteins to DNA", METHODS, ACADEMIC PRESS, NL, vol. 11, no. 2, 1 February 1997 (1997-02-01), pages 225 - 234, XP004466478, ISSN: 1046-2023, DOI: 10.1006/METH.1996.0409 *
NAISH MATTHEW ET AL: "Supplementary materials The genetic and epigenetic landscape of the Arabidopsis centromeres", SCIENCE - AUTHOR MANUSCRIPT, vol. 374, no. 6569, 12 November 2021 (2021-11-12), US, XP093277105, ISSN: 0036-8075, Retrieved from the Internet <URL:https://pmc.ncbi.nlm.nih.gov/articles/instance/10164409/bin/NIHMS1895551-supplement-supplementary_materials_and_methods.pdf> DOI: 10.1126/science.abi7489 *
NEBBIOSO, A. ET AL.: "Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses", SCI REP., vol. 7, no. 1, 15 September 2017 (2017-09-15), pages 11725
ORLANDO V.: "Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation.", TRENDS BIOCHEM SCI., vol. 25, no. 3, 2000, pages 99 - 104, XP004202537, DOI: 10.1016/S0968-0004(99)01535-2
REIM, A. ET AL.: "Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry", NATURE COMMUNICATIONS, vol. 11, 2020, pages 3019
RUSSMANN, C. ET AL.: "Crosslinking of progesterone receptor to DNA using tuneable nanosecond, picosecond and femtosecond UV laser pulses", NUCLEIC ACIDS RES., vol. 25, no. 12, 15 June 1997 (1997-06-15), pages 2478 - 84
RUSSMANN, C. ET AL.: "Two wavelength femtosecond laser induced DNA-protein crosslinking", NUCLEIC ACID RESEARCH, 1998
VALENZUELA-GÖMEZ, F. ET AL.: "Nanopore sensing reveals a preferential pathway for the cotranslocational unfolding of a conjugative relaxase-DNA complex", NUCLEIC ACIDS RESEARCH, vol. 51, no. 13, 2023, pages 6357 - 6869

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