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WO2019081680A1 - Immobilisation d'acides nucléiques à l'aide d'un mimétique d'étiquette histidine enzymatique pour des applications de diagnostic - Google Patents

Immobilisation d'acides nucléiques à l'aide d'un mimétique d'étiquette histidine enzymatique pour des applications de diagnostic

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Publication number
WO2019081680A1
WO2019081680A1 PCT/EP2018/079350 EP2018079350W WO2019081680A1 WO 2019081680 A1 WO2019081680 A1 WO 2019081680A1 EP 2018079350 W EP2018079350 W EP 2018079350W WO 2019081680 A1 WO2019081680 A1 WO 2019081680A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
imidazole
probe
solid support
enzymatic
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PCT/EP2018/079350
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English (en)
Inventor
Marcel HOLLENSTEIN
Pascal RÖTHLISBERGER
Fabienne LEVI-ACOBAS
Original Assignee
Institut Pasteur
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Publication date
Application filed by Institut Pasteur filed Critical Institut Pasteur
Publication of WO2019081680A1 publication Critical patent/WO2019081680A1/fr

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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the invention relates to an in vitro method of diagnosis or assessment of a disease or therapeutic response, comprising the immobilization of nucleic acid on a solid surface by using an enzymatic imidazole nucleotide tag.
  • the invention relates also to a kit for performing the method, comprising nucleic acid immobilized on a solid surface via an enzymatic imidazole nucleotide tag.
  • Prominent examples include:
  • imidazole modified nucleoside triphosphates are excellent substrates for polymerases such as TdT leading to efficient incorporation of imidazole modified nucleotides at the 3 '-end of nucleic acid molecules.
  • This polymerization reaction was exploited to develop an enzymatic his-tag mimic for nucleic acid for their facile immobilization on solid supports ( Figure 1).
  • the inventors have shown that nucleic acid equipped with an imidazole nucleotide tag could efficiently be immobilized on solid support charged with metal ions including Ni-NTA agarose and even better on Eu-NTA agarose resins, at least for DNA.
  • the invention relates to an in vitro method of diagnosis or assessment of a disease or therapeutic response in a biological sample from a subject, comprising the immobilization of nucleic acid on a solid surface charged with metal ions by using an enzymatic imidazole nucleotide tag added to the 3 '-end of the nucleic acid.
  • the invention relates also to a kit for performing the method, comprising nucleic acid immobilized on a solid surface charged with metal ions via an enzymatic imidazole nucleotide tag added to the 3 '-end of the nucleic acid.
  • the method of the invention can be used for the diagnosis, prognosis, prediction of susceptibility or monitoring of a disease, or for the prediction or monitoring of therapeutic response in a subject, based upon the detection of the presence or absence of a biomarker of the disease or therapeutic response in a biological sample from the subject.
  • a biomarker refers to a distinctive biological or biologically derived indicator of a pathogenic process and/or response to a therapeutic intervention.
  • a biomarker includes a nucleic acid marker, a protein marker and other molecular marker.
  • a nucleic acid biomarker or nucleic acid marker refers to a measurable
  • DNA and/or RNA characteristic that is an indicator of a pathogenic process and/or response to a therapeutic intervention.
  • DNA characteristics include but are not limited to: single nucleotide polymorphisms (SNPs), variability of short sequence repeats, haplotypes, DNA modifications (e.g. methylation), deletions or insertions of single nucleotide(s), copy number variations and cytogenetic rearrangements (translocations, duplications, deletions or inversions).
  • SNPs single nucleotide polymorphisms
  • haplotypes haplotypes
  • DNA modifications e.g. methylation
  • copy number variations cytogenetic rearrangements (translocations, duplications, deletions or inversions).
  • RNA characteristics include but are not limited to: RNA sequences, RNA expression levels, RNA processing (e.g. splicing and editing) and microRNA levels.
  • a nucleic acid biomarker includes nucleic acid from infectious pathogens (virus, bacteria, and others), for the diagnosis of infectious diseases, as well as cell-free circulating fetal mRNA and fetal DNA for non-invasive prenatal diagnosis of a disease, in particular a genetic disease.
  • mRNA biomarker may be detected and their expression levels measured by suitable methods that are well-known in the art, such as for example, direct mRNA counting technology or hybridization to a specific probe, eventually labeled with a detectable label.
  • a protein biomarker or protein marker refers to a measurable protein characteristic that is an indicator of a pathogenic process, and/or response to a therapeutic intervention.
  • Protein characteristics include but are not limited to protein state or protein expression levels.
  • Protein state includes but is not limited to variant protein having altered (e.g. upregulated or downregulated) biological activity in comparison to the non-variant or wild- type protein.
  • the biological activity can be, for example, a binding activity or enzymatic activity.
  • Protein biomarker may be detected and their expression levels measured using several different techniques, many of which are antibody -based.
  • Example of such techniques include with no limitations immunoassays (Enzyme-linked immunoassay (ELISA), radioimmunoassay, chemiluminescence- and fluorescence-immunoassay) and antibody microarray-based assays.
  • ELISA Enzyme-linked immunoassay
  • radioimmunoassay radioimmunoassay
  • chemiluminescence- and fluorescence-immunoassay chemiluminescence- and fluorescence-immunoassay
  • antibody microarray-based assays Several biomarkers may be measured simultaneously using multiplex assays.
  • a disease is any disease that can be detected or assessed by detecting the presence or absence of a biomarker in a biological sample from a subject.
  • a disease includes but is not limited to a genetic disease, an inflammatory or allergic disease, cancer, and an infectious disease such as for example, a viral, bacterial, fungal or parasitic disease.
  • a biological sample refers to any material comprising nucleic acids (DNA and/or RNA) and/or proteins that is derived from living or dead individual (human or animal).
  • the biological material may be derived from any biological source that contains nucleic acids and/or proteins, including tissue or body fluid.
  • body fluids include blood (whole-blood), serum, plasma, cerebral spinal fluid (CSF), amniotic fluid, urine and mucosal secretions.
  • Tissue sample can be from any tis sue or organs including tumors . Sample includes swab.
  • Samples include also processed samples that have been treated to disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain reagents (buffers, salts, detergents, enzymes and the like) which are used to prepare, using standard methods, a biological sample for analysis.
  • processed samples include samples that have been treated by standard methods used for the isolation of nucleic acids and/or proteins from biological samples.
  • the biological material is removed from the patient by standard methods which are well-known to a person having ordinary skill in the art.
  • the biological sample is also named “sample”, “nucleic acid sample” or "protein sample”.
  • the term subject includes human or animal individual.
  • nucleic acid includes natural and synthetic nucleic acid such as DNA, RNA and mixed sequence nucleic.
  • nucleic acid is understood to represent one or more nucleic acids.
  • the nucleic acid immobilization comprises the steps of:
  • step b) contacting the nucleic acid having an imidazole nucleotide tag added at its 3 '-end obtained in step a) with the solid surface charged with metal ions.
  • the imidazole modified nucleotide is a modified deoxy- or ribo-nucleoside triphosphate (dN*TP or N*TP, respectively) comprising an imidazole residue (Im).
  • the imidazole residue may be non-substituted or substituted with appropriate group(s) which do not alter dramatically the ability of the imidazole derivative to chelate metal ions.
  • Non- limiting examples of imidazole derivatives that can be used in the present invention include: modifications at positions 2, 4, and 5 of the imidazole moiety with groups such as nitro, amino, carboxylic acids, carboxamides, pyridine, bipyridine, thiols, imines, or hydroxamates.
  • a first type of preferred imidazole modified nucleotide comprises the substitution of the natural nucleobase (purine or pyrimidine) of the nucleotide with an imidazole residue.
  • first type of compounds include the compound ImTP (l ' -(N- imidazol-l-yl)-D-ribofuranose) on Figure 2 or Figure 8; the compound dlmTP (l '-(N- imidazol-l-yl)-D-2'-deoxyribofuranose) on Figure IE of Rothlisberger et al. (Org. Biomol.
  • a second type of preferred imidazole modified nucleotide comprises an imidazole residue linked to the nucleobase directly or via a spacer.
  • a non-limiting example of such second type of compounds includes the compound dA Hs TP (7-[lH-imidazol-5-yl-ethylamino-3-(carbamido)- propynyl]-7-deaza-2'-deoxyadenosine 5 '-triphosphate) on Figure 3).
  • the imidazole modified nucleotide comprises the substitution of the natural nucleobase (purine or pyrimidine) of the nucleotide with a substituted or non- substituted imidazole residue, as defined above (first type of imidazole modified nucleotide).
  • the tailing reaction (step (a)) is performed by incubating the nucleic acid with imidazole modified nucleotides and the polymerase under conditions suitable for polymerization of nucleotides, including modified nucleotides, by polymerases that are well- known in the art.
  • the appropriate polymerase for the tailing reaction is a polymerase capable of catalyzing a non-template directed incorporation of modified deoxy- and/or ribo-nucleotides onto the 3'-OH end of nucleic acid (DNA or RNA).
  • Non-limiting examples of such polymerases include various Terminal nucleotidyltransferases such as Terminal Deoxynucleotidyl Transferase (TdT), PolyA polymerase (PAP), Terminal uridyltransferase Cidl, DNA polymerase ⁇ (pol ⁇ or pol theta), and engineered polymerases.
  • Engineered polymerases for use in the present invention include mutants of the TdT and pol ⁇ such as for example the pol ⁇ mutants disclosed in Randrianjatovo-Gbalou et al., Nucleic Acids Research, 2018, 46, 6271-6284. Wild- type TdT is capable of accepting rNTPs as substrates and typically incorporates up to 5 ribonucleotides. It is thus plausible that mutants of the TdT polymerase will accept ImTP and related compounds and will incorporate enough modifications to allow for the immobilization on the solid support.
  • pol ⁇ has recently been shown to have a better terminal transferase activity than the TdT as well as a better substrate tolerance (Kent et al., eLife, 2016, 5, el3740). Furthermore, mutants of pol ⁇ have recently been shown to enable the incorporation of ribonucleotides on single- stranded primers (Randrianjatovo-Gbalou et al., Nucleic Acids Research, 2018, 46, 6271-6284). Consequently, mutants of both polymerases are expected to readily accept ImTP and related nucleotides as substrates.
  • the imidazole nucleotide tag comprises at least three imidazole modified nucleotides, preferably three to five or more imidazole modified nucleotides; more preferably five to nine or more imidazole modified nucleotides.
  • the imidazole nucleotide tag that is added to the 3 '-end of nucleic acid by an enzymatic reaction catalyzed by a polymerase is named enzymatic imidazole nucleotide tag.
  • the imidazole tag is added to the 3 '-end of nucleic acid by a tailing reaction which is a polymerase extension reaction performed in the absence of template. Therefore, the imidazole nucleotide tag is added to the 3 '-end of nucleic acids by a template- independent polymerase extension reaction.
  • the length of the imidazole nucleotide tag can be optimized by using appropriate co- factor for the polymerase, such as for example Co 2+ or Mn 2+ .
  • TdT is used in combination with Mn 2+ to improve the incorporation efficiency of imidazole modified nucleotides (tailing reaction).
  • imidazole modified deoxyribonucleotides are added to the 3 '-end of DNA using Terminal Deoxynucleotidyl Transferase (TdT), to obtain an imidazole modified DNA tag added to the 3 '-end of DNA.
  • TdT Terminal Deoxynucleotidyl Transferase
  • imidazole modified ribonucleotides are added to the 3 '-end of RNA using Terminal uridyltransferase Cidl, PolyA polymerase (PAP), DNA polymerase ⁇ (pol ⁇ ), or engineered polymerases as defined above, to obtain an imidazole modified RNA tag added to the 3 ' -end of RNA.
  • Terminal uridyltransferase Cidl PAP
  • DNA polymerase ⁇ polymerase ⁇
  • engineered polymerases as defined above
  • the nucleic acid that is immobilized is single-stranded (ss) nucleic acid, for example mRNA, viral RNA, synthetic ss DNA such as cDNA or oligodeoxyribonucleotide probe, synthetic RNA such as oligoribonucleotide probe, and ssDNA obtained by denaturation of double-stranded genomic or mitochondrial DNA using appropriate means that are well-known in the art, such as for example heat-denaturation.
  • the nucleic acid is preferably selected from the group consisting of mRNA, viral RNA, cDNA, oligodeoxyribonucleotide probe and oligoribonucleotide probe.
  • the nucleic acid that is immobilized is double- stranded (ds) nucleic acid, for example ds DNA.
  • step (b) contacting the nucleic acid having an imidazole nucleotide tag with the solid surface charged with metal ions allows the immobilization of the nucleic acid through interaction of imidazole residues of the tag with metal ions of the solid support.
  • the solid support is any support suitable for nucleic acid immobilization.
  • Such supports which are well-known in the art can be made of a material including but not limited to: polystyrene, silica, gold, glass, agarose and others.
  • the support can be, for example, in the form of plate, slide, well, dish, cup, strip, strand, chip, fiber, gel or particle including bead, microparticle and nanoparticle.
  • supports for use in the invention include polystyrene beads, agarose beads, silica particles, glass slides and gold particles. Gold particles enable labelling of oligonucleotides for Electron microscopy (EM) detection or direct visualization.
  • EM Electron microscopy
  • the solid support is charged with metal ions using appropriate means that are well- known in the art, for example using an appropriate metal-chelating agent coupled to the support.
  • the metal-chelating agent such as nitrilotriacetic acid (NTA)
  • NTA nitrilotriacetic acid
  • the solid support is charged with metal ions chosen from the group comprising: Ni 2+ , Eu 3+ ' Ca 2+ , Cu 2+ , Hg 2+ , Pb 2+ , Gd 3+ , La 3+ , Tb 3+ , Zn 2+ , Fe 2+ , Fe 3+ , Ru 2+ , and Sn 2+ ; more preferably Ni 2+ or Eu 3+ .
  • metal ions chosen from the group comprising: Ni 2+ , Eu 3+ ' Ca 2+ , Cu 2+ , Hg 2+ , Pb 2+ , Gd 3+ , La 3+ , Tb 3+ , Zn 2+ , Fe 2+ , Fe 3+ , Ru 2+ , and Sn 2+ ; more preferably Ni 2+ or Eu 3+ .
  • Non-limiting examples of solid supports for use in the present invention include polystyrene beads, gold nanoparticles, glass slides or agarose beads, preferably charged with Ni 2+ or Eu 3
  • the nucleic acid is a nucleic acid from the biological sample or a nucleic acid probe for the diagnosis or assessment of the disease or therapeutic response in the biological sample.
  • nucleic acid from the biological sample includes DNA, such as genomic DNA, mitochondrial DNA or DNA from an infectious agent; RNA such as mRNA or viral RNA; cDNA derived from mRNA. Nucleic acid from the biological sample includes also cell-free circulating fetal mRNA and fetal DNA from maternal plasma.
  • the nucleic acid probe is specific for a biomarker, which means that the nucleic acid probe is capable of binding specifically to the protein biomarker and/or hybridizing to the nucleic acid biomarker.
  • the nucleic acid probe for the detection of the protein biomarker is a nucleic acid ligand of proteins, such as for example, an aptamer.
  • the nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, of at least 10, 15, 30, 50, 100, 250 or 500 nucleotides in length that is sufficient to specifically hybridize under stringent conditions to appropriate R N A , D N A .
  • the nucleic acid probe is advantageously substantially complementary (e.g., at least 80 % identical) to the sequence of the nucleic biomarker.
  • the nucleic acid probe can be DNA, RNA, PNA or mixed, and may comprise modified nucleotides such as for examples LNA (Locked Nucleic Acids), modified internucleotide linkages and/or modified 5' and/or 3' termini.
  • Nucleic acid probes are usually synthesized using any of a variety of well-known enzymatic or chemical methods.
  • the probe is a ribo- or deoxyribo- nucleotide or oligonucleotide probe from 10 to to 100 nucleotides in length, preferably up to 15, 30 or 50 nucleotides in length.
  • the nucleic acid probe is advantageously labeled with a suitable label to facilitate the detection of the biomarker.
  • the label is a moiety that can be detected directly or indirectly by the production of a detectable signal such as for example, radioactive, colorimetric, fluorescent, chemiluminescent, electrochemoluminescent signal or others.
  • Directly detectable labels include radioisotopes and fluorophores. Indirectly detectable labels are detected by labelling with additional reagents that enable the detection.
  • Indirectly detectable labels include, for example, chemiluminescent agents, enzymes that produce visible or coloured reaction products, and a ligand-detectable ligand binding partner, where a ligand (hapten, antibody, antigen, biotin) may be detected by binding to a labelled ligand- specific binding partner.
  • the label is for example, a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label or an epitope label.
  • the detectable label is advantageously added to the 5 '-end of the nucleic acid probe. Such probes can be prepared using standard methods.
  • the method comprises:
  • the nucleic acid biomarker is for example indicative of the disease, the subject being diagnosed with said disease when the presence of nucleic acid biomarker in the sample is detected.
  • the method comprises the detection of several different nucleic acid biomarkers, for example simultaneously.
  • the nucleic acid biomarker immobilized on the surface of the support is detected by appropriate means, for example, using a nucleic acid probe specific for the biomarker (e.g. capable of hybridizing to the nucleic acid biomarker), preferably a labeled nucleic acid probe to facilitate the detection of the nucleic acid biomarker.
  • the method comprises:
  • the nucleic acid biomarker is for example indicative of the disease, the subject being diagnosed with said disease when the presence of nucleic acid biomarker in the sample is detected.
  • the method comprises the detection of several different nucleic acid biomarkers, for example simultaneously.
  • Such embodiments may comprise a step of isolating nucleic acid (DNA and/or RNA) from the biological sample, prior to the immobilization step.
  • Such embodiments may also comprise a step of synthesizing cDNA from the mRNA of the biological sample, prior to the immobilization step. Nucleic acid isolation and cDNA synthesis are performed using standard methods that are well known in the art.
  • the nucleic acid from the biological sample that is immobilized on the support is RNA, preferably mRNA or viral RNA.
  • mRNA is fetal mRNA, in particular cell-free circulating fetal mRNA from maternal plasma, for non-invasive prenatal diagnosis of a disease, in particular a genetic disease.
  • the method comprises: - immobilizing at least one nucleic acid probe specific for a protein and/or nucleic acid biomarker on the solid support,
  • nucleic acid biomarker(s) hybridized to the at least one nucleic acid probe and/or protein biomarker(s) bound to the at least one nucleic acid probe.
  • the method comprises the detection of several different nucleic acid and/or protein biomarkers, for example simultaneously.
  • the biomarker(s) are detected using any of the various methods available for this purpose, which are well-known in the art.
  • the detection method is fluorescence, bioluminescence, colorimetry, immunoenzymatic or others.
  • the detection may be semi-quantitative or quantitative.
  • the detection may also be real-time detection.
  • the method comprises the detection of the level of biomarker(s) in the biological sample.
  • the subject is a human individual.
  • the method of the invention is for the diagnosis or monitoring of an infectious disease, in particular a disease caused by an RNA virus, such as for example Astrovirus, Calicivirus, Picornavirus (Cocksakie virus, Hepatitis A virus, Poliovirus, Rhinovirus), Coronavirus, Flavivirus (Hepatitis C virus, Yellow Fever Virus, Dengue virus, West Nile virus, TBE virus, Chikungunya virus, Zika virus), Togavirus (Rubella virus), Hepevirus (Hepatitis E virus), Retrovirus (HTLV-1, Human Immunodeficiency Virus (HIV)), Orthomyxovirus (Influenza virus), Arenavirus (Lassa virus), Bunyavirus (Hantaan virus), Filovirus (Ebola virus, Marburg virus), Paramixovirus (Measles virus, Mumps virus, Parainfluenza virus, Respiratory Syncytial virus (RSV)), Rhabdovirus (Rabies virus), Reovirus
  • Astrovirus
  • the method of the invention is for prenatal diagnosis of a disease, in particular a genetic disease, comprising the immobilization of fetal DNA or mRNA, preferably cell-free DNA or mRNA from maternal blood or plasma (non-invasive prenatal diagnosis), or a nucleotide probe specific for the fetal DNA or mRNA, preferably a labeled probe, on the solid support.
  • a disease in particular a genetic disease, comprising the immobilization of fetal DNA or mRNA, preferably cell-free DNA or mRNA from maternal blood or plasma (non-invasive prenatal diagnosis), or a nucleotide probe specific for the fetal DNA or mRNA, preferably a labeled probe, on the solid support.
  • kits for performing the diagnosis or assessment method of the invention comprising at least: a solid support, metal ions, imidazole modified nucleotides, and an appropriate polymerase for incorporating the imidazole modified nucleotides at the 3 '-end of a nucleic acid, as defined above.
  • the kit optionally comprises a metal-chelating agent, co-factors and/or buffers for the polymerase- mediated tailing reaction.
  • the kit further comprises at least one nucleic acid probe for the diagnosis or assessment of the disease or therapeutic response in a biological sample from a subject, preferably a labeled nucleic acid probe.
  • the kit comprises a solid support charged with metal ions.
  • nucleic acid immobilized on the solid support is a nucleic acid probe for the diagnosis or assessment of the disease or therapeutic response in a biological sample from a subject as defined above, preferably a labeled nucleic acid probe.
  • kits optionally comprise reagents for nucleic acid and/or protein detection.
  • Reagents available for this purpose are well-known in the art and include nucleic acid probes for the diagnosis or assessment of the disease or therapeutic response, preferably labelled probes.
  • the kits optionally include instructions for performing at least one specific embodiment of the method of the invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques, which are within the skill of the art. Such techniques are explained fully in the literature.
  • Figure 1 Schematic illustration of the enzymatic immobilization of oligonucleotides using the TdT mediated-polymerization of dlmTP
  • Figure 2 Synthetic route to nucleotide analog ImTP
  • Figure 4 Characterization of the products from the TdT-mediated polymerization reaction of dlmTP (200 ⁇ ) on the 5 ' -FAM-labeled primer PI and with Mn 2+ as a cofactor.
  • A Gel analysis (PAGE 20%) of the binding to and elution from different solid supports.
  • P primer PI
  • R TdT reaction with primer PI and dlmTP
  • lanes 1 flow through after binding
  • lanes 2 washes after binding
  • lanes 3 elution of the modified oligonucleotides from the solid support with 250 mM imidazole
  • lanes 3 1 and 32 two consecutive elution steps with 100 mM EDTA
  • Ni-NTA* Ni NTA on agarose magnetic particles.
  • A Schematic representation of the immobilization of DNAzyme PS2.M on Eu-NTA agarose using the dim-tag.
  • A Schematic representation of the immobilization of the sulforhodamine B-aptamer complex on Eu-NTA agarose using the dim-tag.
  • Example 1 Enzymatic incorporation of an imidazole modified nucleotide tag at the 3'- end of a nucleic acid molecule
  • the modified triphosphate dlmTP was synthesized as reported previously (P. Rothlisberger et al., Org. Biomol. Chem., 2017, 15, 4449-4455) and the corresponding phosphoramidite dim was synthesized by application of literature protocols (Johannsen et al., Nat. Chem., 2010, 2, 229-234).
  • Imidazole modified ribonucleoside triphosphate ImTP is synthesized according to the synthetic route shown in Figure 2.
  • the synthesis of compounds 2 and 3 of Figure 2 has been described previously (AlMourabit et al., Tetrahedron- Asymmetry, 1996, 7, 3455-346).
  • the synthesis of dA Hs TP ( Figure 3) has been described previously (Hollenstein, M., Org. Biomol. Chem. 2013, 11, 5162-5172).
  • DNA oligonucleotides without imidazole modifications were purchased from Microsynth. DNA oligonucleotides with imidazole modifications were synthesized on an H-8 DNA synthesizer from K&A on a 0.2 ⁇ scale. Natural DNA phosphoramidites (dT, dC4bz, dG2DMF, dA6Bz) and solid support (dA6Bz-lcaa-CPG
  • oligonucleotides were purified by anion exchange HPLC (Dionex - DNAPac PA 100). Buffer solutions of 25 mM Tris- HC1 in H 2 0, pH 8.0 (buffer A) and 25 mM Tris-HCl, 1.25 M NaCl in H 2 0, pH 8.0 (buffer B) were used. The purified oligonucleotides were then desalted with SepPack C-18 cartridges. Oligonucleotide concentrations were quantitated by UV spectroscopy using a UV5Nano spectrophotometer (Mettler Toledo). The chemical integrity of oligonucleotides was assessed by UPLC-MS analysis: UPLC was performed on a BEH
  • the terminal deoxynucleotidyl transferase was purchased from New England Biolabs.
  • Ni-NTA Agarose was purchased from Macherey-Nagel and Ni-NTA Agarose magnetic particles were obtained from Yena Bioscience.
  • Metal salts (EuCl 3 , CoCl 2 , NiCl 2 ), ABTS, H 2 0 2 , hemin, and sulforhodamine B were all purchased from Sigma Aldrich.
  • Acrylamide/bisacrylamide (29: 1, 40%) was obtained from Fisher Scientific. Visualization of PAGE gels was performed by fluorescence imaging using a Storm 860 phosphorimager with the ImageQuant software (both from GE Healthcare).
  • the reactions mixtures were quenched by addition of 10 ⁇ of loading buffer (formamide (70%), ethylenediaminetetraacetic acid (EDTA; 50 mM), bromophenol (0.1%), xylene cyanol (0.1%)).
  • loading buffer formamide (70%), ethylenediaminetetraacetic acid (EDTA; 50 mM), bromophenol (0.1%), xylene cyanol (0.1%)).
  • the reaction products were then resolved by electrophoresis (PAGE 20%) containing trisborate-EDTA (TBE) lx buffer (pH 8) and urea (7 M). Visualization was performed by fluorescence imaging using a Storm 860 phosphorimager.
  • the polymerase was heat deactivated (20 min at 75 °C) after the tailing reaction and the modified oligonucleotides bound to the solid support by application of the general protocol for the fixation of modified oligonucleotides on Eu-NTA Agarose.
  • the TdT In presence of the preferred cofactor Co 2+ , the TdT incorporated one dim nucleotide at the 3 -end of the 5 ' -FAM-labeled 19-nucleotide long single- stranded DNA primer PI (F AM-T ACG ACTC ACT AT AGCCTC ; SEQ ID NO: 1). Increasing the reaction time and the concentration of the modified triphosphate led to a higher tailing reaction efficiency since the TdT was capable of incorporating up to five dim nucleotides at the 3 -end of the primer in 8 hours. Even longer reaction times and increasing the concentration of the polymerase did not yield any significant improvement of the efficiency of the tailing reaction.
  • oligonucleotide SI FAM-TAC GAC TCA CTA TAG CCT CImlm Imlmlm; SEQ ID NO: 4
  • TdT-mediated tailing reaction with SI led to the incorporation of an additional three modified nucleotides, suggesting that the tailing reaction was indeed limited to the addition of only 3-5 dim units before stalling.
  • Ni-NTA Agarose 200 ⁇ ⁇ of Ni-NTA Agarose were centrifuged and the flow-through was discarded.
  • the agarose was washed with 10 bed volumes (1 mL) of H 2 0.
  • the Ni 2+ ions were stripped off by washing the agarose with 10 bed volumes (1 mL) of EDTA 100 mM (pH 8.0). After a wash with 10 bed volumes (1 mL) of H 2 0, the agarose was incubated with 10 bed volumes of an aqueous solution of EuCl 3 (100 mM) for 10 min at room temperature. After removal of the flow-through, the resin was washed with 10 bed volumes (1 mL) of H 2 0.
  • the resin was equilibrated with 10 volumes of equilibration buffer (100 mM Tris-HCl, pH 8.0).
  • the Eu 3+ -NTA resins can also be stored in 30% EtOH and stored at 4°C.
  • the resin was incubated and constantly mixed at 37°C for 60 min with the TdT tailing reaction (40 ⁇ ) and 360 ⁇ of equilibration buffer. The resin was then washed twice with 10 bed volumes of equilibration buffer.
  • Elution of the bound oligonucleotides was done by incubation of the resin with 10 bed volumes of EDTA 100 mM (pH 8.0) for Eu-NTA agarose resins and with 10 bed volumes of an imidazole buffer (250 mM imidazole, 150 mM NaCl, 100 mM Tris-HCl, pH 8.0).
  • the eluted oligonucleotides were purified with NucleoSpin (Macherey-Nagel) clean-up kit.
  • NucleoSpin Macherey-Nagel
  • the reactions were initiated by addition of 3 ⁇ of H 2 O 2 (60 mM) and the color of the reaction mixtures was recorded by a digital camera, while the absorption intensity was monitored using a UV5Nano (Mettler Toledo) UV-Vis spectrophotometer at room temperature. The experiment was carried out in triplicate.
  • the unbound sulforhodamine B dye was then washed off with multiple additions of 500 ⁇ ⁇ of KCl (10 mM) until disappearance of the color.
  • the color of the immobilized aptamer-target complex was recorded with a digital camera.
  • the aptamer- target complex was eluted from the resin with EDTA (see general protocol C).
  • the color of the eluted dye was recorded by a digital camera, while the absorption intensity was monitored using a UV5 Nano (Mettler Toledo) UV-vis spectrophotometer at room temperature.
  • a 1 ⁇ solution of the fluorescein labelled oligonucleotide S2 (FAM-TAC GAC TCA CTA TAG CCT CImlm I m l m i ni Imlm; SEQ ID NO: 5) was prepared in buffer (100 mM Tris-HCl pH 8.2, 150 mM NaCl). 10 ⁇ (10 pmol) of this solution were incubated for 5 min. with 10 ⁇ of the Ni-NTA magnetic agarose beads that were diluted prior to use in the concentration range of 2 mM down to 4.57 nM (i.e. 16 times a 2/1 dilution of a 2 mM stock solution).
  • the resulting suspension was thoroughly stirred and then transferred into standard MST capillaries.
  • the MST measurements were performed at 25 °C with LED power of 80% and MST power of 20 % on a Monolith NT.115 blue/red Microscale Thermophoresis instrument from Nanotemper technologies. 5 independent repeats of this experiment were carried out.
  • sequences modified with more than 5 dim units were only removed from the Ni-NTA agarose during the elution step with 250 mM imidazole.
  • the binding of the modified strands was highly dependent on the presence of the metal complex since no product was retained on an underivatized beaded agarose resin or an NTA agarose resin.
  • immobilization on the resin was not observed for oligonucleotides that lacked the dim-tag, thus suggesting that interaction of the imidazole units with the metal cation was responsible and necessary for the binding event and precluded a simple ionic bonding between the phosphate units of DNA and the metal cations.
  • oligonucleotides equipped with a dim tail did not elute from Eu-NTA agarose after a standard imidazole elution (250 mM imidazole, 150 mM NaCl, 100 mM Tris-HCl, pH 8.0) and required complexation of Eu 3+ with EDTA for their complete removal from the solid support, suggesting a strong binding to the Eu 3+ -NTA complex.
  • oligonucleotides without a dim-tag did not bind on Eu-NTA agarose resin, confirming the necessity of the modification for immobilization on the solid support and excluding unspecific electrostatic interactions.
  • Eu 3+ improved binding of the modified oligonucleotide no increase in yield of the tailing reaction was observed when Eu 3+ served as a cofactor.
  • the resulting modified oligonucleotide was then bound to Eu- NTA agarose and incubated with ABTS, hemin, and KC1 at room temperature for 60 min to allow for the formation of the hemin-DNA complex (Li et al., Chem. Eur. J., 2009, 15, 1036-1042), as shown on ( Figure 6A).
  • the reaction was followed by UV-Vis absorption spectroscopy as well as visually. After 5 min of reaction, the distinctive green color was observed (Figure 6B) and the formation of the free-radical cation was confirmed by a strong increase of the absorption at 420 nm ( Figure 6C). No reaction was observed by UV-Vis spectroscopy when the same experiment was conducted with P2 lacking the dim-tag.
  • oligonucleotide P3 corresponding to the aptameric sequence was synthesized and subjected to the TdT tailing reaction in the presence of dlmTP.
  • the resulting modified oligonucleotide was then incubated first with KC1 to enable the formation of the G-quadruplex-like structure and then with a large excess of the dye to ensure that the majority of the aptamers are in a bound state (K d value of the aptamer is 660 nM; Zhang et al., ACS Appl. Mater. Interfaces, 2013, 5, 5500-5507).
  • the resulting aptamer-target complex was then immobilized on Eu-NTA agarose and after multiple wash steps eluted from the resin (Figure 7A).
  • Visual and UV/Vis spectroscopy analysis ( Figure 7B and 7C) revealed that most of the aptamer- target complex remained bound on the resin, which is not the case in a control sample lacking the dim-tag.
  • the sulforhodamine B dye can also be captured by an immobilized dim-tagged aptamer, albeit not as efficiently as when the aptamer-target complex is bound on the Eu-NTA agarose first.
  • the inventors have demonstrated that the imidazole modified triphosphate dlmTP is an excellent substrate for polymerase such as TdT polymerase in the presence of Mn 2+ as a cof actor, leading to efficient tailing reactions.
  • This polymerization reaction was exploited to develop an enzymatic his-tag mimic for oligonucleotides for their facile immobilization on solid supports. It is shown here that oligonucleotides equipped with a dim-tag could efficiently be immobilized on Ni- NTA agarose and even better on Eu-NTA agarose resins.
  • the usefulness of this method was highlighted by immobilizing two types of functional nucleic acids on a solid support without any loss in their respective activities.
  • RNA triphosphate ImTP was synthesized according to the synthetic route shown in
  • P-D-ribofuranose-l,2,3,5-tetraacetate 1 (2g, 6.0 mmol, leq) was dissolved in DCM (40 mL) at room temperature under N2. It was added to a solution of sylilimidazole (0.94 mL, 6.4 mmoles, 1.1 eq) and trimethylsilyltriflate (1.4 mL, 6.4 mmol, 1.1 eq) in DCM (80 mL) within 6 min. The reaction mixture was heated to reflux for 15h. The reaction was quenched with 100 mL of saturated NaHC03 and extracted with DCM (3 x 60 mL). The organic phase was dried over MgS04, concentrated under reduced pressure to give 1.2 g of a light brown oil (61%).
  • Nucleoside 3 (1.5 g, 4.6 mmol, leq) was dissolved in 7N ammonia in methanol (5 mL) at room temperature. The reaction mixture was stirred for 16h. It was then concentrated under reduced pressure, coevaporated with pyridine and purified by flash chromatography (DCM/MeOH 80:20) to give 700 mg of a brown powder (76%).
  • Nucleoside 4 (0.4 g, 1.9 mmol, 1 eq) was dissolved in anhydrous pyridine (10 mL) and put under N2- To this solution, 4-dimethylaminopyridine (23 mg, 0.19 mmol, 0.1 eq) was added and 4,4'-dimethoxytrityl chloride (1.24 g, 2.2 mmol, 1.2 eq) was added in 4 portions over one hour. After 12h, the reaction mixture was quenched with methanol (3 mL), the solvent was evaporated and the residue was purified by flash chromatography (DCM/MeOH 99:1 to 95:5) to give 600 mg of a yellow foam (63%).
  • nucleoside analog 6 161 mg, 0.274 mmol, 1 eq
  • TFA 1.2 mL
  • the reaction mixture was stirred for 30 mn at room temperature.
  • the solvent was removed in vacuo and the residue was purified by flash chromatography (DCM/MeOH 96:4) to yield 7 as a white solid (70 mg, 90%).
  • Nucleoside 7 (70 mg, 0.246 mmol, 1 eq) was coevaporated twice with pyridine and dried under reduced pressure overnight before the reaction. Tributylammonium pyrophosphate was dried under reduced pressure overnight before the reaction.
  • Example 4 Synthesis of dIm c TP and enzymatic incorporation of dIm c TP nucleotide tag at the 3 'end of a nucleic acid molecule
  • Nucleoside analog 8 (S. Pochet, L. Dugue, Imidazole -4-carboxamide and l,2,4-triazole-3- carboxamide deoxynucleotides as simplified DNA building blocks with ambiguous pairing capacity, Nucleosides Nucleotides, 17 (1998) 2003-2009) (220 mg, 0.38 mmol) was dissolved in dry pyridine (10 mL) at RT under N 2 .
  • the starting material 9 (230mg, 0.38 mmol) was dissolved in dry chloroform (15 mL) at RT under a N 2 atmosphere. To this solution, dichloroacetic acid (0.32 mL, 38 mmol, 10 eq.) was added and the resulting orange solution was stirred for 20 min at RT. The reaction mixture was quenched with NaHC0 3 sat. (10 mL), extracted with DCM (3 x 20 mL), dried over MgS0 4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM/MeOH 2-5%) to yield 90 mg (78%) of compound 10 as a white solid.
  • Nucleoside 10 (40 mg, 0.13 mmol) was dissolved in dry pyridine (0.2 mL) and dry dioxane (0.4 mL) at RT under N 2 .
  • 2-chloro-l,3,2-bonzodioxaphosphorin-4-one 38 mg, 0.18 mmol, 1.4 eq.
  • a solution of tributylammonium pyrophosphate 95 mg, 0.17 mmol, 1.3 eq.
  • dry DMF (0.17 mL) and tributylamine (60 ⁇ ) was added dropwise and the reaction mixture stirred for another 45 min.
  • the reaction mixture was then oxidized by the addition of iodine (56 mg, 0.21 mmol, 1.6 eq.) in pyridine (0.98 mL) and H 2 0 (0.02 mL). After 30 min of stirring, the excess of iodine was quenched with a sodium thiosulfate solution (10% w/v in water) and the resulting clear solution was concentrated under reduced pressure at 30°C. The concentrated mixture was treated with ammonium hydroxide 30% (12 mL) for 2h. The yellow suspension was again concentrated under reduced pressure at 30°C. The yellow residue was dissolved in H 2 0 (2 mL) and precipitated by the addition of NaC10 4 2% in acetone (12 mL).
  • TdT-mediated tailing reaction with dim TP was performed according to the general protocol described in example 1. The results show that dim TP is a better substrate for the TdT than dlmTP and thus improves the efficiency of nucleic acid tailing with imidazole modified nucleotides ( Figure 9B).
  • Example 5 Enzymatic Incorporation of dlmTP nucleotide tag at the 3 'end of a double-stranded DNA molecule
  • TdT-mediated tailing reaction was performed with dlmTP and a dsDNA substrate, according to the general protocol described in example 1.
  • the ds DNA was a DNA duplex consisting of the 5 '-FAM-labelled 19 nt long primer used for experiments with ssDNA and the complementary sequence 5'-labelled with a Cy5 dye ( Figure 10A).
  • TdT and dlmTP were added and the tailing reactions were left at 37°C for given time points.
  • the reactions were then analyzed by gel electrophoresis (PAGE 20%) and by scanning with a filter for fluorescein (Figure 10B) or for Cy5 ( Figure 10B) on the phosphorimager.

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Abstract

L'invention concerne un procédé in vitro de diagnostic ou d'évaluation d'une maladie ou d'une réponse thérapeutique dans un échantillon biologique provenant d'un sujet, comprenant l'immobilisation d'acide nucléique sur une surface solide chargée d'ions métalliques à l'aide d'une étiquette nucléotidique d'imidazole enzymatique ajoutée à l'extrémité 3' de l'acide nucléique. L'invention concerne également un kit pour mettre en œuvre le procédé, comprenant un acide nucléique immobilisé sur une surface solide chargée d'ions métalliques par l'intermédiaire d'une étiquette nucléotidique d'imidazole enzymatique ajoutée à l'extrémité 3' de l'acide nucléique.
PCT/EP2018/079350 2017-10-25 2018-10-25 Immobilisation d'acides nucléiques à l'aide d'un mimétique d'étiquette histidine enzymatique pour des applications de diagnostic WO2019081680A1 (fr)

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WO2024168196A1 (fr) * 2023-02-08 2024-08-15 University Of Washington Systèmes et procédés de synthèse enzymatique de polynucléotides contenant des paires de bases nucléotidiques non standard

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