WO2019081680A1

WO2019081680A1 - Immobilization of nucleic acids using an enzymatic his-tag mimic for diagnostic applications

Info

Publication number: WO2019081680A1
Application number: PCT/EP2018/079350
Authority: WO
Inventors: Marcel HOLLENSTEIN; Pascal RÖTHLISBERGER; Fabienne LEVI-ACOBAS
Original assignee: Institut Pasteur
Priority date: 2017-10-25
Filing date: 2018-10-25
Publication date: 2019-05-02

Abstract

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. A kit for performing the method, comprising a 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.

Description

IMMOBILIZATION OF NUCLEIC ACIDS USING AN ENZYMATIC

HIS-TAG MIMIC FOR DIAGNOSTIC APPLICATIONS FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

The immobilization of nucleic acids on solid supports is an important and routine activity in numerous chemical, biochemical, and biological protocols and applications, in particular for diagnostic applications (Sassolas et al., Chem. Rev., 2008, 108, 109-139; Tjong et al., Chem. Soc. Rev., 2014, 43, 1612-1626; Ham et al., Chem. Int. Ed., 2011, 50, 732-736; Singh et al., J. Mater. Chem., 2011, 21, 10602-10618).

Prominent examples include:

- the immobilization of thiol-containing oligonucleotides on gold surfaces in DNA chip technology (Heme et al., J. Am. Chem. Soc, 1997, 119, 8916-8920; Demers et al., Science, 2002, 296, 1836-1838; Jambrec et al., Angew. Chem. Int. Ed., 2015, 54, 15064- 15068),

- adsorption of oligonucleotides on graphene oxide for the development of DNA- optical biosensors (Lu, et al., Angew. Chem. Int. Ed., 2009, 48, 4785-4787),

- deposition of nucleic acids on glass slides for gene expression monitoring, sequencing purposes and microarray fabrication (Zhao et al., Nucleic Acids Res., 2001, 29, 955-959; Lockart et al., Nature, 2000, 405, 827-836; Nimse et al., Sensors, 2014, 14, 22208- 22229), and

- binding of products stemming from primer extension reactions (PEX) during the process of preparation of functional nucleic acids via SELEX (Joyce et al., Angew. Chem. Int. Ed., 2007, 46, 6420-6436; Silvermann et al., Angew. Chem. Int. Ed., 2010, 49, 7180-7201; Hottin et al., Accounts Chem. Res., 2016, 49, 418-427).

Most strategies used for the immobilization of nucleic acids proceed via: - a phys- or chemisorption on the relevant solid support (e.g. through electrostatic interactions, adsorption, hydrogen bonding, or p-p stacking; Sassolas et al., Chem. Rev., 2008, 108, 109-139; Jambrec et al., Angew. Chem. Int. Ed., 2015, 54, 15064-15068; Zheng et al., Nat. Mater., 2003, 2, 338-342),

- bioaffinity coupling (mainly by formation of biotin-avidin complexes; Pan et al., Langmuir, 2005, 21, 1022-1027; Fang et al., J. Am. Chem. Soc, 1999, 121, 2921-2922 ; Yan et al., J. Am. Chem. Soc, 2001, 123, 11335-11340), or

- covalent attachment (e.g. thiol-Au interactions or amide bond formation; Shrestha et al., Angew. Chem. Int. Ed., 2007, 46, 3855-3859; El-Sagheer et al., Chem. Soc. Rev., 2010, 39, 1388-1405; Banuls et al., Bioconjugate Chem., 2017, 28, 496-506; Palla et al., J. Am. Chem. Soc, 2017, 139, 1967-1974.).

However, the vast majority of methods require oligonucleotides functionalized by solid-phase synthesis with adequate groups that enable attachment to the solid support (Min et al, Nucleic Acids research, 1996, 24, 3806-3810) and only very few post-synthetic or enzymatic methods have been reported so far (Langer et al., Proc. Natl. Acad. Sci. U. S. A., 1981, 78, 6633-6637 ; Cook et al., Nucleic Acids Res., 1988, 16, 4077-4095; Cole et al., Nucleic Acids Res., 2004, 32, 9; Paredes et al., Methods, 2011, 54, 251-259; Riley, et al., DNA, 1986, 5, 333-337).

Therefore, a faster, universal, and selective method for the immobilization of nucleic acids such as DNA oligonucleotides that does not require prior functionalization of nucleic acid strands by solid-phase oligonucleotide synthesis is highly desirable (Ham et al., Chem. Int. Ed., 2011, 50, 732-736; Banuls et al., Bioconjugate Chem., 2017, 28, 496-506).

SUMMARY OF THE INVENTION

The inventors have shown that 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 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. This enzymatic technique for the immobilization of nucleic acids is facile and broadly applicable and does not require any pre-functionalized DNA oligonucleotides.

Therefore, 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.

DETAILED DESCRIPTION OF THE INVENTION

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.

As used herein, 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. As used herein, 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).

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.

As used herein, 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. Several biomarkers may be measured simultaneously using multiplex assays.

As used herein, 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.

As used herein, 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. Non-limiting examples of 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. In particular, 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". As used herein, the term subject includes human or animal individual.

As used herein, the term nucleic acid includes natural and synthetic nucleic acid such as DNA, RNA and mixed sequence nucleic.

As used herein, the terms "a", "an", and "the" include plural referents, unless the context clearly indicates otherwise. For example, "a marker" as used herein is understood to represent one or more markers. As such, the term "a" (or "an"), "one or more" or "at least one" can be used interchangeably herein. As used herein, the term "nucleic acid" is understood to represent one or more nucleic acids.

In some preferred embodiments of the invention, the nucleic acid immobilization comprises the steps of:

a) incorporating an imidazole nucleotide tag at the 3 '-end of the nucleic acid, in the presence of imidazole modified nucleotides and an appropriate polymerase; and

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. Non- limiting examples of such 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. Chem., 2017, 15, 4449-4455); and the compound dIm^cTP (l,2-dideoxy-l-(4- (methylcarboxy)-lH-imidazol-l-yl)-D-ribofuranose) on Figure 9A. 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^HsTP (7-[lH-imidazol-5-yl-ethylamino-3-(carbamido)- propynyl]-7-deaza-2'-deoxyadenosine 5 '-triphosphate) on Figure 3). In some preferred embodiments, 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 Θ (pol Θ or pol theta), and engineered polymerases. Engineered polymerases for use in the present invention include mutants of the TdT and pol Θ such as for example the pol Θ 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. In addition, pol Θ 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 Θ 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. In the various embodiments of the invention, 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. As mentioned above, 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 Co²⁺ or Mn²⁺.

In some preferred embodiments, TdT is used in combination with Mn²⁺ to improve the incorporation efficiency of imidazole modified nucleotides (tailing reaction).

In some preferred embodiments, 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.

In some other preferred embodiments, imidazole modified ribonucleotides are added to the 3 '-end of RNA using Terminal uridyltransferase Cidl, PolyA polymerase (PAP), DNA polymerase Θ (pol Θ), or engineered polymerases as defined above, to obtain an imidazole modified RNA tag added to the 3 ' -end of RNA.

In some preferred embodiments, 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.

In some other preferred embodiments, the nucleic acid that is immobilized is double- stranded (ds) nucleic acid, for example ds DNA. In 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.

In the various embodiments of the invention, 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. Non-limiting examples of 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.

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), is advantageously linked to the support via an appropriate spacer designed to provide optimal access to metal ions.

In some preferred embodiments, the solid support is charged with metal ions chosen from the group comprising: Ni²⁺, Eu³⁺' Ca²⁺, Cu²⁺, Hg²⁺, Pb²⁺, Gd³⁺, La³⁺, Tb³⁺, Zn²⁺, Fe²⁺, Fe³⁺, Ru²⁺, and Sn²⁺; more preferably Ni²⁺ or Eu³⁺. 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 Ni²⁺ or Eu³⁺, more preferably using NTA as metal-chelating agent.

In some advantageous embodiments, 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.

As used herein, 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. In the various embodiments of the invention, 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. In some embodiments 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.

In some embodiments, the method comprises:

- immobilizing nucleic acid from the biological sample on the solid support, and - detecting the presence or absence of at least one nucleic acid biomarker in the nucleic acid from the biological sample immobilized on the solid support.

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.

In a preferred embodiment, the method comprises:

- immobilizing nucleic acid from the biological sample on the solid support,

- contacting at least one nucleic acid probe specific for a nucleic acid biomarker with nucleic acid from the biological sample immobilized on the solid support, and

- detecting the presence or absence of at least one nucleic acid biomarker hybridized to the nucleic acid probe.

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.

In some preferred embodiments, the nucleic acid from the biological sample that is immobilized on the support is RNA, preferably mRNA or viral RNA. In some more preferred embodiments; 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.

In other embodiments, the method comprises: - immobilizing at least one nucleic acid probe specific for a protein and/or nucleic acid biomarker on the solid support,

- contacting a biological sample containing nucleic acids and/or proteins with the at least one nucleic acid probe immobilized on the solid support, and

- detecting the presence or absence of 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.

In the various embodiments of the method, the biomarker(s) are detected using any of the various methods available for this purpose, which are well-known in the art. For example, 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.

In some embodiments, the method comprises the detection of the level of biomarker(s) in the biological sample.

In some embodiments of the invention, the subject is a human individual.

In some embodiments, 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 (Rotavirus) and Hepatitis D virus. The diagnosis or monitoring method of the viral disease, comprises the immobilization of viral RNA or a nucleotide probe specific for the viral RNA, preferably a labeled probe, on the solid support.

In some embodiments, 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.

Another aspect of the present invention is a (first) kit 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. In some preferred embodiments, 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. In some preferred embodiments, the kit comprises a solid support charged with metal ions.

Another aspect of the present invention is a (second) kit for performing the diagnosis or assessment method of the invention, comprising at least nucleic acid immobilized on a solid surface charged with metal ions via an imidazole nucleotide tag added to the 3 '-end of the nucleic acid, as defined above. In some preferred embodiments, the 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.

The (first and second) 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.

The invention will now be exemplified with the following examples, which are not limitative, with reference to the attached drawings in which:

FIGURE LEGENDS

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 3: Chemical structure of dA^HsTP

Figure 4: Characterization of the products from the TdT-mediated polymerization reaction of dlmTP (200 μΜ) on the 5 ^' -FAM-labeled primer PI and with Mn²⁺ as a cofactor.

A. Gel analysis (PAGE 20%) of the products of the TdT tailing reaction as a function of time.

B SIM of the reaction products at the charge state -4.

C. ESTMS spectrum corresponding to the main n+5 product. The insert shows the deconvoluted mass spectrum (calcd: 7476; found: 7476).

Figure 5

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.

B: Validation of oligonucleotide S2 binding affinity to Ni-NTA agarose magnetic particles by microscale thermophoresis at 20% MST power with the corresponding standard deviations. Data represents the average of five independent trials. Figure 6

A: Schematic representation of the immobilization of DNAzyme PS2.M on Eu-NTA agarose using the dim-tag.

B: Photographs of the color change.

C: UV/Vis absorption spectra for analysis of the formation of the oxidation product (ABTS^"+):

1) DNAzyme (primer P2) with dim-tag on solid support (Eu-NTA agarose) prior to addition of H₂0₂;

2) DNAzyme with dim-tag on solid support after addition of H₂0₂;

3) DNAzyme without dim-tag prior to addition of H₂0₂;

4) DNAzyme without dim-tag after addition of H₂0₂. All experiments have been carried out in triplicate.

Figure 7

A: Schematic representation of the immobilization of the sulforhodamine B-aptamer complex on Eu-NTA agarose using the dim-tag.

B: Photographs of samples prior (left) and after (right) elution from the solid support.

C: UV/Vis absorption spectra of eluted sulforhodamine B on sample with (1) and without (2) the dim-tag. All experiments have been carried out in triplicate.

Figure 8: Synthesis of triphosphate ImTP.

Figure 9: TdT-mediated tailing using dIm^cTP.

A: Chemical structure of dIm^cTP

B: Gel (PAGE 20%) analysis of the TdT tailing reaction with dim TP and a 15-mer 5'-FAM labelled DNA primer with different cof actors.

Figure 10: TdT-mediated tailing of a dsDNA substrate using dlmTP

A: Schematic representation of the experiment (Fluorescein label (5'-FAM) shown in grey. Cy5 label shown in black).

B: Fluorescein signal after Gel (PAGE 20%) analysis of the TdT tailing reaction with dlmTP and the double-labelled (5'-FAM/5'-Cy) dsDNA primer.

C: Cy5 signal after Gel (PAGE 20%) analysis of the TdT tailing reaction with dlmTP and the double-labelled (5'-FAM/5'-Cy) dsDNA primer. EXAMPLES

Example 1: Enzymatic incorporation of an imidazole modified nucleotide tag at the 3'- end of a nucleic acid molecule

Materials and methods

General Procedures

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^HsTP (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 μπιοΐ scale. Natural DNA phosphoramidites (dT, dC4bz, dG2DMF, dA6Bz) and solid support (dA6Bz-lcaa-CPG

500A) were all purchased from ChemGenes. Natural DNA phosphoramidites as well as the dim phosphoramidite were prepared as 0.07 M solutions in MeCN and were coupled using 50 sec and 490 sec steps, respectively. 5-(ethylthio)-lH-tetrazole (0.25 M in MeCN) was used as coupling agent. Capping, oxidation, and detritylation were performed using standard conditions. Cleavage from the solid support and deprotection of oligonucleotides was achieved by treatment with concentrated ammonia at 55 °C for 16 h. After centrifugation, the supernatants were collected and the resulting solutions were evaporated to dryness on a speed-vac. Crude oligonucleotides were purified by anion exchange HPLC (Dionex - DNAPac PA 100). Buffer solutions of 25 mM Tris- HC1 in H₂0, pH 8.0 (buffer A) and 25 mM Tris-HCl, 1.25 M NaCl in H₂0, 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

C18 column (130 A, 1.7 μπι, 2.1 mm x 50 mm) from Waters, installed on an ACQUITY UPLC H-Class System (SQ Detector 2). A Buffer containing 20 mM TEA and 400 mM HFIP in H₂0 was used with a linear gradient from 18 to 31% Methanol within 5 minutes and a flow rate of 0.3 niL/min.

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 (EuCl₃, CoCl₂, NiCl₂), ABTS, H₂0₂, 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).

General Protocol for the TdT tailing reactions

A solution containing 40 pmol of the appropriate single- stranded DNA primer and 10 U of TdT, was added to a mixture composed of of dlmTP (200 μΜ final), lOx TdT reaction buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, pH 7.9), the adequate metal cof actor (0.25 mM Co²⁺ or 1 mM Mn²⁺, final concentrations), and H₂0 (for a final reaction volume of 10 μΕ). The reaction mixtures were then incubated at 37°C for various time periods. For gel analysis, the reactions mixtures were quenched by addition of 10 μΕ of 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. For fixation on the agarose resin or magnetic particles, 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.

Results

Numerous nucleoside triphosphates modified with imidazole units (T. Gourlain et al.,

Nucleic Acids Res., 2001, 29, 1898-1905 ; Hollenstein et al., Angew. Chem. Int. Ed., 2008, 47, 4346-4350; Perrin et al., J. Am. Chem. Soc, 2001, 123, 1556-1563), particularly dlmTP, have been shown to be tolerated by a number of DNA polymerases (P. Rothlisberger et al., Org. Biomol. Chem., 2017, 15, 4449-4455). Based on these findings, it was questioned whether dlmTP could act as a substrate for the TdT polymerase which is known to readily accept modified nucleoside triphosphates (Hollenstein et al., Bioorg. Med. Chem. Lett., 2012, 22, 4428-4430; Horakova et al., Org. Biomol. Chem., 2011, 9, 1366-1371; Cho et al., ChemBioChem, 2006, 7, 669-672; Hollenstein, Org. Biomol. Chem., 2013, 11, 5162-5172; Kobayashi et al., Chem. Commun., 2016, 52, 3762-3765; Takezawa, et al., Int. J. Mol. Sci., 2016, 17, 10; Winz et al., Nucleic Acids Res., 2015, 43, 10; Sorensen et al., ACS Nano, 2013, 7, 8098-8104).

In view of the high capacity of the imidazole nucleoside dim to form stable Ag⁺-mediated base pairs, it was reasoned that this nucleoside analog should be capable of binding to immobilized Ni²⁺ in analogy to histidine residues of protein tags and that the TdT-mediated homopolymerization of the corresponding triphosphate dlmTP would enable the construction of a His-tag mimic for DNA (Johannsen et al., Nat. Chem. , 2010, 2, 229-234; Petrovec et al., Chem. Commun. , 2012, 48, 11844-11846; Schweizer et al., J. Biol. Inorg. Chem. , 2015, 20, 895-903; Rothlisberger et al., Org. Biomol. Chem. , 2017, 15, 4449-4455; Leon et al., Angew. Chem. Int. Ed. , 2017, 56, 6098-6102).

In presence of the preferred cofactor Co²⁺, 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. In order to assess whether the polymerase stalled after incorporating five nucleotides, as observed for other related substrates (Kobayashi et al., Chem. Commun., 2016, 52, 3762-3765), oligonucleotide SI (FAM-TAC GAC TCA CTA TAG CCT CImlm Imlmlm; SEQ ID NO: 4) that comprises 5 dim nucleotides at its 3 ^' -terminus was chemically synthesized. The 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. Considering that the incorporation of only 3-5 dim nucleotides might not be sufficient for strong binding to the metal cation of the Ni-NTA complex (Knecht et al., J. Mol. Recognit., 2009, 22, 270-279), the Co²⁺ cofactor was substituted with Mn²⁺, which is known to improve the incorporation efficiency of modified nucleoside triphosphates by the TdT (Motea et al., BBA- Proteins Proteomics, 2010, 1804, 1151-1166). Indeed, under these conditions, a highly efficient tailing reaction was observed leading to the incorporation of up to 9 modified dim residues in 4 hours and to a characteristic product distribution similar to that of natural dNTPs with longer reaction times (Figure 4A). The products stemming from a 4 hour long TdT tailing reaction were also analyzed by UPLC-MS (Figures 4B). In this context, selected ion monitoring (SIM) of the reaction mixture showed the same product distribution as observed in the gel electrophoresis analysis where the n+4, n+5, and n+6 products are the dominating species (Figure 4B). Next, the chemical identity of each species was confirmed by ESI-MS (Figures 4C).

Example 2: Immobilization of the imidazole-tagged nucleic acid molecule

Material and methods

General Protocol for the fixation of modified oligonucleotides on Eu-NTA Agarose

200 μΐ_^ of Ni-NTA Agarose were centrifuged and the flow-through was discarded. The agarose was washed with 10 bed volumes (1 mL) of H₂0. The Ni²⁺ 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 H₂0, the agarose was incubated with 10 bed volumes of an aqueous solution of EuCl₃ (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 H₂0. For an immediate use, the resin was equilibrated with 10 volumes of equilibration buffer (100 mM Tris-HCl, pH 8.0). The Eu³⁺-NTA resins can also be stored in 30% EtOH and stored at 4°C. After equilibration, the resin was incubated and constantly mixed at 37°C for 60 min with the TdT tailing reaction (40 μΕ) and 360 μΕ 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. A similar protocol was applied using the Ni-NTA agarose magnetic particles (using 150 μΐ. of the slurry) instead of the resin.

Fixation of DNAzyme and catalytic oxidation ofABTS

After the TdT tailing reaction with primer P2 (TTGTGGGTAGGGCGG

GTTGGG; SEQ ID NO: 2) according to the general protocol for the TdT tailing reactions, a total of 240 pmol of the modified oligonucleotide were immobilized on the Eu-NTA agarose magnetic particles by application of the general protocol C). The slurry was then incubated at room temperature for 60 min in the presence of 20 mM KC1, 25 mM Tris-HCl (pH 8.0), 100 mM ABTS, 1 mM hemin (dissolved in DMSO), 200 mM NaCl, and 0.05% Triton X-100 in a total volume of 17 μΕ. The reactions were initiated by addition of 3 μΕ of H₂O₂ (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.

Capture of the anti- sulforhodamine B ap tamer on solid support

Method A:

After the TdT tailing reaction with primer P3 (CCGGCCAAGGGTGGGAGGGAGGGGGCCGG; SEQ ID NO: 3) according to the general protocol for the TdT tailing reactions, a total of 160 pmol of the modified oligonucleotide was evaporated to dryness on a speed- vac and incubated at 37°C for 30 min in 100 mM KC1 and 100 mM Tris-HCl (pH 8.0). A large excess of sulforhodamine B (4 mg, 7.16 μπιοΐ)— to ensure that a large proportion of the aptamers are bound to the target (K_d = 660 nM; Zhang et al., ACS Appl. Mater. Interfaces, 2013, 5, 5500- 5507)— was then added to the slurry which was allowed to stir at 37°C for another 30 min. The aptamer-dye complex was then incubated with 50 μΕ of Eu-NTA agarose resin at 37°C for 60 min. The unbound sulforhodamine B dye was then washed off with multiple additions of 500 μΕ of KC1 (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 UV5Nano (Mettler Toledo) UV- Vis spectrophotometer at room temperature. The experiment was carried out in triplicate. Method B

After the TdT tailing reaction with primer P3 according to the general protocol for the TdT tailing reactions, a total of 160 pmol of the modified oligonucleotide was immobilized on Eu-NTA agarose magnetic particles (50 μΐ.) by application of the general protocol C). The bound aptamer was then incubated in 70 μΐ_^ of incubation buffer (100 mM KCl, 100 mM Tris-HCl, pH 8.0) for 30 min at 37°C. A large excess of sulforhodamine B (12 mg, 21.5 μπιοΐ) was then added to the immobilized aptamer and incubated at 37°C for 30 min to ensure complete formation of the aptamer-dye complex. The unbound sulforhodamine B dye was then washed off with multiple additions of 500 μΐ_^ 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.

Biophysical analysis of binding

Microscale Thermophoresis

A 1 μΜ 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 μΐ (10 pmol) of this solution were incubated for 5 min. with 10 μΕ 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.

Similar experiments were performed using primer PI devoid of the dim-tag as a negative control. Results

Encouraged by these preliminary results, the possibility of immobilizing the dim-modified oligonucleotides on a Ni-NTA agarose solid support was investigated. Therefore, primer PI equipped with 4-9 dim nucleotides at its 3 -terminus was allowed to bind to Ni-NTA agarose and the fractions of the different steps (i.e. flow- through, wash, elution) were recovered and analyzed by gel electrophoresis (Figure 5A). As expected, the modified strands possessing dim-tags shorter than 5 nucleotides did not bind or at least were not well retained on the Ni-NTA agarose resin, presumably due to their low affinity for the metal complex. However, sequences modified with more than 5 dim units were only removed from the Ni-NTA agarose during the elution step with 250 mM imidazole. Moreover, 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. In addition, 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. The immobilization of oligonucleotides on Ni-NTA was significantly improved when longer dim-tags were used. In order to confirm these results, the capacity of oligonucleotide S2 modified with seven dim nucleotides at the 3 -end and a fluorescein at the 5 'end to bind to Ni-NTA magnetic agarose beads was assessed by microscale thermophoresis (MST). These experiments clearly showed that dim- modified S2 was able to increase the thermophoretic mobility of the beads, while the unlabeled primer PI, lacking the dim-tag, had no effect (Figure 5B).

In addition, a gel electrophoretic analysis revealed that dim-tagged oligonucleotides had a strong preference for Ni²⁺ over Co²⁺ since practically no modified sequences could be observed in the elution step after binding to Co-NTA agarose (Figure 5A). Surprisingly, when Ni²⁺ was stripped off from the resin and replaced with Eu³⁺, oligonucleotides possessing as little as three dim nucleotides at the 3 -end were completely retained on the solid support. What is more, 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 Eu³⁺ with EDTA for their complete removal from the solid support, suggesting a strong binding to the Eu³⁺-NTA complex. Again, 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. Lastly, while Eu³⁺ improved binding of the modified oligonucleotide, no increase in yield of the tailing reaction was observed when Eu³⁺ served as a cofactor.

Having identified conditions that enabled immobilization of oligonucleotides equipped with an enzymatic dim-tag on a solid support, it was next tried to bind functional nucleic acids on Eu-NTA agarose without impairing their specific activities. Versatile methods for the immobilization of DNAzymes and aptamers on solid supports are in high demand for the development of potent biosensors (Dellafiore et al., PLoS One, 2015, 10, 11.), tools for affinity chromatography for protein purification and analysis (Hathout et al., Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 7153-7158) and as drug delivery systems with improved therapeutic applications (Lee et al., Adv. Drug Deliv. Rev., 2010, 62, 592-605).

In this context, it was first envisioned to immobilize the peroxidase mimicking DNAzyme PS2.M on a Eu-NTA agarose resin (Travascio et al., Chem. Biol., 1998, 5, 505-517). This hemin-dependent DNAzyme is capable of oxidizing ABTS to the corresponding radical cation ABTS^"+ which has a distinctive green color and represents a key player in multiple biosensing applications. Sequence P2 that contains the catalytic motif of PS2.M was synthesized and used as a template in the TdT tailing reaction with dlmTP. 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). After addition of H₂O₂, 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.

Lastly, it was proceeded to immobilize a functional aptamer on an agarose solid support without impairing its binding capacity. To do so, the anti-sulforhodamine B aptamer (Wilson and J. W. Szostak, Chem. Biol., 1998, 5, 609-617) was chosen as a model since target binding can easily be followed visually as well as by UV/Vis absorption spectroscopy. Thus, 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.

Herein, the inventors have demonstrated that the imidazole modified triphosphate dlmTP is an excellent substrate for polymerase such as TdT polymerase in the presence of Mn²⁺ 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. Lastly, 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. The advantages of this method compared to the conventional biotinylation of oligonucleotides are i) the absence of chemical derivatization of the sequence prior to immobilization, ii) ease of removal of the bound oligonucleotide from the solid support by a simple imidazole or EDTA elution step, iii) cost affordable solid support, and iv) broad applicability to any nucleic acid such as single stranded DNA molecule. Unlike the method based on the enzymatic polymerization of biotinylated nucleoside triphosphates (Langer et al., Proc. Natl. Acad. Sci. U. S. A., 1981 , 78, 6633-6637; Riley et al., DNA, 1986, 5, 333-337), no prior enzymatic treatment for the creation of nick sites is necessary and the incorporation of multiple dim nucleotides is clearly beneficial for the immobilization efficiency of modified oligonucleotides. This method is expected to be of broad and general use, particularly for the immobilization of nucleic acids for diagnostic applications.

Example 3: Synthesis of the RNA triphosphate ImTP RNA triphosphate ImTP was synthesized according to the synthetic route shown in

Figure 8 and detailed as follows.

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%).

½ NMR (400.13 MHz, CDC13): 2.15 (s, 3H), 2.17 (s, 3H), 2.18 (s, 3H), 4.38 (dd, / = 12.4 Hz, 1H), 4.50- 4.57 (m, 2H), 5.32-5.41 (m, 3H), 6.29 (d, / = 4.40 Hz, 1H), 7.73 (d, / = 1.60 Hz, 1H), 9.77 (s, 1H).

Riboimidazoie 4 CJ¾g¾C¾, M 200.08 g/mol.

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%).

AH NMR (400.13 MHz, MeOD): 3.37 (bs, 1H), 3.72-3.83 (m, 2H), 4.06-4.07 (m, 1H), 4.08- 4.27 (m, 2H), 5.97 (d, / = 5.60 Hz, 1H), 7.03 (bs, 1H), 7.38 (bs, 1H), 7.94 (bs, 1H. ¹³C NMR (100.62 MHz, MeOD): 61.5, 70.7, 76.0, 85.6, 90.4, 117.0, 127.9, 136.1.

PMT-RiboimidmzQle 5. C_jgH^ O_fr M ~ 502.57 g/mol

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%).

½ NMR (400.13 MHz, CDC13): 2.02 (s, 6H), 3.14-3.20 (m, 2H), 4.23-4.38 (m, 3H), 5.54 (bs, 2H), 5.63 (d, / = 5.60 Hz, 1H), 6.83 (d, / = 8.80 Hz, 4H), 6.96, (bs, 1H), 7.16-7.33 (m,

9H), 7.43 (d, / = 7.60 Hz, 1H), 7.53 (bs, 1H). HRMS (ESI) for C29H3lN206⁺ m/z calcd: 503.2182; found: 503.2192.

DMT-l'J'-bisacetylated -R.i.iwimidazole 6, C^H^O*. M = 586.64 g/mol

Compound 5 (150 mg, 0.3 mmol, 1 eq) was dissolved in dry pyridine (2.5 mL) with DMAP (7 mg, 0.06 mmol, 0.2 eq) and Et3N (100 μί, 0.7 mmol, 2.3 eq). To this solution under N2, acetic anhydride (84 μί, 1.1 mmol, 3.5 eq) was added dropwise at 0°C and the reaction mixture was stirred for 12h. Saturated NaHC03 was then added to quench the reaction and the mixture was extracted with DCM (3 x 20 mL), dried over MgS04 and concentrated under reduced pressure. The product was then purified by flash chromatography (DCM/MeOH 95:5) to yield 160 mg of a clear oil (91%).

^AH NMR (400.13 MHz, CDC13): 2.11 (s, 3H), 2.12 (s, 3H), 2.16 (s, 3H), 2.17 (s, 3H), 4.35- 4.43 (m, 2H), 5.37-5.40 (m, 1H), 5.52-5.54 (m, 1H), 5.67-5.70 (m, 1H), 5.83-5.86 (m, 1H), 6.85 (d, / = 9.20 Hz, 4H), 7.16, (bs, 1H), 7.29-7.46 (m, 9H), 7.45 (d, / = 7.20 Hz, 1H), 8.63 (bs, 1H).

2' '-bisacety ated riboirolriazole 7, C^H^N^O^ M = 2S4.27 E m.oi

To a stirred solution of nucleoside analog 6 (161 mg, 0.274 mmol, 1 eq) in anhydrous DCM (3 mL) under N2 was added 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%).

Rf = 0.12 (DCM/MeOH 95:5)

1H NMR (400.13 MHz, MeOD): 1.33 (bs, 1H), 2.11 (s, 3H), 2.15 (s, 3H), 3.80-3.95 (m, 2H), 4.39-4.45 (m, 1H), 5.47-5.53 (m, 1H), 5.55-5.62 (m, 1H), 6.20 (t, / = 5.20 Hz, 1H), 7.62 (d, / = 5.20 Hz, 1H), 7.95 (d, / = 5.60 Hz, 1H), 9.21 (s, 1H).

¹³C NMR (100.62 MHz, MeOD): 18.8, 19.0, 60.3, 70.9, 75.6, 85.2, 90.1, 119.3, 120.7, 134.6, 169.8, 170.0.

HRMS (ESI) for C12H19N204⁺ m/z calcd: 285.1087; found: 285.1088. RibotiiiidiKole triphoiphitc ImTP, C HJ^N^O^P^ M ^~ 440.1.3 e/mol

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.

Compound 7 was then dissolved in dry pyridine (0.3 mL) and dried dioxane (0.6 mL) at room temperature under inert atmosphere. To this solution, 2-chloro- 1,3,2- benzodioxaphosphorin-4-one (71 mg, 0.345 mmol, 1.4 eq) were added and the reaction mixture was stirred for 45 min.

A solution of tributylammonium pyrophosphate (175 mg, 0.319 mmol, 1.3 eq), in dry DMF (0.3 mL) and tributylamine (0.1 mL) was added dropwise and the reaction mixture was stirred for another 45 min. It was then oxidized by the addition of iodine (100 mg, 0.394 mmol, 1.6 eq) in pyridine (1.6 mL) and H20 (0.4 mL). After 30 min of stirring, the excess of iodine was quenched with a sodium thiosulfate solution (10% w/v in water) and the solution was concentrated under reduced pressure at 30°C. The residue was treated with aqueous ammonia (10 mL) for 2h. The suspension was then concentrated under reduced pressure at 30°C. The residue was dissolved in H20 and precipitated by the addition of NaC104 2% in acetone. The crude product was purified by HPLC (TEAB 2M, 50% in 30min).

HRMS (ESI) for C35H34F3N407⁺ m/z calcd: 438.9709; found: 438.9706.

Example 4: Synthesis of dIm^cTP and enzymatic incorporation of dIm^cTP nucleotide tag at the 3 'end of a nucleic acid molecule

1. Synthesis of dIm^cTP

The imidazole modified nucleotide dIm^cTP according to Figure 9A was synthesized and the details are given below.

Synthesis of ethyl 5'-0-(4,4 ' -dimethoxytrityl)-3 '-0 -acetyl- 1-( 2-deoxy- -D-ribofuranosyl)-imidazole-4- carboxylate (9):

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 N₂. To this solution, 2-(dimethylamino)pyridine (15 mg, 0.11 mmol, 0.3 eq.) was added followed by triethylamine (134 μΐ, 0.95 mmol, 2.5 eq.) and subsequently acetic anhydride (55μ1, 0.6 mmol, 1.5eq.). After 4h of stirring, the reaction mixture was quenched with NaHC0₃ sat. (20 mL) and extracted with DCM (3 x 30 mL). The organic layers were combined dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography (DCM/MeOH 2% with Et₃N 1%) to yield 230 mg of 9 as a yellowish oil (quant.).

R_f (DCM/MeOH 5%) = 0.7

HR-MS C₃4H3₇N₂0₈ ⁺ calculated: 601.2544; found: 601.2538. H NMR (400 MHz, CDC1₃) δ = 8.64 (dt, / = 4.3, 1.7, 1H), 7.78 (d, / = 1.4, 1H), 7.73 - 7.64 (m, 2H), 7.45-7.39 (m, 2H), 7.33-7.27 (m, 4H), 7.26-7.20 (m, 1H), 6.90-6.74 (m, 4H), 6.01 (dd, / = 8.5, 5.5, 1H), 5.42 (dt, / = 5.9, 1.9, OH), 4.34 (qd, / = 7.1, 1.8, 2H), 4.23 (td, / = 3.8, 1.9, 1H), 3.62-3.18 (m, 2H), 2.76-2.45 (m, 2H), 2.11 (s, 3H), 1.33 (t, / = 7.1, 3H).

¹³C NMR (101 MHz, CDC1₃) δ = 170.1, 162.6, 158.7, 144.3, 136.5 , 135.5, 135.4, 134.7, 130.0, 129.9, 129.0, 128.2, 128.1, 128.0, 127.0, 122.7, 113.3, 86.9, 86.6, 86.5, 85.7, 84.5, 75.0, 63.6, 62.6, 60.5, 55.2, 20.9, 14.4, 14.3.

Synthesis of ethyl 3 '-0-acetyl-l-(2-deoxy-fi-D-ribofuranosyl)-imidazole-4-carboxylate (10):

The starting material 9 (230mg, 0.38 mmol) was dissolved in dry chloroform (15 mL) at RT under a N₂ 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 NaHC0₃ sat. (10 mL), extracted with DCM (3 x 20 mL), dried over MgS0₄ 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.

R_f (DCM/MeOH 5%) = 0.3

MS C₁₃H₁₉N₂0₆ ⁺ calculated: 299.1238; found: 299.1249

*H NMR (400 MHz, CDC1₃) δ = 7.87 (d, / = 1.4, 1H), 7.81 (d, / = 1.4, 1H), 6.04 (dd, / = 8.2, 5.8, 1H), 5.61-5.19 (m, 1H), 4.39 (q, / = 7.1, 2H), 4.19 (q, / = 3.0, 1H), 3.91 (s, 2H), 2.67 (ddd, / = 14.3, 8.2, 6.2, 1H), 2.55 (ddd, / = 14.0, 5.8, 2.2, 1H), 2.14 (s, 3H), 1.61 (s, 2H), 1.40 (t, / = 7.1, 3H).

¹³C NMR (101 MHz, CDC1₃) δ = 170.4, 136.6, 134.6, 122.8, 86.7, 85.8, 75.0, 62.6, 60.6, 39.5, 20.9, 14.4.

Synthesis ofdIm^cTP:

Nucleoside 10 (40 mg, 0.13 mmol) was dissolved in dry pyridine (0.2 mL) and dry dioxane (0.4 mL) at RT under N₂. To this clear solution, 2-chloro-l,3,2-bonzodioxaphosphorin-4-one (38 mg, 0.18 mmol, 1.4 eq.) was added and the reaction mixture was stirred for 45 min. A solution of tributylammonium pyrophosphate (95 mg, 0.17 mmol, 1.3 eq.) in dry DMF (0.17 mL) and tributylamine (60 μί) 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 H₂0 (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 H₂0 (2 mL) and precipitated by the addition of NaC10₄ 2% in acetone (12 mL). The crude product was purified by RP-HPLC (30% B in 20 min; Buffer A) TEAB 50mM pH=8; Buffer B) TEAB 50mM pH=8, 50% ACN) to give 3.7 mg (6%) of the pure triphosphate dIm^cTP.

ESI-MS C₉H₁₄N₂O₁₄P₃ ^" calculated: 466.9663; found: 465.9564.

¾ NMR (400 MHz, D₂0) δ = 7.99 (s, 1H), 7.94 (s, 1H), 6.13 (t, J = 6.7, 1H), 4.15 (s, 1H), 4.06 (dt, / = 8.6, 5.3, 2H), 2.53 (dt, / = 13.3, 6.5, 1H), 2.48-2.37 (m, 1H).

³¹P NMR (162 MHz, D₂0) δ = -6.02 (d, / = 19.2), -9.99 (d, / = 19.6), -20.68 (t, / = 19.3).

2. Enzymatic incorporation of dIm^cTP nucleotide tag at the 3 'end of a nucleic acid molecule

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). After hybridization of the duplex, 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. The tailing reaction with dlmTP was very efficient on the Cy5-labelled strand of the duplex (comparable or even better than with the ssDNA substrate) but did not work on the FAM-labelled oligonucleotide (Figure 10B (Fluorescein) and Figure IOC (Cy5)). This could be used for the immobilization of dsDNA substrates.

Claims

1. 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.

2. The method according to claim 1, wherein the nucleic acid immobilization comprises the steps of:

a) incorporating an enzymatic imidazole nucleotide tag at the 3 '-end of the nucleic acid, in the presence of imidazole modified nucleotides and an appropriate polymerase; and b) contacting the nucleic acid having an enzymatic imidazole nucleotide tag added at its 3'-end obtained in step a) with the solid surface charged with metal ions.

3. The method according to claim 2, wherein the polymerase is selected from the group consisting of: Terminal Deoxynucleotidyl Transferase (TdT), PolyA polymerase (PAP), Terminal uridyltransferase Cidl, DNA polymerase theta and engineered polymerases.

4. The method according to any one of claims 1 to 3, wherein the enzymatic imidazole nucleotide tag comprises five to nine or more imidazole modified nucleotides.

5. The method according to any one of claims 1 to 4, wherein the solid support is charged with metal ions chosen from the group comprising: Ni²⁺, Eu³⁺' Ca²⁺, Cu²⁺, Hg²⁺, Pb²⁺, Gd³⁺, La³⁺, Tb³⁺, Zn²⁺, Fe²⁺, Fe³⁺, Ru²⁺, and Sn²⁺.

6. The method according to any one of claims 1 to 5, wherein the solid support is selected from the group consisting of polystyrene beads, gold nanoparticles, glass slides and agarose beads.

7. The method according to any one of claims 1 to 6, wherein 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.

8. The method according to claim 7, wherein the nucleic acid probe further comprises a detectable label at its 5 '-end.

9. The method according to any one of claims 1 to 8, wherein the nucleic acid is single- stranded nucleic acid selected from the group consisting of mRNA, viral RNA, cDNA, oligodeoxyribonucleotide probe, oligoribonucleotide probe and double-stranded nucleic acid such as double- stranded DNA.

10. The method according to any one of claims 1 to 9, which comprises:

- immobilizing nucleic acid from the biological sample on the solid support, and

- detecting the presence or absence of at least one nucleic acid biomarker in the nucleic acid from the biological sample immobilized on the solid support.

11. The method according to any one of claims 1 to 9, which comprises:

- immobilizing at least one nucleic acid probe specific for a protein or nucleic acid biomarker on the solid support,

- detecting the presence or absence of nucleic acid biomarker hybridized to the at least one nucleic acid probe or protein biomarker bound to the at least one nucleic acid probe.

12. The method according to claim 11, wherein the nucleic acid probe for the protein biomarker is an aptamer.

13. The method according to any one of claims 1 to 12, which is for the diagnosis or monitoring of a viral disease caused by an RNA virus, comprising the immobilization of viral RNA or specific nucleotide probe thereof, on the solid support.

14. The method according to any one of claims 1 to 12, which is for non-invasive prenatal diagnosis of a disease, comprising the immobilization of cell-free fetal DNA or mRNA or specific nucleotide probe thereof, on the solid support.

15. A kit for performing the diagnosis or assessment method according to claims 1 to 14, comprising at least one nucleic acid probe for the diagnosis or assessment of the disease or therapeutic response, wherein said nucleic probe(s) is immobilized on a solid surface charged with metal ions via an enzymatic imidazole nucleotide tag added to the 3 '-end of the nucleic acid probe.