BE1030246B1 - POLYMER ASSISTED BIOMOLECULE ANALYSIS - Google Patents

Abstract

Methods and compounds for the analysis of biomolecules are revealed. The method involves combining a specific marker of a biomolecule with a polymer capable of increasing in size, providing access to improved information about the structure and identity of the biomolecule.

Description

POLYMER ASSISTED BIOMOLECULE ANALYSIS

Technical field of the invention

Embodiments herein generally involve grafting biomolecules onto a polymer matrix, wherein the polymer and its physical behavior enable genomic analysis

References — Gottfried A. et al. “Sequence-specific covalent labeling of DNA,” Biochemical Society

Transactions, 39(2), 623-628 — Compounds and processes for single-pot attachment of label to nucleic acid,

US2006/0188927 — Proudnikov D., et al, (1996), Chemical methods of DNA and RNA fluorescent labeling, Nucleic

Acids Research, , Vol. 24, 4535-4532 — Biomolecular labeling, US6.657.052 B1 — Selection of individual nucleic acids based on optical signature, US2014/0011686 — Methods for specifically labeling nucleic acids with CRISPR/CAS, US 2016/0168621 — Methods and apparatus for the whole-genome analysis based on single molecules

US8628919 — Covalent labeling of nucleic acids, Nils Klöcker, Florian P. Weissenboeck and Andrea

Rentmeister; 10.1039/DOCS00600A, Chem. Soc. Rev., 2020, 49, 8749-8773.

Background of the invention

The study of biopolymers is greatly facilitated by methods that utilize the spatial organization of specific monomers or specific combinations thereof, with the aim of locating the presence, position, abundance or sequence of such specific sites. Examples of such specific sites may be individual nucleobases, amino acid side chains, or combinations thereof.

Examples of methods that enable such observations of sequence-specific domains are Fiber-FISH and Genomic Mapping, both of which have extensive applications in human diagnostics.

In pursuit of longitudinal information, these approaches quickly reach their limits when it comes to distinguishing a large number of sequence-specific domains, where the resolution of the underlying sequence information is inadequate. Furthermore, longitudinal information is an essential part of several of the methods, requiring long stretches of biopolymer to obtain sufficient information for species identification, genome scaffolding, or structural variant analysis.

[0005] Therefore, there is a need for methods with improved resolution, which allow the observed spatial arrangements and patterns to be analyzed in more detail and accuracy.

Summary of the Invention

The present invention relates to and includes methods and compositions for biopolymer analysis

One aspect of the present disclosure concerns the grafting of a linearized biopolymer, either as such or with a specific label bound to the linearized biopolymer, onto a polymeric matrix. This polymeric matrix is then increased in size. This increase in size occurs in the dimension of the linear biopolymer, but can extend across multiple dimensions. As size increases according to certain models, the site-specific markers on the grafted biopolymer or the grafted associated markers maintain their relative order, and/or distance patterns can be reconstructed. Measuring order and/or distance on the polymer matrix at its increased size provides improved order and/or distance resolving power so that the underlying biopolymer structure and identity can be analyzed with increased resolution and accuracy.

In one aspect the invention also relates to grafting the biopolymer onto a hydrogel, wherein the size increase is achieved by swelling of the hydrogel upon addition of water.

The present invention is very suitable for the analysis of polynucleotides, such as

DNA and RNA and polypeptides.

Other aspects of the invention will become apparent from the description and examples below, and can be summarized by the following numbered embodiments. 1) A biopolymer analysis method, consisting of; 1. Grafting the combination of a linearized biopolymer and biopolymer site-specific markers onto a polymer 2. Increasing the spatial dimensions of the cross-linked polymer along the linearization axis 3. Obtaining position information on the site-specific labels

Brief description of the drawings

Drawing 1 is a schematic representation of the invention

Drawing 2 is a schematic representation of an embodiment of the invention

Drawing 3 schematic representation of one embodiment of the invention

Drawing 4 are some of the chemical compounds used in the invention

Definitions

The following terms and associated definitions are used in this text.

[0012] "Stochastic" means a random probability distribution or pattern that can be statistically analyzed but cannot be precisely predicted

[0013] "Optical reading" is defined here as: a method that uses light signals to read specific information about the sample.

[0014] "Biorthogonal" is defined here as: chemical reactions that can be used in biological systems, in which a reactive group is specifically linked to another reactive group: without side reactions; in neutral, aqueous solution; and under additional conditions that are compatible with the biological system. Selective reaction between bioorthogonal binding partners can minimize side reactions with other binders, biological compounds, or other non-complementary bioorthogonal binders or non-complementary bioorthogonal functional groups.

Bioorthogonal functional groups of bioorthogonal binders include, but are not limited to, an azide and alkyne for the formation of a triazole via Click-chemistry reactions, trans-cyclooctene (TCO) and tetrazine (Tz) (e.g. 1,2,4,5 -tetrazine), and others. The binders useful in the present disclosure can have high reactivity with the corresponding binder so that the reaction proceeds rapidly.

The term "complementary" as used herein refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified .

Complementary nucleotides are generally A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared and with the appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and preferably from about 98 to 100%. Complementarity also occurs when an RNA or DNA strand hybridizes to its complement under selective hybridization conditions. Typically, selective hybridization occurs when there is at least 65% complementarity over a stretch of at least 14 to 25 nucleotides, preferably at least 75%, and preferably at least 90% complementarity.

The term "affinity ligand" refers to a molecule capable of binding to a specific molecule with a specific affinity, and in the present invention the term refers to molecules capable of selectively binding to a protein or to bind aptamer. Note that the affinity ligand can simply be called "ligand".

The terms "binding" and "binder" refer to a chemical or biological agent that specifically binds to a target (e.g., a targeted biomolecule), forming a stable association between the binder and the specific target. "Stablely associated" or “stable association” means that a molecule is bound or otherwise associated with another molecule or structure under standard physiological conditions. Bonds may include covalent bonds and non-covalent interactions, such as, but not limited to to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g. London dispersion forces), dipole-dipole interactions, and the like. A targeting agent can be a member of a specific binding pair, such as, but not limited to: member of a receptor/ligand pair; a ligand-binding part of a receptor; member of an antibody/antigen pair; an antigen-binding fragment of an antibody; a hapten; member of a lectin/carbohydrate pair; member of an enzyme /substrate pair; biotin/avidin

The term "contact" or "contact" refers to the process of bringing at least two different species into contact with each other so that they can interact with each other, such as in a non-covalent or covalent bonding interaction or bonding reaction. However, the resulting complex or reaction product may be produced directly from an interaction or reaction between the added reactants or from an intermediate of one or more of the added reactants or compounds, which may be produced in the contact mixture.

The term "position information" refers to position values in a coordinate system.

The presence and combination of such values create spatial patterns, which can be interpreted to obtain information about the system.

The term "site specific" refers to processes, binding events, reactions, docking events and the like that occur depending on the presence or absence of specific combinations of biomonomers. Examples include reactions to specific nucleobases, amino acids and combinations thereof. Locations can consist of specific combinations of nucleobases, amino acids and combinations thereof. Such combinations can consist of any number of monomers, but preferably between 2 and 20.

The term "linker", "linked" or "linker" refers to a chemical moiety that bonds two moieties together, such as a compound of the present disclosure to a biological material that targets a specific cell type, such as a cancer cell , another type of diseased cell or a normal cell type. The coupling can occur via covalent bonds, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g. London dispersion forces), dipole-dipole interactions, and the like. The link can be a direct link between the two constituent parts being linked, or an indirect one, such as via a linker. Linkers useful in embodiments of the present disclosure include linkers with 30 carbon atoms or less in length. In some embodiments, the linkers are 1-15 carbon atoms in length, such as 1-12 carbon atoms, or 1-10 carbon atoms, or 5-10 carbon atoms in length. The representative linkers can have from 1 to 100 connecting atoms, and can include, but are not limited to, ethylene oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, linkers can have 1 to 50 bond atoms, or 1 to 30 bond atoms. Other types of bindings can also be used in embodiments of the current revelation.

The terms "binder" or "binder" refer to a process or agent having a functional group capable of forming a covalent bond with a complementary functional group of another binder in a biological environment. Bonding between binders in a biological environment can also be referred to as bioconjugation.

Representative binders include, but are not limited to, an amine and an activated ester, an amine and an isocyanate, an amine and an isothiocyanate, thiols for disulfide formation, an aldehyde and an amine for enamine formation, an azide for the formation of an amide via a Staudinger ligation. Binders also include bioorthogonal binders, that is, binders with bioorthogonal functional groups. [0023] The "nucleic acids" or "polynucleotides" of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), closed nucleic acids (LNAs, including LNA with a B-D-ribo configuration, α-LNA with an α-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA with a 2'-amino functionalization, and 2'-amino -a-

LNA with a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.

The term "oligonucleotide", as used in the invention, refers to a short nucleic acid molecule of about 8 to about 50, optionally up to about 90, nucleotides in length, natural or synthetic, that can function as a starting point of complementary nucleic acid hybridization. The oligonucleotide itself can also be labeled, if desired, by incorporating a compound detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include fluorescent dyes, electron-dense reagents, biotin, or small peptides for which antisera or monoclonal antibodies are available.

A "reactive group" is a chemical moiety that can react with a chemical partner moiety to form a covalent or non-covalent bond. A group can be considered a reactive group based on its high reactivity with a single partner molecule, a set of partner molecules, or by virtue of its reactivity with many partners.

"DNA mapping" refers to a process in which sequence-specific markers are introduced into a polynucleotide, and in which the distance information between these markers provides information about the genetic makeup of the polynucleotide. DNA mapping can include all polynucleotides in a sample, including but not limited to genomic DNA, plasmid DNA, mRNA, tRNA, and genomic RNA.

Detailed description of the invention

The present invention will be described with reference to particular embodiments and with reference to certain drawings, but the invention is not limited thereto, but only by the claims. Furthermore, the terms first, second and the like are used in the description and in the claims to distinguish between similar elements and not necessarily to describe an order, whether temporal, spatial, serial or otherwise. It is understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein may operate in other sequences than those described or illustrated herein.

It is noted that the term "consisting of" used in the claims is not to be construed as being limited to the means mentioned below; he does not exclude other elements or steps. The term should therefore be interpreted as specifying the presence of the mentioned features, integers, steps or components, but does not exclude the presence or addition of one or more other features, integers, steps or components, or groups thereof . The scope of the expression "a device consisting of parts A and B" should therefore not be limited to devices consisting only of parts A and B. It means that, as far as the present invention is concerned, the only relevant parts of the device A and B are.

Reference in this specification to "an embodiment" or "an embodiment" means that a particular feature, structure or feature described in connection with the embodiment is included in at least one embodiment of the present invention . Thus, the expressions "in an embodiment" or "in an embodiment" appearing at various places in this specification do not necessarily all refer to the same embodiment, but may all refer to the same embodiment. Furthermore, the particular properties, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

Likewise, it should be understood that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped into a single embodiment, figure or description thereof for the purpose of streamlining the disclosure and aiding in understanding of one or more of the various inventive aspects. However, this method of disclosure should not be interpreted as meaning that the claimed invention requires more features than those expressly stated in each claim. On the contrary, as the following conclusions show, the inventive aspects lie in less than all the features of a single aforementioned revealed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, each claim standing alone as a separate embodiment of this invention.

Numerous specific details are set forth in the description herein. However, it is clear that embodiments of the invention can also be practiced without these specific details. In other cases, known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this description.

The method of the invention involves obtaining a linearized biopolymer in or on a polymeric matrix, where the polymeric matrix contributes to analysis of the linearized biopolymer

An embodiment of the described method 100 is shown in Fig. 1 and includes 3 different steps, [10, 20, 30], which can be divided as follows

A. Obtaining a linearized biopolymer marked at specific sites and bound to a polymeric matrix

B. Increasing the dimensions of the polymer, at least in line with the linearized biopolymer

C. Analysis of the signal

For the methods of the invention, the first step itself may consist of several sub-steps. For example, a biomolecule can react in a site-specific manner to place labels at known locations within the polymer, followed by linearization of the biomolecule along with the bound labels. Another possibility is that the biomolecule is first linearized, followed by specific binding at selected locations within the biomolecule.

Linearization of the biomolecule can be performed by linearization on a surface or in a flow system. Examples of linearization are linearization on surfaces (Kaykov et al. Sci Rep 6, 19636 (2016) or in flow systems (Kim et al, Lab Chip, 2011,11, 1721-1729).

Increasing the size of the polymer can be achieved by merely physically stretching the polymer, by applying a force. Since the direction of the linearized biopolymers is known, the direction of the force can be consistent with the direction of the biopolymer.

In a specific embodiment, the polymer matrix forms a swelling matrix. A well-known example of such an intumescent matrix are hydrogels, which consist of a polymer mesh and an aqueous solution. With further stimulation, the physical dimensions of the hydrogel increase. Traditional stimuli that elicit a hydrogel reaction are pH, temperature, solvents, and ionic strength. With different hydrogel recipes, sizes 4-20 times larger can easily be achieved.

In yet another embodiment, the swelling is limited in some of its dimensions by a container. This can force the gel to limit swelling in a preferred direction, increase swelling in a preferred direction, or ensure proper handling of the gel.

Preferably, the size expansion of the polymer follows predetermined patterns, so that the original biological information can be traced at high resolution. This pattern may be a linear size expansion. For applications where only the presence and sequence of specific locations is important, this previously known expansion pattern is of less importance. In a specific embodiment, a measurement is included in the workflow to analyze the size expansion profile.

Groups that can effect such grafting onto the polymer matrix include, but are not limited to, vinyl or vinyl monomers such as styrene and its derivatives (e.g. divinylbenzene), acrylamide and its derivatives, butadiene, acrylonitrile, vinyl acetate, maleimides, alkynes, epoxides , aldehydes or acrylates and acrylic acid derivatives.

In a further embodiment, the methods of the invention may include a step that disrupts the biopolymer upon grafting the specific signals onto the polymeric matrix. This can be beneficial for the uniformity of the size gain, as the covalent bonds in the biopolymer can hinder the local size gain if not broken.

Examples of such steps are a UV treatment to disrupt the polynucleotide chains or an enzymatic step, with a nuclease or proteinase enzyme.

In embodiments of the invention, site-specific labeling of DNA is a key element of the methods. Such sequence-specific labeling of DNA is a rapidly developing area, discussed by Gottfried A. et al. "Sequence-specific covalent labeling of DNA", Biochemical Society Transactions, 39(2), 623-628" and the sequence-specific methods described are herein incorporated by reference.

Enzyme-targeted methods include labeling with methyltransferase enzymes, nicking

enzymes, polymerases and CRISPR-CAS methods. Chemical methods involve the use of sequence-specific ligands, which can be small molecules or oligomers.

In general, DNA nucleobases are buried in the DNA helix, with only limited availability for chemical modification at the nucleobase. However, in a specific embodiment of the invention, DNA is linearized on a surface using molecular combing. This molecular combing causes partial melting of the DNA, making nucleobases more accessible to chemical reactions. For example, bisulfite-mediated transamination of cytosines is capable of generating a DNA signature within a polymeric matrix. In such an embodiment, it may be beneficial to graft the DNA onto the polymeric matrix in a non-specific manner after linearization. The polymer matrix can then be subjected to the required chemicals and solutions without the risk of the DNA becoming detached from its position on the surface.

The methods of the invention are easily translatable to some existing ones

DNA mapping approaches, in which sequence-specific markers are applied to DNA before analysis. By further modifying this DNA with a polymerizable moiety, the DNA can be subjected to grafting with a polymer matrix, the polymer can be extended, and DNA mapping can be performed with improved resolution.

A certain property of DNA can be used to efficiently generate signals or incorporate monomer molecules. Agents that bind to the major or minor groove of DNA can induce a sequence-specific signal (Dervan et al, . Proc Natl

Acad Sci U S A. 2006; 103(4):867.), serve as a way to identify monomeric units along the

DNA backbone or a combination of both.

In another embodiment, the DNA is denatured to obtain single-stranded DNA, followed by hybridization with random oligomers. The oligomers can then be modified oligomers containing polymerizable groups or labels, or a combination thereof.

In some embodiments, the method further includes detecting a relative distance between the labels on the linearized polynucleotide, thereby barcoding a portion of the genomic information. This relative distance can be obtained by fluorescent microscopy.

It is clear that the linearized biomolecules do not need to be in the polymeric matrix, but that the sequence specific marker only needs to be bound or transferred to the polymeric matrix. The biopolymers can therefore be on or against the polymeric matrix, and the sequence specific marker is bound to or in the polymeric matrix.

Thus, in such embodiments of the methods of the invention, the signal for analysis no longer becomes a direct signal of the biomolecule, but of its identity. The biomolecule should therefore no longer maintain its integrity after binding of the specific markers to or in the polymeric matrix.

Sequence-specific marking of RNA can be achieved by sequence-specific interactions or direct reactions with specific nucleobases.

Sequence-specific interactions, or interactions that depend on the sequence of more than one nucleobase, that can be used for the methods of the invention are, for example, complementary hybridization probes or sequence-specific enzymes.

For these embodiments, the hybridization probes can preferably be selected for a high melting temperature so that the secondary and tertiary structures of the RNA are disrupted. Examples of such hybridization probes with favorable binding are peptide nucleic acids (PNA), closed nucleic acids (LNA) and other non-natural polynucleotides.

The length of the hybridization probes is chosen depending on the application, but is preferably between 4 and 400 nucleotides.

Importantly, in the methods described in the invention, the sequence specific signature is transposed onto a polymeric matrix. The integrity of the biomolecule does not need to be maintained to analyze the sequence-specific signature.

[0055] To facilitate linearization, substances that disrupt the secondary and tertiary RNA structure and which would otherwise hinder the linearization process can be added to the linearization mixture. Such agents can be random oligonucleotides (e.g. random hexamers or nonamers), hydrogen bond breaking agents (e.g. formamide, urea) or charge neutralizing salts.

Functionalization of the RNA species according to the methods of the invention can also be accomplished by targeting specific nucleobase interactions on RNA. For example, bisulfite-mediated cytosine transamination rapidly and completely converts all cytosines to an amine exchanged variant. In this way, all cytosines in an RNA can be functionalized with both signaling molecules and monomer functionalities. The sequence-specific signal generated upon binding to the polymer matrix and expansion is therefore a linear image of the cytosine content of the RNA. Guanine labeling can be accomplished by the action of reagents such as the electrophilic nitrogen mustards, platinum reagents, which react preferentially with the nitrogen of guanine nucleobases.

In a specific embodiment, nucleobase specific labeling is combined with hybridization probes. For example, bisulfite-mediated transamination is hindered when parts of the RNA are hybridized. As such, RNA signals can be prepared that combine both a signal at unhybridized sites, indicating the presence of cytosine, and a “dark signal” at the position of specific hybridization probes.

Alternatively, a first cytosine transamination is performed in the presence of hybridization probes, and signal is generated in one channel (e.g., one color), with hybridization probes in a second channel (e.g., another color).

A unique advantage of the methods of the invention is that the combination of sequence-specific labeling of RNA with an extended polymer matrix brings RNA within the reach of high-resolution genomic mapping and Fiber FISH, methods previously blocked by the limited scope of most RNA. There are no other methods that allow the direct imaging and identification of mRNA and rRNA.

In a specific embodiment of the invention, there are hybridization or affinity probes in the polymerization mixture, polymerization medium or linearization medium that contribute to the linearization of the biopolymer. These probes can also have a signaling function. A possible example is a hybridization probe for RNA, such as a poly-T tail for hybridization of poly-A containing RNA.

In particular, direct imaging of rRNA and its genetic signature will have extensive applications in species identification, lineage and phylogenetics and the analysis of complex mixtures, or microbiomes. Currently, these signals are measured indirectly through the analysis of the DNA encoding the rRNA, by targeted amplification of the genes followed by sequencing, microarray analysis, qPCR and other methods.

[0061] In specific embodiments, the method is extended to include mapping

Protein. By tagging specific amino acid side chains with signals, linearizing the polypeptide, and binding it to the polymer matrix, a linear signal can be obtained. This signal corresponds to a coarse representation of the total protein sequence.

However, this rough representation of the sequence of amino acids contains sufficient information to determine the identity of the protein.

It is clear to one trained in the art that with diffraction-limited microscopy methods the resolution of the genetic information obtained can be further increased.

The methods of the invention have broad application potential in the fields of biotechnology, genomics, medicine and beyond.

In particular, the methods enable unprecedented resolution in genomic mapping approaches, which use sequence-specific signatures of linearized DNA to determine DNA identity and origin. Such genomic mapping is used in species identification, genome mapping, genomic and genetic analysis, and structural variant analysis. Here the sequence-specific signature is directly related to the length, as the DNA piece becomes more unique as a function of the length. In current genomic mapping approaches, sequence-specific elements are focused on 4 to 8 base pairs, and DNA stretches are longer than 10 kbp up to MBp. The expansion of the genetic signature leads to a greater resolution of the genetic signature, and therefore to a more specific attribution.

To date, no genetic mapping methods have been extended to RNA species. This is largely attributed to the length requirements mentioned above. With mRNA several hundred to several thousand bases long, the signature obtained in genomic mapping approaches would not contain sufficient information for further use. However, building on the methods of the invention, high-density labeling of RNA can be physically separated by the extension of the polymer to yield a useful trace. As such, genomic mapping of mRNA from tissues and cells becomes feasible. rRNA from different organisms and mixtures thereof can be directly visualized, studied and assigned.

These methods can also be applied to other enzymes and proteins that bind to specific sites on a polynucleotide or to specific states of a polynucleotide. Examples of such enzymes and proteins that bind to polynucleotides in a sequence-specific manner beyond methyltransferases are transcription factors or proteins containing DNA-binding domains such as Helix-turn-helix,

Zinc finger, Leucine zipper, Winged helix, Winged helix-turn-helix, Helix-loop-helix, HMG box,

Wor3 domain, OB fold domain, Immunoglobulin fold, B3 domain or TAL effector. A specific case of DNA-binding proteins are proteins guided by RNA. Cas9, for example, can be used as a customizable RNA-directed DNA-binding platform, and when combined with ligands such as described in this invention, can achieve targeted DNA modification.

Another example is enzymes that bind to accessible chromatin, transfer functionality to said chromatin and allow specific analysis of chromatin accessibility and its use in genetic analysis.

A specific embodiment is the use of the methods of the inventions for the analysis of epigenetic and post-translational marks on polynucleotides and polypeptides.

Examples of this are the site-specific labeling of, for example, DNA hydroxymethylation, DNA carbonylation, specific RNA bases such as pseudouridine or protein modifications such as prenylation or phosphorylation. Specific chemical, enzymatic, or chemoenzymatic approaches exist for transferring functionality to these specific sites, and when combined with linearization and grafting onto a polymeric matrix, the expansion of the signal can improve the analysis of these marks.

Further embodiments of the inventions may replace the enzyme with synthetic macromolecules capable of recognizing specific docking positions on the biomolecule. An example of such macromolecules could be an aptamer. Another example of such macromolecules could be an antibody. Specific antibodies and aptamers have been described in the literature for genetic signatures such as DNA methylation and specific sequence combinations, and can therefore be easily incorporated into the methods of the invention.

In each method described above, a) the biopolymer labeling reactions can be performed in the same reaction vessel, or b) biopolymer from the sample can be aliquoted and added to different reaction vessels, with biopolymer labeling reactions performed in each reaction vessel. The different reactions can then be recombined or analyzed independently of each other.

In the kits of the invention, at least one labeling entity may be a fluorophore or a molecule to which a fluorophore can be attached.

Each of the kits of the invention may further contain at least one affinity chromatography column.

Each of the kits of the invention may further contain at least one buffer.

Each of the kits of the invention may further contain one or more other enzymes for carrying out reactions required in methods of the invention. The enzymes can

DNA methyltransferases, DNA nickase or a DNA polymerase.

Particularly useful are labels that are optically detectable. Examples are chromophores, luminophores and fluorophores. Fluorophores are particularly useful and include, for example, fluorescent nanocrystals, quantum dots, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarins, pyrene, malachite green, Cy3, Cy5, stilbene, Matchstick yellow, Cascade blue, Texas red, Alexa dyes,

SETA dyes, Atto dyes, phycoerythin, bodipy and analogues thereof. Useful optical probes are described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; the Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence

Spectroscopy, 2nd Ed., Plenum Press New York (1999), or WO 98/59066; WO 91/06678 of US

Pat. Appl. Publ. No. 2010/0092957 A1, all of which are incorporated herein by reference. Optical labels offer the advantage of rapid, relatively non-invasive detection.

Other labels, some of which are non-optical labels, can be used in various applications of the methods and compositions described here. Examples include, without limitation, an isotopic label such as a naturally occurring non-abundant radioactive or heavy isotope; magnetic substance; electron-rich material such as a metal; electrochemiluminescent label such as Ru(bpy); or a part that can be detected based on a nuclear magnetic, paramagnetic, electrical, charge-mass, or thermal characteristic.

Labels can also include magnetic particles or optically encoded nanoparticles.

Such labels can be detected using suitable methods known to those skilled in the art. For example, a charged label can be detected with an electrical detector such as those used in commercially available sequencing systems from lon Torrent (Guilford, Conn., a subsidiary of

Life Technologies) or detection systems described in US Pat. App. Publ. Nos. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; and 2010/0282617 A1, all of which are incorporated herein by reference. It will be clear that for some applications a nucleotide analogue does not need to have a label.

In one embodiment, the reporter is an oligonucleotide. By specific hybridization with complementary oligonucleotides, this reporter can serve as a barcode for the biomolecule originally present at the site mentioned. The complementary oligonucleotides may be fluorescently labeled (i.e. conjugated to a fluorophore). Fluorophores conjugated to imager strands of different nucleotide sequences may be identical to each other, or they may have an emission profile that overlaps or does not overlap with that of other fluorophores. The fluorescently labeled imager strand may contain at least one fluorophore. Both the reporter oligonucleotides and the signaling oligonucleotides can possess a hairpin secondary structure.

In one embodiment, the complementary oligonucleotide hybridization is transient, with binding times on a long enough time scale to cause a "blinking" event.

In yet another embodiment, the reporter group includes multiple separate signals

For some embodiments of the invention, some form of signal amplification may be useful. Such signal amplification provides good reading of low abundance signals or allows the technique to be used in less demanding analytical setups, as the need for expensive detectors and sensitive reading is reduced. Examples of such amplification methods can be hybridization based, such as Hybridization-Chain-Reaction (HCR), SABER or Rolling Circle amplification. Depending on the type of amplified signal, this amplification can be performed before or after the swelling. Another example of signal amplification is the use of antenna dyes, which amplify the signal generated by one dye by harvesting excitation light.

Another type of label that may be useful is a secondary label that is detected indirectly, for example via interaction with a primary label, binding to a receptor or conversion to a detectable product by an enzyme catalyst or other substance. An example of a secondary label is a ligand such as biotin or analogues thereof, which can be detected via binding to a receptor such as avidin, streptavidin or analogues thereof.

Other useful ligands include epitopes that can bind to receptors such as antibodies or active fragments thereof, and carbohydrates that can bind to receptors such as lectins.

The receptors can be labeled, for example with an optical label, so that they can be detected.

In particular applications, the ligand can be attached to a nucleotide analog in such a way that its affinity for a receptor is reduced or prevented.

The release of the ligand can then be detected based on the affinity of the ligand for its respective receptor when released from the nucleotide analog.

The ligand can be further linked to a blocking group or can itself act as a blocking group, as explained more generally above for labeling groups.

Thus, removal of the ligand from a nucleotide analog can function to unblock the nucleotide analog and provide a detectable event.

Examples

[0082] Example 1

Lambda phage DNA is incubated in the presence of Product 1 (Figure 4) at 55°C for 60 minutes. The DNA is deposited on a polymer surface in accordance with literature examples (Kaykov et al. Sci Rep 6, 19636 (2016)). The surface is covered with a polymerization mixture (aqueous solution of sodium acrylate, bisacrylamide, TEMED and APTS, in line with standard literature recipes), and polymerization is carried out for 40 minutes. The polymeric gel is gently detached from the substrate and allowed to incubate for 30 minutes at 37°C in a solution containing TAMRA-DBCO (Jena Biosciences) in PBS. The gel is washed extensively with PBS, followed by swelling in double distilled water overnight. The swollen gel is mounted on a coverslip and exposed. The linear features are analyzed in accordance with the literature (Torche PC, PLoS ONE 12(6): e0179041) to reveal the sequence-specific signal.

Claims (10)

Conclusions 1. A method for characterizing biopolymers, the method consisting of: ij) Grafting the combination of a linearized biopolymer and biopolymer site-specific markers onto a polymer ii) Increasing the spatial dimensions of the polymer along the linearization axis iii) Obtain positional information from the location-specific labels 2. The method according to claim 1 where increasing the spatial dimensions of the polymer is achieved by swelling of a hydrogel 3. The method according to claim 1 where increasing the spatial dimensions of the polymer is achieved by applying a force to the polymer The method of claim 1 where the biopolymer is a polynucleotide The method of claim 2 where the biopolymer is DNA The method of claim 2 where the biopolymer is RNA The method according to claim 2 where the positional information is obtained by fluorescence microscopy 8. The method according to claim 7 where it is used to identify the origin of the biopolymer The method of claim 7 where applied in cellular analysis The method of claim 7 where it is applied in the identification or treatment of a disease