WO2007087653A1

WO2007087653A1 - Method for detecting 5-methylcytosine

Info

Publication number: WO2007087653A1
Application number: PCT/AT2006/000546
Authority: WO
Inventors: Rong Zhu; Peter Hinterdorfer; Hermann Gruber
Original assignee: Universität Linz
Priority date: 2006-02-03
Filing date: 2006-12-29
Publication date: 2007-08-09
Also published as: AT503189B1; AT503189A1

Abstract

The present invention provides a method for the detection of 5-methylcytosine, characterized in that either a 5-methylcytosine specific binding moiety or a nucleotide analyte is attached to the tip of a single molecule force spectroscopy (SMFS) probe and the other of the 5-methylcytosine specific binding moiety or the nucleotide analyte is attached to a carrier, wherein the SMFS probe is brought into contact to the carrier and is detached and the detachment force is measured through an SMFS detection means; as well as an SMFS probe and an SMFS apparatus for said method.

Description

Method for detecting 5-methγlcytosine

The present invention relates to the detection and measurement of methylated poly- or oligonucleotides.

DNA methylation plays an important role in the regulation of gene expression (1) , which has strong impact on tissue development (2), aging (3-4), and some diseases (5-7). Many kinds of cancer correlate with abnormal DNA methylation, including genome-wide hypomethylation (8) and site-specific hypermethylation (9) which switches off some cancer suppressor genes or DNA repair genes. Therefore, detecting the pattern of methylation of gene promoters is of great importance for diagnosis and basic research (10-12) .

Known techniques include restriction landmark genome scanning, representational difference analysis, methylation-sensit- ive arbitrarily primed PCR and methylated CpG islands amplification, COBRA (35) and MethyLight (11-12) . Present chip based techniques like the MethyLight (12) technique rely on PCR and do not yield high-resolution methylation information because of cross-reactivities and aim only at a relative amount of prevalence of DNA methylation patterns. Methylation-sensitive restriction enzyme digestion followed by PCR is prone to false- positive results since even low levels of spurious incomplete digestions in cleavage resistant DNA stretches can result in a PCR product. Other problems, such as incomplete cutting, normal cell contamination and the need for considerable quantities of DNA for analysis are further drawbacks. Improper design of PCR primers, which have to be adapted to each methylation site (of e.g. 50 millions CpG sites in the human haploid genome), can also result in false positive m⁵C identification in the bisulfite method, where cytosine is converted to uracil and m⁵C remains nonreactive (11) . The bisulfite method also requires DNA treatment at an acidic pH (~pH5) for a long time, which can lead to aquirinic sites.

PCR based methods also suffer from biased PCR amplification artifacts. The same primer pair can preferentially amplify either the methylated or unmethylated sequence, even though the sequence to which the primers anneal and the lenghts of the PCR products are identical.

All these methods for analysing methylation of nucleic acids described in the art have drawbacks because they do not relate to the single molecule as information provider, but are mainly based on high-throughput-analyses or bisulfite-PCR based methods .

The US 2003/0186311 Al describes the measurement of molecular interactions with an AFM array. Among others protein-nucleic acid interactions can be determined by reaction parameters .

Stroh et al . (Biophys J. 87(3) (2004): 1981-9) describe in- teractios between lysozyme, immobilized on a carrier, and an anti-lysozyme-antibody on an AFM tip. The AFM tip scans the carrier in an oscillating mode.

In the US 5 372 930 an arrangement to determine AFM interactions between molecules is described, in particular between complementary nucleic acid interactions and base pairing.

EP 1 233 259 Al contemplates an AFM probe with a monolayer. A multitude of nucleic acids can be fastened on the monolayer, e.g. thymine or guanine for the detection of complementary bases on an immobilized DNA sample.

According to Boland et al. (Proc Natl Acad Sci U S A 92(12) (1995): 5297-301) layers of nucleic acids were scanned by AFM. Hydrogen-bonds have been measured between nucleic acids with a tapping mode scan.

The publication Kienberger et al. (Ace Chem Res. 39(1) (2006) : 29-36) discloses measuring of molecular interactions between avidin-biotin and RanGDP/GTP-importin-beta by AFM.

It is a goal of the present invention to provide new means for the analysis of nucleic acid-methylation patterns with high precision (preferably at single base resolution) .

The present invention provides a method for the detection of 5-methylcytosine, characterized in that either a 5-methyl- cytosine specific binding moiety or a nucleotide analyte is attached to the tip of a single molecule force spectroscopy (SMFS) probe and the other of the 5-methylcytosine specific binding moiety or the nucleotide analyte is attached to a carrier, wherein the SMFS probe is brought into contact to the carrier and is detached and the detachment force is measured through an SMFS detection means. A nucleotide analyte is any nucleic acid or polynucleic acid potentially comprising one or more 5-methylcytosine (s) .

On the tip of the SMFS probe or the carrier preferably one or more single 5-inethylcytosine specific binding molecule is (are) bound. A 5-methylcytosine comprising analyte and the 5- methylcytosine specific moiety have strong attractive molecular forces after contact, which can be measured by SMFS. The measured force or the force profile of the detachment reaction can be used to detect and distinguish single 5-methylcytosine nucleotides. This result was completely surprising since it was not expected to achieve a specific distinction based on single molecule measurements because of specificity problems associated with common 5-methylcytosine specific binding moieties.

Force spectroscopy has been widely used for studying the interaction between or within biological molecules, such as ligand and receptor (13-15), antibody and antigen (16), single proteins (17), DNA (18-22), RNA (23-24), cells (25), etc., by using atomic force microscopy, laser tweezers, optical traps, or biomem- brane force probe (26,27). Force measurements can quantify the interaction and reveal further information, such as dissociation rate, energy landscape, contour length, persistence length, and structural information, however, antibodies have not been used before in SMFS or AFM techniques to analyse nucleic acids, especially the methylation of nucleic acids, or vice versa.

The 5-methylcytosine specific binding moieties are preferably 5-methylcytosine specific antibodies, which are generally known in the state of the art and presently used in fluorescent assays to detect methylated DNA (30-32, WO 2004/104582) and are commercially available (e.g. from Serotec) . 5-methylcytosine (In⁵C) is also referred to as simply methylcytosine and is present in biological methylated DNA. The 5-methylcytosine specific binding moitey is generally also specific for 5-methylcytosidine (e.g. the antibody developed by serotec). E.g. the antibody clone 33D3 by Serotec has been developed to discriminate between the modified base m⁵C and the normal counterpart cytosine. Antibodies can be obtained from both monoclonal or polyclonal stocks, preferably after immunoaffinity selection for optimisation. For the present invention any 5-methylcytosine specific antibody or antibody fragment can be used, preferably Fab (antigen binding fragment), Fab' or scFv (single chain variable fragment) fragments. Also, the antigen binding determinant region alone, specific for 5-methylcytosine (optionally provided in a suitable scaffold anchoring) can be used. These fragments can be produced by common recombinant techniques, protein engineering or purchased commercially.

In preferred embodiments the SMFS is an atomic force microscopy (AFM) , wherein preferably the detection means is a laser deflection means, most preferred a cantilever. For example, a laser is deflected from the cantilever and the reflection is sensible to small position alteration, effected by molecular interaction between the molecules attached to the probe and the carrier. Preferably the AFM is a contact or dynamic force microscopy (DFM) method, wherein the probe tip is oscillated over the sample carrier.

Another preferred SMFS is the optical tweezer method also called laser tweezer or optical trap method. For this spectroscopy the carrier in form of a bead is brought into an optical trap. By contacting the carrier with the probe the forces between the attached molecules on the carrier and the probe can be measured via the trapping constant (33) . Both standard AFM and optical tweezers are especially preferred for the detection of the interaction of the probe and carrier attached moieties. Both techniques are thoroughly reviewed in (34) and can be routinely adapted for the present invention.

Another preferred SMFS technique is the use of a force apparatus. Herein the SMFS probe and the carrier are opposing surfaces, wherein each have attached several molecules of one either the analyte or the 5-methylcytosine specific binding moiety to the respective surface. The interaction and detachment forces are measured by standard techniques, e.g. laser deflection.

A further SMFS method is the biomembrane force probe method. In this method the probe is a biomembrane, e.g. from an erythrocyte or a whole erythrocyte, which is used as a spring cantilever similar to standard AFM (26,27) .

In another preferred embodiment the 5-methylcytosine specific binding moiety is bound to the SMFS probe, preferably by a linker, most preferred by a glycosidic linker. In the case of an antibody the linker is for example the Fc part of said antibody, which can be bound, e.g. by its lysine residues or by its glyc- osylation structures. Immobilization via glycosylation structures is especially preferred since optimal spacial symmetry and flexibility is achieved thereby. Other chemical or biological linker can also be used to immobilize the 5-methylcytosine specific binding moiety, wherein the linker molecules can be branched, prolate or globular. Especially preferred is a linker with well defined branching, e.g. di- or tri-branched linkers. Such linkers can also be used consecutively resulting in multiple defined branchings, e.g. 4-fold or 9-fold or any combination. At each end a 5-methylcytosine specific binding moiety is preferably attached. The branching is preferably a tree-type branching (Figs. 4 and 6), which can be facilitated by standard chemical synthesis, e.g. by amide bonding between the branching monomers .

The carrier is preferably a solid carrier, especially a glass slide. Preferably, the carrier is an aldehyde glass slide. Aldehyde glass is preferably used to immobilize biomolecules, like proteins or nucleotides, which can be aminated at one end. The nucleotide analytes can be for example RNA or DNA, double or single strand and are preferably denaturated prior to SMSF measurement. Denaturation is preferably a chemical denaturation e.g. by use of lysolecithine .

Preferably the nucleotide analyte is immobilized in spots onto the carrier. This allows a systematic measurement of many analytes on one carrier and its easy handling.

Further preferred is the method, wherein the nucleotide analyte on the carrier is analyzed by fluorescent specific 5- methylcytosine assay prior to contacting and detachment by the SMFS probe. By this method a faster screening technique is first applied before the time consuming SMFS measurement. After the presence of 5-methylcytosine is confirmed, e.g. by standard assays, the SMFS measurement provides further information, like the binding forces (and, optionally, the binding length (s)) of the attached molecules. Small DNA stretches, which form base pairs at at least one portion of the sequence can be untangled by the pulling force on the SMFS probe. This behaviour can be measured selectively on spots with 5-methylcytosine. Therefore, preferably more than one nucleotide analyte spot is on the carrier and the SMFS measurement is carried out for the spots, which were 5-methylcytosine positive in the fluorescence assay.

According to a preferred embodiment, the method according to the present invention also uses recognition imaging (28, 29) .

Most preferred the SMFS probe comprises two, three, four, five, six or more 5-methylcytosine specific binding moieties and is capable of binding two or more 5-methylcytosines on the nucleotide analyte. Two such moieties, e.g. antibody Fab portions, can bind two 5-methylcytosines on one nucleotide analyte, which is preferably a oligo- or polynucleotide molecule. With SMFS it is possible to measure the distance of these (at least) two 5- methylcytosines through the atomic force profile. Therefore the present invention also provides the method, wherein through the measured SMFS force profile, the distance of the two or more 5- methylcytosines on the nucleotide analyte is detected at a single base level.

Force spectroscopy has been used to obtain the distance information of methylcytosines in single-stranded DNA (ssDNA) . For example, two Fab-domains of an anti-methylcytosine antibody can bind two methylcytosines in an ssDNA. If one end of the DNA is fixed to a solid support and the Fc-domain of the antibody is pulled, the Fab-domains are separated from the DNA one after the other, with a distance equal to the contour length of nucleotides between two methylcytosines. In this way, the distance between methylcytosines in the DNA can be measured.

Preferably, the nucleotide analyte is a polynucleotide, bound to the SMFS probe or the carrier through base pairing to an oligonucleotide, which is immobilized onto the SMFS probe or the carrier (whereto the nucleotide analyte is to be attached to) , preferably the base pairing is reinforced by a nucleotide intercalating agent, which is preferably psoralen. The nucleotide analyte may be a nucleic acid molecule from a sample of a patient being suspected of having a disease which correlates with an abnormal methylation (e.g. an abnormal methylation of chromosomal DNA) . The nucleic acid analyte may also be a nucleic acid with one or more 5-methylcytosine residues (e.g. for analysing the degree of methylation and/or for analysing the position of the methylated residues within that nucleic acid molecule) . The nucleic acid analyte will in most cases be DNA, preferably DNA extracted from biological sources, especially from human tissue samples or samples of human body fluids (containing cells) . The analyte is attached to the probe or the carrier via a short oligonucleotide, for example an unspecific poly-T or poly-A stretch. Thus affixed the nucleotide analyte can easily be removed by denaturating agents and another analyte can be in turn attached to the oligonucleotide on the probe tip or carrier. For short nucleotides the base pairing can be reinforced to achieve stability during the contacting and detachment steps of the SMFS measurement. A reinforcing agent is for example the intercalating agent psoralen, which can be reactively activated by UV light. The oligonucleotide acts as a linker to the nucleotide analyte by double helix formation. The analyte can be detached form the oligonucleotide after SMFS measurement by denaturating conditions, e.g. rinsing with water and formam- ide .

In another aspect the present invention also provides a single molecule force microscopy (SMFS) probe, comprising a 5- methylcytosine specific binding moiety attached to the SMFS probe, preferably by a linker moiety. The linker is for example the Fc portion of an antibody or a polyvalent crosslinker. Polyvalent crosslinkers for (specific) multiple molecule attachment are generally known in the art and include small polymers, glycosides and amides.

Preferably the SMFS probe comprises two or more 5-methyl- cytosine specific binding moieties. As mentioned above this has the advantage that the distance between methylcytosines can be measured. Preferably the probe comprises 2, 3, 4, 5, 6, 7, 8 or 9 5-methylcytosine specific binding moieties. These moieties are preferably attached to the probe tip by a (multi-branched) linker, as mentioned above.

Preferably the SMFS probe is an AFM probe, preferably a tipped cantilever.

In a further aspect the present invention also provides a SMFS apparatus, e.g. standard, AFM apparatus, comprising an SMFS probe with the 5-methylcytosine specific binding moiety and a carrier, preferably the carrier is a chip. The chip has most preferred many spots or sites for the immobilization of nucleotide analytes. The chip is exchangeable for automated measurement of many analytes, preferably including a 5-methylcytosine screening, e.g. by fluorescent methods and the SMFS measurement on the 5-methylcytosine positive sites to analyse the methyla- tion properties, e.g. the distances between 5-methylcytosines from which the 5-methylcytosine density can be deduced. The chip to be used according to the pressingle resolutionent invention is preferably a chip, on which nucleic acids are immobilised (or are iirimobiIisable) which play an important role in methylation, e.g. human suppressor genes.

The present invention is further illustrated by the following figures and examples, without being restricted thereto. Figures :

Fig. 1. Anti-methylcytosine antibody is conjugated onto the cantilever tip via PEG crosslinker . ssDNA containing multiple methylcytosines is conjugated onto aldehyde glass via amino group on its 3 '-end. Two Fab-domains of the antibody can bind with two methylcytosines of the DNA. Pulling Fc-domain separates two Fab-domains from the DNA one after the other, causing two unbinding peaks in the force-distance curve, with which the distance between two methylcytosines can be measured.

Fig.2. Example force-distance curves with two unbinding events measured on the first DNA sample (with 9 methylcytosines separated by 6 nucleotides, panel A) and the second DNA sample (with 5 methylcytosines separated by 4 nucleotides, panel B) are shown with arrows indicating positions of unbinding events. The sketches show the possible binding position of antibody on DNA with the measured distance between two unbinding events. Red sticks in sketches are methylcytosines while blue ones are other nucleotides . Statistic distribution of the measured distance between two unbinding events of 150 force-distance curves from the first DNA sample (C) and of 259 curves from the second DNA sample (D) display guasi-equidistant peaks, the positions of which are listed in Table 1, which are in agreement with the distance between methylcytosines bound by Fab-domains.

Fig.3. The third DNA sample has six methylcytosines separated by 3, 8, 1, 8 and 3 nucleotides. The distance between methylcytosines can be 1, 3, 8, 9, 11, 12, 17, 20 or 23 nucleotides. From 298 force distance curves with two unbinding events, a distance distribution with eight peaks is obtained, corresponding to 3, 8, 9, 11, 12, 17, 20 or 23 nucleotides respectively.

Fig.4. Schematic representation of a multi-branched linker with 9 methylcytosine specific proteins on a AFM cantilever bound to methylated DNA. The ssDNA on the genechip functions as an anchor for the analyte DNA.

Fig.5. Schematic representation of a a SMFS set-up, wherein one polynucleotide analyte is immobilized on the probe tip and a - g _ multitude of methylcytosine specific antibody fragments is immobilised on the carrier via a lipid membrane.

Fig.6. Schematic representation of a a SMFS set-up, where a branched linker binds four methylcytosine specific antibody fragments to the probe tip and the polynucleotide analyte is immobilized on the carrier.

Example s :

Force spectroscopy measurements using anti-methylcytosine antibody tethered cantilevers on single-stranded DNA with methylcytosines conjugated on aldehyde glass revealed that two Fab-domains of the antibody could bind with two methylcytosines in the DNA. Pulling the Fc-domain separated Fab-domains from the DNA one after the other with a distance identical to the contour length of nucleotides between two methylcytosines. The system works like flexible molecular callipers, which can measure the distance between two particular bases .

Example 1. DNA samples

Three ssDNA samples were studied. The first one has nine methylcytosines separated by six nucleotides, the second one has five methylcytosines separated by four nucleotides, while the third one has six methylcytosines with different distances. The sequence of the DNA (5 '-3') is: AXTATGTXTATGTXTATGTXTATGTXTAT- GTXTATGTXTATGTXTATGTXA (synthesized by Metabion) for the first sample, ATXGATXGATXGATXGATXGTCCAGGAGCGCCC (VBC-Genomics) for the second one, ATGTXTTXTATGATGXXTATGATGXTGXTGATGATGATG (Metabion) for the third one, where X is methylcytosine and the 3 ' -end of the DNA is modified with an amino group which can be coupled to an aldehyde group on a glass slide.

Example 2. Method fox conjugation of DNA onto aldehyde glass

The DNA was diluted to a concentration of 50μM in SSC buffer (15OmM NaCl, 15mM tri-sodium citrate, pH7) with 2.5% glycerol. Sodium cyanoborohydride was added to the DNA solution at a concentration of 1OmM. The DNA solution was spotted onto the aldehyde glass and incubated in a humid chamber with an argon atmosphere for 6 hours. The unreacted aldehyde groups on the glass were inactivated by adding Tris into the reaction solution at a concentration of 5OmM. After 30min, the sample was washed with 10OmM NaHCO₃ (pH8.2), 0.1% SDS in 2^χSSC (pH7) and 0.1% SDS in 0.2*SSC (pH7) respectively, rinsed with water, and stored in argon. According to fluorescence data, the surface density of conjugated DNA on the aldehyde glass was about 200 molecules/μm².

Example 3. Method for conjugation of antibody through its lysine residue to cantilever tip

Anti-methylcytosine antibody (Serotec) was conjugated onto the cantilever (Thermomicroscopes, coated sharp microlevers) tip through the reaction between some lysine residue on the antibody and an aldehyde group of a cantilever-bound polyethyleneglycol (PEG) crosslinker. For this reaction, the antibody was diluted in buffer A (10OmM NaCl, 5OmM NaH₂PO₄, ImM EDTA, pH7.5) to a concentration of 0.2mg/ml. Sodium cyanoborohydride was added to the antibody solution as described above. The cantilever tip was immersed in the antibody solution for 1 hour. After incubation, the unreacted aldehyde groups on the cantilever tip were inactivated by adding ethanolamine into the reaction solution at a concentration of 5OmM.^~ After 30min, the cantilever was washed with buffer A. Cantilever tips prepared in this way were used to measure the DNA sample with 9 or 5 methylcytosines (Fig.2 in the report) .

Example 4. Method for conjugation of antibody through its carbohydrate residue to cantilever tip lOμl lmg/ml antimethylcytosine antibody (Serotec) was dia- lyzed with mini dialysis tube (Pierce) against 800ml 10OmM sodium acetate (pH5.5) at 4⁰C for 8 hours, and against the second 800ml 10OmM sodium acetate (pH5.5) at 4⁰C for 12 hours. The antibody solution (a little more than 60μl) was collected from the dialysis tube into a 0.5ml tube, βμl 10OmM NaIO₄ (freshly prepared in water) and 3μl 30OmM SPDP-Hydrazide (Molecular Bios- cience) in DMSO were applied to antibody solution. The reaction was kept in dark at 4⁰C for 80 min and afterwards quenched with ^βμl 5% glycerol in water for 10 min. Then, 7.5μl 0.5M DTT was applied for 10 min to cleave the disulfide. The solution was transferred into the same mini dialysis tube and dialysed against 500ml buffer A under argon protection at 0⁰C for 9.5 hours. Then, dialysis was continued in clean buffer A for 14.5 hours. About 130μl antibody solution was collected from the dia- lysis tube. Now, the antibody has reactive thiol group at the end of its carbohydrate residue on the Fc-domain. The coupling between antibodies and cantilever tips with PEG-PDP was performed under argon protection at room temperature for 1 hour, and subsequently at 4⁰C for 20 hours. Then, the cantilevers were washed in buffer A. Cantilever tips prepared in this way were used to measure the DNA sample with 6 methylcytosines (Fig.3) .

Example 5. Atomic force microscopy (AFM)

AFM cantilever tips with antibody conjugated through lysine residue were used for measurement in Fig.2, while cantilever tips with antibody conjugated through carbohydrate residue as linker were used for measurement in Fig.3.

Force spectroscopy was performed with an atomic force microscope (Molecular Imaging) in PBS (15OmM NaCl, 5mM Na₂HPO₄, pH7.5) containing 30μg/ml lysolecithin (Sigma) , which was used to prevent the unspecific binding between the antibody and the glass surface (16). The spring constant of cantilevers ranges from 0.01 to 0.03N/m. The scan range for force-distance curve measurement was fixed at 200nm, while cycle time was 0.25-4s. The quantity of data points for one cycle is 1000 for Fig.2 and 2000 for Fig.3.

The unbinding force between methylcytosine and its antibody was measured to be 58+13pN at a force-loading rate of 2nN/s. Some of the force-distance curves show only one unbinding event, while some curves contain two. From 17 cantilevers, the average percentage of curves containing two unbinding events is 2+1%. The percentage of curves containing one unbinding event is typically 33%. Some example curves with two unbinding peaks are shown in Fig.2. The arrows indicate the position of unbinding events. Curves in Fig.2A are from measurements on the first DNA sample, while curves in Fig.2B are from the second sample. The sketch beside the curve depicts the possible binding position of antibody on DNA. For ssDNA, the contour length of one nucleotide is 0.59nm for C3-endo structure, or 0.70nm for C2-endo structure (20) . Since the first DNA sample has nine methylcytosines with the separation of 6 nucleotides, the measured distance between two methylcytosines can range from 3.5nm to 33.6nm. From 150 curves with two unbinding events, the statistic distribution of the measured distance between two unbinding events is obtained in Fig.2C. There are 6 peaks in the distribution, the position of which is listed in Table 1. From the peak position, the contour length of single nucleotide was calculated. For 9 methyl- cytosines there should be theoretically 8 peaks in the distribution. Only 6 peaks were obtained in the experiment. Methylcytosines with shorter distance might be easier for two Fab-domains to bind together. They have higher probability also simply due to more number of pairing, e.g. two methylcytosines with a distance of 6 nucleotides have 8 pairs in the DNA, while with a distance of 12 nucleotides they have only 7 pairs, etc. The calculated average contour length of single nucleotide is 0.59nm to 0.65nm, which is in the reasonable range of prediction.

The second DNA sample has 5 methylcytosines separated by 4 nucleotides. Fig.2D displays the statistic result of data from 259 curves with two unbinding events measured by 4 cantilever tips. The position of peak 2, 3 and 4 coincides well with the contour length of nucleotides between methylcytosines. However, from the position of the first peak, the average contour length of one nucleotide was calculated as 0.88nm, which is much longer than the contour length of a C2-endo nucleotide. The abnormal longer length might be caused by the fact that the Fab-domain of the antibody has a certain width, so that there might be a minimum distance limit for the two antigen binding sites of the antibody when they get close. The maximum contour length of four stretched nucleotides is only 2.8nm. However, the orientation of cytosines can rotate freely. The molecular modeling and analysis software (CS Chem3D Pro, CambridgeSoft) reveals that the distance between two methyl groups separated by four nucleotides can be more than 4 nm, which may be large enough for two antigen binding sites of the antibody to bind together. When the antibody is pulled from the DNA, the lower methylcytosine and Fab- domain are pulled to approach the orientation of the pulling force direction. However, before this is achieved, the distance between two antigen binding sites of the antibody has already reached the minimum, so that the lower Fab-domain has to detach from the methylcytosine before it reaches the orientation of the pulling force. Thus, the distance between two unbinding events in this case measures the minimum distance limit of two epitopes of the antibody, which is reflected by the position of the first peak in Fig . 2D .

The third DNA sample has 6 methylcytosines separated by 3, 8, 1, 8 and 3 nucleotides. Therefore, the distance between methylcytosines can be 1, 3, 8, 9, 11, 12, 17, 20 or 23 nucleotides. The force curve measurements show that the distance between two unbinding events has a distribution with eight peaks (Fig.3, from 298 force distance curves with two unbinding events measured with one cantilever tip) , the position of which corresponds well to the contour length of nucleotides (Table 1), except the position of peak 1, which is similar to the case of the second DNA sample. Two methylcytosines separated by single nucleotide can not be directly detected from the force distance curve. However, from the statistic distribution, distance of 8 or 11 nucleotides can be distinguished from 9 or 12 nucleotides, which demonstrates the single nucleotide resolution of this method.

Table 1. Peak position in Fig.2 and 3 and average contour length of nucleotide

In conclusion, distance information of methylcytosines in ssDNA was obtained by using antibody tethered cantilever tips in force spectroscopy. The method developed in this study is further applicable to obtain sequence information in DNA. The antibody can be considered as a 1:2 crosslinker. For sequencing, one can use a 1:N crosslinker (Fig. 4). On the end of every crosslinker, a single chain variable fragment (scFv) or a Fab fragment of anti-methylcytosine antibody can be conjugated. Cantilever tip can be moved to the specific spot with the help of fluorescence microscope and atomic force microscope with recognition imaging function (28, 29). Therefore, the sequence information can be obtained directly on gene chips with single molecular sensitivity.

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Claims

Claims :

1. Method for the detection of 5-methylcytosine, characterized in that either a 5-methylcytosine specific binding moiety or a nucleotide analyte is attached to the tip of a single molecule force spectroscopy (SMFS) probe and the other of the 5-methylcytosine specific binding moiety or the nucleotide analyte is attached to a carrier, wherein the SMFS probe is brought into contact to the carrier and is detached and the detachment force is measured through a SMFS detection means.

2. Method according to claim 1, characterized in that the SMFS is an atomic force microscopy (AFM) , wherein preferably the detection means is a laser deflection means.

3. Method according to claim 1, characterized in that the SMFS is an optical tweezer method.

4. Method according to claim 1, characterized in that the SMFS probe and the carrier form a force apparatus.

5. Method according to claim 1, characterized in that the SMFS is a biomembrane force probe method.

6. Method according to any one of claims 1 to 5, characterized in that the 5-methylcytosine specific binding moiety is an, optionally modified, 5-methylcytosine specific antibody or antibody fragment, preferably a Fab, Fab' or scFv fragment.

7. Method according to any one of claims 1 to 6, characterized in that the 5-methylcytosine specific binding moiety is bound to the SMFS probe, preferably by a linker, most preferred by a glycosidic linker.

8. Method according to any one of claims 1 to 7, characterized in that the carrier is a glass slide, preferably an aldehyde glass slide.

9. Method according to any one of claims 1 to 8, characterized in that the nucleotide analyte is immobilized in spots onto the carrier .

10. Method according to any one of claims 1 to 9, characterized in that the nucleotide analyte on the carrier is analyzed by a fluorescent specific 5-methylcytosine assay prior to SMFS measurement .

11. Method according to claim 10, characterized in that more than one nucleotide analyte spot is on the carrier and the SMFS measurement is carried out for the spots, which were 5-methylcytosine positive in the fluorescence assay.

12. Method according to any one of claims 1 to 11, characterized in that the SMFS probe comprises two or more 5-methylcytosine specific binding moieties and is capable of binding two or more 5-methylcytosines on the nucleotide analyte.

13. Method according to claim 12, characterized in that through a measured SMFS force profile of the contacting and detachment of the SMFS probe, the distance of the two or more 5-methylcytosines on the nucleotide analyte is detected.

14. Method according to any one of claims 1 to 13, characterized in that the nucleotide analyte is a polynucleotide bound to the SMFS probe or the carrier through base pairing to an oligonucleotide, which is immobilized onto the SMFS probe or the carrier.

15. Single molecule force microscopy (SMFS) probe, comprising a 5-methylcytosine specific binding moiety attached to the SMFS probe, preferably by a linker moiety.

16. SMFS probe according to claim 15, characterized in that the 5-methylcytosine specific binding moiety is attached to the SMFS probe by a linker, preferably the linker is the Fc portion of an antibody or a polyvalent crosslinker.

17. SMFS probe according to claim 15 or 16, characterized in that the SMFS probe comprises two or more 5-methylcytosine specific binding moieties.

18. SMFS probe according to claim 15 or 16, characterized in that the SMFS probe is an AFM probe, preferably a tipped cantilever.

19. SMFS apparatus comprising an SMFS probe according to any one of claims 15 to 18 and a carrier, preferably being a chip.