RU2764807C1 - SET FOR THE DIAGNOSIS OF BLADDER CANCER USING C228T AND C250T MUTATIONS IN THE hTERT GENE PROMOTER AND THE METHOD FOR ITS IMPLEMENTATION - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. A set and method for the diagnosis of bladder cancer using C228T and C250T mutations in the promoter of the hTERT gene are proposed. The set includes a sensor and solutions of dye-labeled oligonucleotides with a detection limit of no more than 1 micron, GnuS-C228T-label and GnuS-C250T-label. The method involves taking a biological sample, where one part of the sample is mixed with a solution of the oligonucleotide GnuS-C228T-label, and the other part with a solution of GnuS-C250T-label, carrying out their hybridization, followed by the determination of DNA mutations C228T and C250T.EFFECT: inventions ensure the achievement of high accuracy in the diagnosis of bladder cancer in the early stages of tumor development.18 cl, 16 dwg, 2 tbl, 2 ex

Description

The present invention relates to the field of analytical and bioorganic research, is intended for the diagnosis of aggressiveness of glioma and the initial detection and recurrence of a bladder tumor, as well as the development of the field of translational research in the future for other applications by detecting mutations in the human telomerase reverse transcriptase gene promoter. The presented invention can be used in hematology and oncological diagnostics, in biochemical studies for molecular recognition, qualitative and quantitative detection of mutations in the human telomerase catalytic subunit gene promoter with high sensitivity, selectivity and rapidity.

State of the art

Human telomerase reverse transcriptase (hTERT) is an enzyme whose main function is to add specific repetitive nucleotide sequences to the ends of chromosomes that are shortened due to DNA underreplication. Normally, telomerase is active in stem, germ, and some other body cells that need to constantly divide, but in differentiated somatic cells, the expression level and telomerase activity are limited. Telomerase activation in somatic cells is a critical event for their transformation into tumor cells and is observed in 85% of cancer cases [Shay J. W., Wright W. E. Senescence and immortalization: role of telomeres and telomerase // Carcinogenesis. 2005. V.26. P. 867-874]. Bladder cancer is the most common cancer of the urinary system, and recent studies have shown that mutations in the hTERT promoter are the most common mutations at all stages of the disease. [Allory Y. et al. Telomerase reverse transcriptase promoter mutations in bladder cancer: high frequency across stages, detection in urine, and lack of association with outcome // Eur. Urol. 2014. V. 65. P. 360-366]. From the prior art solutions are known that allow molecular diagnostics of oncological diseases. One of the causes of cancer is the appearance of somatic mutations in the cell that disrupt its normal functioning. Some genetic changes are characteristic of a certain type of tumor lesion and can serve as markers of the disease. Detection and identification of damaged DNA regions allows for the prediction and reliable diagnosis of a wide class of diseases at an early stage. For example, it is known that the presence of certain somatic mutations in oncogenes determines the rate of development of certain oncological lesions. For example, in the presence of somatic mutations in the KRAS and BRAF oncogenes, colorectal cancer develops in the shortest possible time, quickly metastasizes, and is difficult to respond to chemotherapy. The presence of certain markers makes it possible to determine the exact area of the lesion and the stage of the disease, to predict the response to a certain type of therapy, and also provides the possibility of monitoring the course of the disease and recurrence. Currently, tissue biopsy is the standard procedure for selecting biological material for tumor analysis for the presence of specific markers, but this approach has a number of limitations. First, surgical intervention is necessary to obtain the material, which limits the frequency of this procedure. Secondly, the spatial heterogeneity of the tumor can lead to unreliable results in the detection of tumor markers, especially with a single test. Finally, there is a need to monitor the course of the disease over time, and for these purposes, frequent invasive intervention is not possible. It is also known that late diagnosis of cancer is one of the reasons that reduces the patient's chances of survival. The urgent need to use more accessible biomaterials for analysis, as well as the need to develop non-invasive diagnostic methods, led to the development of diagnostic approaches using freely circulating DNA (ssDNA), and more precisely, circulating tumor DNA (ctDNA) present in physiological fluids. Thus, the study of easily accessible fsDNA using highly sensitive methods opens up great opportunities for diagnosing patients with cancer. Early diagnosis of diseases and timely treatment require the determination of DNA with damaged fragments with high sensitivity and accuracy, since even a small change in the qualitative and quantitative composition of biological fluids can be caused by the onset of a wide range of serious diseases.

, U. Schneider, I. Lurje, K. Guricova, N.T.Gaisa, E. Lorsy, K. Lindemann-Docter, A. Heidenreich, R

. Next-generation sequencing reveals potential predictive biomarkers and targets of therapy for urothelial carcinoma in situ of the urinary bladder. Pathology. 2020. V. 190. P. 323-332], основано на секвенировании нового поколения (СНП) и позволяет анализировать одновременно миллионы копий цоДНК. Промотор гена TERT был проверен на наличие двух известных повторяющихся мутаций: С124 и С146C. Частота вариативности аллеля была установлена в 5%. В качестве эталонного генома для дизайна панели использовали hg19; hg38 служил эталонным геномом для аннотации мутаций.Currently used methods are based on the identification of DNA sequences by hybridization of complementary sequences with a fluorescent detection method. Most methods for the determination of small amounts of nucleic acids, as a rule, use the preliminary amplification of DNA fragments in the polymerase chain reaction (PCR). The prior art solution described in [S. Garczyk, N. Ortiz-

, U. Schneider, I. Lurje, K. Guricova, NTGaisa, E. Lorsy, K. Lindemann-Docter, A. Heidenreich, R

. Next-generation sequencing reveals potential predictive biomarkers and targets of therapy for urothelial carcinoma in situ of the urinary bladder. pathology. 2020. V. 190. P. 323-332], is based on next generation sequencing (NGS) and allows simultaneous analysis of millions of copies of ctDNA. The TERT gene promoter was tested for two known repeat mutations: C124 and C146C. The frequency of allele variation was set at 5%. hg19 was used as a reference genome for panel design; hg38 served as a reference genome for mutation annotation.

However, the proposed strategy, such as whole exome and whole genome sequencing, usually allows to generate from 30 to 100 DNA fragments, which leads to too low sensitivity for the analysis of rare mutations in sfDNA in the entire volume of csDNA. The SNP process is complicated in terms of setting up an experiment, time consuming, not very sensitive, and requires complex bioinformatics analysis.

The prior art solution described in [J. J. G. Pritchard, G. Hamilton, C. D. Hurst, S. Fraser, C. Orange, M. A. Knowles, R. J. Jones, H. Y. Leung, T. Iwata. Monitoring of urothelial cancer disease status after treatment by digital droplet PCR liquid biopsy assays. Urologic Oncology: Seminars and Original Investigations. 2020. doi: 10.1016/j.urolonc.2020.05.012], based on Sanger sequencing and ccPCR. Sanger sequencing revealed point mutations in 70% of patients whose biosamples were analyzed by ccPCR. Cases of remission and relapse, monitored by analysis of PIK3CA E542K and TP53 Y163C mutations in plasma and urine, coincided with clinical observations within 48 months from the start of chemotherapy. The developed technique based on ccPCR made it possible to detect a mutation in the telomerase reverse transcriptase (TERT) promoter (-124). The proposed method for detecting this mutation is able to distinguish the mutant from wild-type alleles.

However, for staging ccPCR, it is necessary to know in advance marker genetic and epigenetic changes, in addition, ccPCR has limited potential for multiplex analysis. The limit of detection of DNA isolated from tumor tissues for approaches such as Sanger sequencing does not exceed 5% (5% of the mutant allele against the background of wild-type sequences).

The solution known from the prior art, described in the patent of the Russian Federation No. 2463354 dated October 10, 2012 “A method for diagnosing bladder cancer using the tfdp1 oncomarker (variants) and a kit for its implementation”, consists in obtaining a biomaterial and isolating ribonucleic acids (RNA), synthesizing complementary deoxyribonucleic acids (cDNA) on an RNA template, normalizing the concentration of TFDP1 cDNA by the control gene, performing quantitative PCR amplification of the TFDP1 gene fragment. Next, the amount of the amplified TFDP1 DNA fragment for the biomaterial sample is determined. When the cDNA concentration of the TFDP1 gene in physiological fluids exceeds 1.5% of the cDNA concentration of the ACTV beta-actin gene, the presence of transitional cell carcinoma of the bladder is diagnosed. The proposed solution includes a kit for implementing the described method by PCR, including two primers with a specific sequence at a molar ratio of 1:1. The variant method provides for diagnostics by enzyme-linked immunosorbent assay (ELISA). Urine and blood samples of the patient are obtained, a mixture of protein components of urine and blood is isolated, ELISA is performed with monoclonal antibodies and/or polyclonal antibodies and/or their fragments against the recombinant TFDP1 protein and/or its unique fragments longer than 8 amino acids. Bladder cancer is diagnosed when the concentration of the TFDP1 protein in the test samples exceeds 5-fold the concentration of the TFDP1 protein in the control. The method (version) makes it possible to reliably diagnose bladder cancer, including at an early stage of progression of tumor transformation.

However, in the proposed solution, the diagnosis of bladder cancer is based on the content of matrix ribonucleic acids (mRNA) of the TFDP1 gene and/or the TFDP1 protein in the blood and urine. In addition, the described solution is characterized by all the limitations associated with the use of PCR: (1) sensitivity directly depends on the efficiency of PCR amplification of a DNA fragment of a sample, which decreases sharply in the presence of DNA damage or fragmentation; (2) if it is necessary to determine several target molecules multiplexly and against the background of a large amount of DNA with a similar sequence, the accuracy of PCR becomes unsatisfactory for making a diagnosis with a significant decrease in the sensitivity of the analysis; (3) an increasing detection limit (LOD) of mutations leads to an increase in the probability of obtaining false positive results; (4) significant DNA fragmentation will depend on sampling and sample preparation procedures; (5) for detection of fragments with a length of less than 50 nucleotides, the isolation and specific hybridization of primers for amplification is practically impossible; (6) identification of the DNA sequence by PCR methods prevents the formation of non-canonical structures by the DNA template, such as G(guanine)-quadruplexes.

At the moment, giant Raman spectroscopy (GRS spectroscopy) is a possible effective analytical tool and has great potential in the field of studying biomolecules, in particular nucleic acids. The main advantages of SERS spectroscopy as applied to the analysis of biomolecules, in comparison with existing methods, are (1) the high sensitivity of the method, up to the detection of single molecules; (2) SERS signals contain information about the molecular fingerprints of all components of the analyzed biological system; (3) SERS spectroscopy is resistant to photodegradation and quenching compared to fluorescence methods and is suitable for long-term monitoring; (4) the width of the peaks is 10-100 times smaller compared to the peaks of the fluorescence spectra of organic labels and quantum dots, which makes it possible to perform multiplex analysis at one excitation wavelength; (5) SERS-active nanosubstrates can be selected and designed with different coatings, different shapes and sizes depending on the analytical goals. Many studies have shown the fundamental possibility of using direct Raman spectroscopy to detect single nucleotide substitutions [Xu LJ, Lei ZC, Li J., Zong C., Yang CJ, Ren, B. Label-Free Surface-Enhanced Raman Spectroscopy Detection of DNA with Single -Base Sensitivity // Journal of the American Chemical Society. 2015. V. 137. P. 5149-5154]. However, SERS spectra of DNA without the use of reporter molecules were obtained predominantly using synthetic oligonucleotides. The interpretation of SERS spectra obtained from the analysis of clinical samples is still associated with a number of difficulties, since signals corresponding to a large amount of non-target genetic material can prevail over weak signals of target molecules contained in low concentrations in the sample under study. Another approach to detecting an analyte, indirect SERS spectroscopy, is to use SERS-active reporter molecules and record the signal change that occurs as a result of the interaction of the SERS-active molecule with the analyte. This method overcomes the difficulties that often arise when recording intrinsic vibrational spectra of nucleic acids, namely, the difficulty of interpreting the results in the presence of a large number of non-target molecules or genetic material, if we talk about the analysis of clinical samples. On the other hand, information about the structural rearrangements and chemical features of the analyzed molecules, which is contained in the intrinsic characteristic SERS spectra, is lost.

Closest to the claimed is a method for measuring cancer-related substances, including free DNA derived from cancer cells, using Raman spectroscopy (RU2723160, 06/09/2020). The proposed method for the determination of DNA in biological fluids consists in the preparation of noble metal nanoparticles acting as an SERS enhancer; hybridization of the target with a directionally selected covalently GKR-labeled complementary oligonucleotide; applying a sample with a test compound to the surface of nanoparticles of a liquid sample with a test compound to form a directionally ordered structure due to non-covalent interactions, including a DNA to be determined, a SERS label, and an oligonucleotide; detection of the resulting hybridized SERS-labeled duplex by giant Raman spectroscopy, and the qualitative and quantitative content of DNA is judged by the position and intensity of the bands in the recorded spectra. The method is applicable for the determination of small amounts of DNA against the background of a large number of extraneous interfering components of analyzed real biological fluids: blood, urine, cerebral fluid, etc. This result is achieved due to additional selective binding of DNA fragments with directionally selected covalently SERS-labeled oligonucleotides on the surface of nanoparticles. In addition, due to directional structuring and ordering, as well as control of the distance from the SERS label to the surface of the nanoparticles, an enhanced SERS signal is recorded that is reproduced over the entire sensor surface. Raman signal amplification by 10 ³ - 10 ¹⁴ times is achieved due to additional wrapping of the hybridized DNA duplex around metal nanoparticles acting as an SERS amplifier due to non-covalent interactions. The proposed method allows you to reliably quantify the necessary DNA at the level of 10 ^-14 M.

However, the known method proposes the determination of a mutant DNA sequence that does not allow diagnosing bladder cancer. For reliable detection and determination of C228T and C250T mutations at the level of ultra-low concentrations, which will allow diagnosing the disease at early stages (up to about 6,000 damaged fragments in the analyzed sample), it is necessary to use a certain working distance from the SERS label to the metal nanostructure. In the absence of a mutant DNA sequence, the free probe forms a hairpin. In the presence of a mutant DNA fragment, the determined sequence interacts with the complementary sequence of the probe, the hairpin opens, as a result of which a sharp increase in the intensity of the SERS signal is observed compared to the situation when the label was in the hairpin. At the same time, the use of a complementary probe sequence with any length from the range of 1-5 nm does not lead to the possibility of highly sensitive detection of C228T and C250T mutations at the level of ultra-low concentrations. Moreover, the invention describes an analysis method and its principle without an optimized GKR platform, which makes it possible to reliably determine biomarkers - mutant DNA sequences C228T and C250T.

The technical problem is the lack of kits and simple methods for diagnosing bladder cancer in the early stages of tumor development.

Disclosure of invention

The technical result of the claimed group of inventions is the development of a kit that makes it possible to diagnose bladder cancer in the early stages of tumor development with high accuracy.

The claimed group of inventions allows the identification of two mutant DNA sequences C228T and C250T and their selective determination in a biological sample to detect bladder cancer (1) in patients at early stages: starting from stage I (at the initial stages of the tumor process: stage T1 or more superficial tumors) when the tumor is non-invasive; (2) with high sensitivity (low detection limit from a concentration of 1⋅10 ^-14 M, which is approx. 6,000 damaged DNA fragments); (3) selectivity for other DNA fragments; and (4) rapidity (time for sample preparation of several samples simultaneously - up to 60 min and the analysis itself - 10 s).

Diagnosis of bladder cancer is carried out by selective recognition and detection of C228T and C250T mutations in the DNA sequence, which includes the use of a set of planar nanostructured SERS sensor and aqueous solutions labeled with a dye with a detection limit of no more than 1 μM, for example, rhodamine 6G, oligonucleotides : GnuS-C228T-R6G and GnuS-C250T-R6G (where Gnus is a sequence consisting of 15 to 20 amino acids, of which at least 20% are cytosine (C) and guanine (G), for example, sequences SEQ ID No. 1 - No. 4). Rhodamine, cyanine, xanthene and diazo dyes, methylene blue and crystal violet are used as a dye with a detection limit of not more than 1 μM.

The method consists in taking the patient's urine, then taking 20 μl of a liquid sample for hybridization of the mutant C228T and C250T DNA sequences supposedly contained there with their complementary oligonucleotides GnuS-C228T-tag or GnuS-C250T-tag, covalently modified with a commercially available GKR-tag - for example, rhodamine 6G (R6G). Next, 10 μl of the resulting mixture is applied to the planar sensor surface. The immobilization of the probe in the present invention is based on a specific electrostatic interaction between the Gnus anchor oligonucleotide sequence, which is part of the GnuS-C228T-tag and GnuS-C250T-tag probes, and metal nanoparticles. Due to the high binding energy of silver clusters with cytosine (C) and guanine (G), which are part of the anchor sequence, which is several times higher than the formation energy of Watson-Crick pairs, silver particles are inside the anchor sequence. This interaction, in contrast to covalent crosslinks, provides additional surface structuring, which ensures the formation of “hot spots” and signal amplification. Amplification of the Raman signal by a ^{factor of 10 2} - 10 ^{8 is} achieved by creating additional "hot spots" on the surface due to the fact that the silver particles are inside the anchor sequence. As part of the indicator system for detecting mutations C228T and C250T in the promoter of the hTERT gene, a construct consisting of a deoxyoligonucleotide was used, which ensures the immobilization of the probe on the sequence of silver nanoparticles (Gnus), which is associated with the sequence with the GRS tag (R6G), complementary to the analyte sequence (Fig. eleven). The proposed approach makes it possible to use the SERS spectroscopy method with high sensitivity coefficients (up to 10 ⁵ -10 ⁶ c.u.), low detection limits (LOD up to 3 fM), high signal-to-noise ratio (no effect of the matrix of real samples on useful signals), high selectivity (with respect to intact DNA fragments or mutations of other origin), a wide range of determined concentrations (DOC from 1⋅10 ^-14 to 1 10 ^-7 M), high reproducibility and precision (correlation coefficients of calibration dependences is at least 0.97, relative standard deviation at the lower detectable concentration is not more than 0.10) for the qualitative and quantitative determination of C228T and C250T mutations in order to diagnose oncological diseases. Due to directional structuring and ordering, as well as control of the distance from the SERS label to the surface of the nanoparticles, an enhanced SERS signal reproduced over the entire sensor surface is recorded. The proposed method for determining DNA demonstrates the absence of signals in the giant Raman spectra for normal DNA sequences and mutations of a different type, which makes it possible to reliably selectively quantify C228T and C250T mutations characteristic of bladder cancer with LO up to 10 ^-15 M.

The technical result is achieved by the claimed set, consisting of (1) a solid-phase planar sensor: a dielectric chemically inert material and a nanostructured coating based on noble metal nanoparticles 0.1-10 µm thick, and (2) 1 µM aqueous solutions labeled with a dye with a detection limit of not more than 1 µM oligonucleotides: GnuS-C228T-tag and GnuS-C250T-tag (where Gnus is a sequence consisting of 15-20 amino acids, of which at least 20% are cytosine (C) and guanine (G), for example, the sequences of SEQ ID No. 1 - No. 4) for the detection and determination of C228T and C250T DNA mutations characteristic of bladder cancer in biological samples. The technical result is achieved by a method for detecting and determining C228T and C250T DNA mutations, which consists in (1) applying a preliminarily prepared liquid sample obtained by hybridization of a solution of the analyzed patient's biological fluid with a complementary oligonucleotide GnuS-C228T-tag or GnuS-C250T-tag , covalently modified with a commercially available SERS label; (2) irradiation with a monochromatic laser beam with a wavelength of 485 - 785 nm and registration of a Raman signal, while the qualitative and quantitative content of C228T and C250T DNA mutations in the studied sample is judged by the position and intensity of the bands on the recorded spectra, characteristic of the dye label .

At the same time, silver, gold, and platinum are used as nanoparticles of noble metals.

The nanoparticles are 20–90 nm in size and are characterized by a plasmon resonance band of 420–650 nm.

In this case, silicate glass is used as a carrier material for nanoparticles.

In this case, 10-500 µmol of noble metal is used per 1 mm ^{2 of} the support surface, followed by drying.

At the same time, a surface of 4×4 mm ^{2 is} used for applying a sample and a single analysis.

In this case, the amount of GKR-labeled oligonucleotide is used in a molar ratio of 1:1 to C228T and C250T DNA mutations.

When this hybridization is carried out by heating and keeping for 5-10 min at a temperature of 95°C, followed by cooling to room temperature.

In this case, the prepared liquid samples are applied by dropping onto the surface of the nanoparticles.

In this case, liquid samples with a volume of 10 μl are used with a concentration of C228T and C250T DNA mutations of 10 ^-15 - 10 ^-2 M.

In this case, when performing the analysis, monochromatic laser radiation with a wavelength of 514 nm and a power not exceeding 25% of the nominal radiation value is used for up to 10 - 60 s.

At the same time, the qualitative and quantitative content of C228T and C250T DNA mutations in the sample is judged by the position and intensity of the bands at 1385, 1470 and 1590 cm ^-1 with an allowable error of not more than 10 cm ^-1 .

When using rhodamine 6G as a dye label, the presence and content of C228T and C250T DNA mutations in the sample is judged by the position and intensity of the bands at 1385, 1470 and 1590 cm ^-1 with an allowable error of not more than 10 cm ^-1 .

To determine the quantitative content of mutations, calibration curves are preliminarily built.

The presented method is suitable for use with portable, commercially available, off-the-shelf equipment outside of the laboratory. Thus, using the proposed optical planar sensor, highly sensitive and selective qualitative and quantitative determination of C228T and C250T DNA mutations present in the sample is possible at the bedside.

Thus, the use of the proposed method of analysis allows you to learn about the presence of mutations C228T and C250T and judge their quantitative content with high selectivity, a wide range of detectable concentrations, high reproducibility and precision.

Brief description of the drawings

The invention is illustrated by drawings.

In FIG. 1 shows fragments of cloned inserts. Underlined letters are nucleotides corresponding to the wild type.

In FIG. 2 shows A) selection of conditions for PCR amplification of the hTERT gene promoter with human genomic DNA (the target product is highlighted in a box); B) PCR amplification products of the insert from the genomes of HepG2 and A375 cell lines.

In FIG. 3 is a map of the pUC19 vector used, with HindIII and KpnI cloning sites marked.

In FIG. 4 shows Sanger sequencing chromatograms for insert fragments. Mutant nucleotides are highlighted by a box - 4b; nucleotides corresponding to the wild type are highlighted in box - 4a.

In FIG. 5 shows a scheme for detecting single nucleotide substitutions using fluorescent TaqMan probes. FAM-tagged probes are complementary to mutant-type sequences, R6G-tagged probes are complementary to wild-type sequences.

In FIG. 6 shows the chemical structures of the fluorescent labels used and the wavelengths corresponding to the fluorescence emission maximum.

In FIG. Figure 7 shows a diagram of the structure of a fragment of the promoter region of the hTERT gene containing sites -124 and -146 from ATG (marked with circles), in which mutations C228T and C250T are possible [Zvereva M., Pisarev E., Hosen I., Kisil O., Matskeplishvili S., Kubareva E., Kamalov D., Tivtikyan A., Manel A., Vian E., Kamalov A., Ecke T., Calvez-Kelm FL Activating telomerase TERT promoter mutations and their application for the detection of bladder cancer. Int. J. Mol. sci. 2020, 21(17), 6034]. G-chain is marked in blue, C-chain is marked in red. The regions corresponding to the primers with which this fragment was amplified are underlined. The structure was obtained using the QGRS Mapper program with the following parameters: the maximum length of the G-quadruplex was 30 nucleotides, the minimum size of the nucleotide group was 2 nucleotides, and the loop size was from 0 to 36 nucleotides.

In FIG. 8 shows a) the result of ccPCR, shown as a two-dimensional mapping of the signal in two independent channels. The ratio of mutant-type plasmids to wild-type plasmids in this experiment pUC19_235_C228T : pUC19_235_wt = 1:4; b) ccPCR result for the system with 100% pUC19_235_wt plasmid content. Above the threshold line, there are no droplets corresponding to the mutant plasmid pUC19_235_C228T.

In FIG. Figure 9 shows the levels of analytical sensitivity of the method on the example of the detection of the mutant plasmid pUC19_235_C228T against the background of a high content of the wild-type plasmid pUC19_235_wt. On the abscissa axis, the ratio of the number of mutant-type plasmids to wild-type plasmids. The concentration of the wild-type plasmid in all cases is ~5000 copies in the reaction mixture. Titration by concentrations of the plasmid containing the C228T mutation with an initial concentration of ~2500 copies in the reaction mixture with a 2-fold step. The detection sensitivity was 2.03%.

In FIG. 10 shows optical micrographs of the surface of silver nanoparticles at various objective magnifications.

In FIG. 11 shows a) a schematic representation of a system for detecting fragments of the hTERT gene promoter sequence, consisting of a probe immobilized on the surface of nanoparticles labeled R6G due to the Gnus anchor sequence; b) sequence sequences of Gnus, GnuS-C250T-R6G - a probe for detecting a sequence with a C250T mutation and GnuS-C228T-R6G - a probe for detecting a sequence with a C228T mutation. The sequence providing immobilization is highlighted in blue. Areas that form a duplex with analytes are highlighted in green.

In FIG. Figure 12 shows the SERS spectra obtained using λ _ex = 514 nm for the GnuS-C228T-R6G probe deposited in a volume of 10 μl and at concentrations of 10 ^-8 - 10 ^-14 M on the planar surface of silver nanoparticles. The numbers on the signature correspond to the negative decimal logarithm of the sample concentration: 8 - 10 ^-8 M, 10 - 10 ^-10 M, 12 - 10 ^-12 M, 14 - 10 ^-14 M. The positions of the characteristic peaks are marked with frames.

In FIG. Figure 13 shows the SERS spectra obtained using λ _ex = 514 nm for the GnuS-C228T-R6G system with A228 deposited in a volume of 10 μl and at concentrations of 10 ^-8 - 10 ^-14 M on the planar surface of silver nanoparticles. The numbers on the signature correspond to the negative decimal logarithm of the sample concentration: 8 - 10 ^-8 M, 10 - 10 ^-10 M, 12 - 10 ^-12 M, 14 - 10 ^-14 M. The positions of the characteristic peaks are marked with frames.

In FIG. Figure 14 shows the SERS spectra obtained using λ _ex = 514 nm for the GnuS-C250T-R6G probe deposited in a volume of 10 μl and at concentrations of 10 ^-8 - 10 ^-14 M on the planar surface of silver nanoparticles. The numbers on the signature correspond to the negative decimal logarithm of the sample concentration: 8 - 10 ^-8 M, 9 - 10 ^-9 M, 10 - 10 ^-10 M, 11 - 10 ^-11 M, 12 - 10 ^-12 M, 13 - 10 ^-13 M, 14 - 10 ^-14 M. The positions of the characteristic peaks are marked with blue frames.

In FIG. Figure 15 shows the SERS spectra obtained using λ _ex = 514 nm for the GnuS-C250T-R6G system with A250 deposited in a volume of 10 μl and at concentrations of 10 ^-8 - 10 ^-14 M on the planar surface of silver nanoparticles. The numbers on the signature correspond to the negative decimal logarithm of the sample concentration: 8 - 10 ^-8 M, 9 - 10 ^-9 M, 10 - 10 ^-10 M, 11 - 10 ^-11 M, 12 - 10 ^-12 M, 13 - 10 ^-13 M, 14 - 10 ^-14 M. The positions of the characteristic peaks are marked with blue frames.

In FIG. 16 shows a) secondary structures formed by the GnuS-C228T-R6G probe sequence. The R6G label is located at the 3' end of the chain; b) the place of the most probable formation of the G-quadruplex.

Implementation of the invention

In the present invention, the following designations and terms are adopted:

HPLC - high performance liquid chromatography,

GRS - giant Raman scattering,

SERS spectrum - Raman scattering spectrum with signals amplified due to the effect of surface plasmon resonance on noble metal nanostructures,

DMAA - dimethylamylamine,

DMSO - dimethyl sulfoxide,

DMF - dimethylformamide,

DNA - deoxyribonucleic acid,

DOC - range of determined concentrations,

ELISA - enzyme immunoassay,

cDNA - complementary deoxyribonucleic acid,

mRNA - matrix ribonucleic acid,

SNPs - silver nanoparticles,

RP-HPLC - reverse phase HPLC,

ON - limit of detection,

Promoter - a region of the oligonucleotide sequence recognized by RNA polymerase as a launching pad for the start of transcription,

PCR - polymerase chain reaction,

RNA - ribonucleic acid,

SNP - next generation sequencing,

SERS spectrum - Raman scattering spectrum with signals amplified due to the effect of surface plasmon resonance on noble metal nanostructures,

scDNA - free circulating DNA deoxyribonucleic acid,

Tris-HCl - a solution of trisaminomethane with hydrochloric acid,

ccPCR - digital drop polymerase chain reaction,

cDNA - circulating tumor deoxyribonucleic acid,

EDTA - sodium ethylenediaminetetraacetate,

A - adenine,

ACTB - English. actin, cytoplasmic a - a highly conserved protein that polymerizes to form filaments that form cross-linked networks in the cytoplasm of cells,

BRAF - Serine/threonine protein kinase B-raf; English Serine/threonine-protein kinase B-raf; CF: 2.7.11.25,

C - cytosine,

ESI-MS - electrospray ionization mass spectrometry,

FAM - fluorescein (3',6'-dihydroxy-3H-spiro[2-benzofuran-1,9'-xanthene]-3-one),

G - guanine,

hTERT - reverse transcriptase telomerase,

KRAS is a proto-oncogene, a member of the Ras protein family (this is a family of genes, as well as the proteins they encode - small G proteins),

PAGE - denaturing polyacrylic gel,

R6G - rhodamine 6G (ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate hydrochloride),

SERS - English. surface enhanced Raman spectroscopy,

T - thymine,

TAMRA - tert-butylamine/water solution, 1:3 v/v,

TBTA - tris(benzyltriazolyl)amine,

TFDP1 - English. Transcription Factor Dp-1 is a gene encoding a protein. Diseases associated with TFDP1 include colorectal cancer and hepatocellular carcinoma.

Chemical symbols/abbreviations have their usual meanings: °C (degree(s) Celsius), nm (nanometer(s)), µm (micrometer(s)), cm (centimeter(s)), min(minute(s)) , s (second (seconds)), µl (microliter (microliters)), µg (micrograms (micrograms)), M (moles (moles) per liter), l (liter (liters)), ml (milliliter (milliliters)) , µl (microliter (microliters)), g (gram (grams)), mg (milligram (milligrams)), mol (moles), mmol (millimoles (millimoles)), wt. % (mass percent (mass percent)), m.m. (molecular mass).

The following examples of specific implementation of the invention are provided to provide specialists in the art with a complete description of the conduct and application of the assay according to the invention, but do not limit the scope of the invention intended by the inventors.

All of the following reagents are commercially available. All procedures, unless otherwise noted, were carried out at room temperature or ambient temperature, that is, in the range from 18 to 25°C. In the course of all experiments, high-purity deionized water with a resistivity of at least 18.2 MΩ cm, purified using a Milli-Q, Millipore unit, was used to prepare aqueous solutions and wash suspensions. SERS spectra were recorded using an InVia Reflex microscope (Renishaw, UK) in the confocal mode using an Ar+ laser (wavelength 514.5 nm, 20 mW). The transmission of the broadband neutral density filter was 10% for the SERS spectra. Signal accumulation time - 10 s. The device was adjusted using single-crystal silicon wafers as a standard. The focal length of the lens used is 250 mm, the maximum size of the laser spot on the object is approx. 300 µm. All spectra were taken using a 50x Leica objective.

The claimed test kit consists of two key components: a planar nanostructured SERS sensor and solutions labeled with a dye with a detection limit of not more than 1 μM, for example, rhodamine 6G, oligonucleotides: GnuS-C228T-label and GnuS-C250T-label (where Gnus is the sequence, consisting of 15 to 20 amino acids, of which at least 20% are cytosine (C) and guanine (G), for example, sequences of SEQ ID No. 1 - No. 4). The use of the proposed kit consists in applying to the planar sensor surface a preliminarily prepared liquid sample obtained by hybridization of a solution of the analyzed biological fluid of a patient with a complementary oligonucleotide GnuS-C228T-tag or GnuS-C250T-tag, covalently modified with a commercially available SERS-label; at the same time, the qualitative and quantitative content of C228T and C250T DNA mutations in the studied sample is judged by the position and intensity of the bands on the recorded SERS spectra.

Example 1 Method for Selective Detection of C228T and C250T Mutations by SERS Spectroscopy

Synthesis of nanostructured silver surface. As an SERS amplifier, a planar optical SERS sensor was used, which is silver nanoparticles 0.4–1.5 μm thick deposited on the surface of a thin glass plate. Obtaining a nanostructured silver surface was carried out by pyrolysis of an aerosol of an ammonia complex. To 100 ml of a freshly prepared 0.17 M aqueous solution of silver nitrate (2.93 g) was added dropwise from a glass rod with constant stirring ~30 ml of a 0.1 M aqueous solution of sodium hydroxide until a black-brown precipitate of silver oxide (I) completely precipitated. Then the Ag ₂ O precipitate was thoroughly washed with deionized water (three times with 100 ml of water), then 100 ml of water and 7 ml of a concentrated aqueous ammonia solution were added dropwise with constant stirring to form an aqueous solution of the ammonium silver complex. After complete dissolution of the precipitate, 25 ml of a concentrated aqueous ammonia solution was additionally added to prevent precipitation of silver oxide (I) during the aerosol spraying of the ammonia complex. Larger amounts of ammonia significantly worsened the microstructure of the silver rings formed in the course of pyrolysis. The solution was diluted to 170 ml with water. The resulting transparent 0.1 M solution of the silver complex was filtered through a membrane filter, then diluted 8 times with water to a concentration of [Ag(NH ₃ ) ₂ ]OH of 12.5 mM. 50 ml of the final solution was transferred to the container of the ultrasonic aerosol inhaler. Two coverslips were pre-washed with piranha etching solution (H ₂ SO ₄ : H ₂ O ₂ = 3 : 1) and heated at the bottom of the beaker to 290 - 320°C. An adapter pipe with a diameter of ~1 cm was installed on the nebulizer. The spray parameters were used: 7 for “US” and 1 for “Air”. Thus, silver was deposited on the surface of the coverslip with an ultrasonic silver shower of silver(I) ammonia complex. Aerosol droplets with a diameter of 1-5 mm served as microreactors and ensured the formation of ring clusters of silver nanoparticles with a diameter of 90±5 nm (Fig. 10). The morphology of the obtained sensory nanostructured surfaces was controlled by the method of scanning electron microscopy with field emission. An important property of the proposed silver sensors was the implementation of a wide plasmon resonance excitation band, which covered the entire visible wavelength range, starting from 420 nm. This made it possible to obtain a resonantly enhanced SERS signal from a wide range of labels, including rhodamine 6G (R6G), fluorescein (FAM), and cyanine (Cy3, Cy5, etc.) (Fig. 6). To obtain a resonantly enhanced SERS signal, it is necessary that the region of plasmon resonance excitation frequencies overlap with the absorption band of the SERS label. Since commercially available argon and helium-neon lasers emit in the visible light region with the strongest intensity at wavelengths of 514.5 and 633 nm, respectively, rhodamine 6G (λ _max ≈ 524 nm) was chosen as the most suitable SERS-active reporter molecule.

Any rough metal surface characterized by a roughness of at least 2 nm and having the effect of surface plasmon resonance can be used as a planar sensor element to amplify the Raman signal, which was confirmed by the example of silver and gold.

The most promising strategy for creating systems for detecting nucleic acid sequences using SERS spectroscopy is to use the developed analytical system, in which target DNA is detected due to complementary interactions of the target with SERS-labeled probe immobilized on the surface of metal nanoparticles.

Synthesis of labeled oligonucleotide sequences. To obtain labeled oligonucleotide sequences, an automated synthesis technique was used, previously described and given in the authors' works "Method for detecting and determining DNA with a given sequence by giant Raman spectroscopy" RF Patent No. 2723160 of 06/09/2020; VM Farzan, EA Ulashchik, YV Martynenko-Makaev, MV Kvach, IO Aparin, VA Brylev, TA Prikazchikova, SY Maklakova, AG Majouga, AV Ustinov, GA Shipulin, VV Shmanai, VA Korshun and TS Zatsepin. Automated solid-phase click synthesis of oligonucleotide conjugates: From small molecules to diverse n-acetylgalactosamine clusters. Bioconjug. Chem. 2017. V. 28. P. 2599-2607]. 5'-Alkyne-modified oligonucleotides were obtained in an ASM-2000 (Biosset) or MM-12 (Bioautomation) oligonucleotide synthesizer using the phosphoramide method. Protected 2'-deoxyribonucleoside-3'-phosphoramidites, Unylinker-CPG (500 Å) and S-ethylthio-1H-tetrazole were purchased from ChemGenes. A solution of azide 6-R6G (100 mM) and copper catalyst CuI⋅P(OEt) ₃ (100 mM) in an aprotic bipolar solvent (dimethyl sulfoxide (DMSO), dimethylformamide (DMFA) or dimethylamylamine (DMAA), 100 μl) was added to 5' -alkyne-modified oligonucleotides bound to the solid phase in the column (1 μmol) 5 times every 30 min. After 3 h, the solid support was thoroughly washed (10 x 150 µl DMSO, then 3 x 150 µl acetonitrile) followed by deprotection of the cyanoethyl groups (2 x 10% diethylamine in acetonitrile (10 min) and complete deprotection using a mixture of TAMRA (tert -butylamine/water, 1:3 v/v) at 55° C. overnight The solution was evaporated and an aliquot analyzed by high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS).

The resulting oligonucleotides were purified twice with denaturing polyacrylic gel (PAGE) followed by reverse phase HPLC and (RP-HPLC). Gel electrophoresis of denatured oligonucleotides was performed in 15% PAGE containing 7 M urea in Tris-borate buffer (50 mM Tris-HCl, 50 mM boric acid, 1 mM sodium ethylenediaminetetraacetate (EDTA), pH 8.3). Oligonucleotides were recovered from the gel by electroelution with Elutrap (Whatman) in Tris-borate buffer (5 mM Tris-HCl, 5 mM boric acid, 0.1 mM EDTA, pH 8.3). HPLC analysis and purification of oligonucleotides were performed using a 2.1 × 50 mm Jupiter C18 chromatographic column (5 μm, Phenomenex), buffer A: 10 mM diisopropylamine and 15 mM 1,1,1,3,3,3-hexafluoroisopropanol and buffer B: 10 mM diisopropylamine, 15 mM 1,1,1,3,3,3-hexafluoroisopropanol and 80% acetonitrile. The salts were washed with buffer A (4 cycles) and then with 100% buffer B (2 cycles) at a flow rate of 0.3 ml/min and a temperature of 45°C. The structure of the obtained oligonucleotides was confirmed by ESI-MS using an Agilent 1260-Bruker Maxis Impact system. Mass spectrometric analysis of oligonucleotides was carried out in the negative mode (capillary voltage 3500 V), and the recorded spectra were recalculated by the maximum entropy method.

The resulting 5'-alkyne-modified oligonucleotide was modified with small molecules of SERS indicators. The resulting oligonucleotide was prepared in the solid phase and associated with 5'-alkynephosphoramidite. Click modification was performed on the solid phase by adding 10 mM azide solution, 100 mM CuI⋅P(OEt) ₃ 4×100 μl (azide 2 eq.) for 4 h followed by washing. In most cases, oligonucleotides were cleaved from the carrier and deprotected in the synthesizer using 1,2-ethylenediamine/toluene, 1:1 v/v for 2 h at room temperature followed by a wash (4 x 150 µl acetonitrile) and elution 50% acetonitrile in water or deprotection using an AMA solution (concentrated aqueous ammonia and 40% aqueous methylamine, 1:1 v/v) for 30 min at 65°C. For R6G-labeled oligonucleotides, deprotection was performed using a TAMRA solution at 55° C. overnight. For FAM-labeled oligonucleotides, two deprotection schemes were used (concentrated aqueous ammonia at room temperature for 2 and 18 hours). To switch the phase of the solution, the 5'-alkyne-modified oligonucleotide (2 nmol) was dissolved in 1 M triethylammonium acetate (30 μl). Then 20 µl of a copper catalyst solution in 60% DMSO (15 mM tris(benzyltriazolyl)amine (TBTA) and 13 mM copper(II) sulfate) was added, followed by 4 µl of 10 mM FAM azide in DMSO and 140 µl of 80% DMSO. The solution was degassed and filled twice with argon in a vacuum centrifuge concentrator, and then a degassed solution of ascorbic acid (0.25 M, 6 μl) was added. The reaction mixture was left overnight at room temperature, and the oligonucleotide product was precipitated with 4% LiClO ₄ in acetone (1 ml). Purification and HPLC analysis were performed as described above.

et al. Fluorescence-tunable Ag-DNA biosensor with tailored cytotoxicity for live-cell applications // Sci. Rep. 2016. V. 6. P. 37897]. В приведенных примерах в качестве последовательности GnuS рассматривали 5’-TGCCTTTTGGGGACGGATA-3’. При этом при использовании иных последовательностей GnuS (5’-AATGATCTTAACCAAGTATA-3’, 5’-TCGGTTGGGGAGCCTAT-3’ и 5’-TGGCTAATCCCCTCGGATA-3’), т.е. при сохранении длины зонда от 15 до 20 нуклеиновых кислот и содержании цитозина и гуанина не менее 20% обеспечивались аналогичные чувствительность, селективность, экспрессность и достоверность анализа.Usually, to immobilize a probe on a metal surface, covalent interactions between the modified oligonucleotide and metal nanoparticles are used. Immobilization of the probe in the present invention was based on a specific electrostatic interaction between the Gnus anchor oligonucleotide sequence, which is part of the probe and contains at least 20% of cytosine and guanine nucleotides, and metal nanoparticles. Due to the high binding energy of silver clusters with cytosine (C) and guanine (G), which are part of the anchor sequence, which is several times higher than the formation energy of Watson-Crick pairs, silver particles are inside the anchor sequence. Such interaction, in contrast to covalent crosslinks, provides additional surface structuring, which ensures the formation of hot spots and signal amplification [Bossert N., de Bruin D.,

et al. Fluorescence-tunable Ag-DNA biosensor with tailored cytotoxicity for live-cell applications // Sci. Rep. 2016. V. 6. P. 37897]. In the examples given, 5'-TGCCTTTTGGGGACGGATA-3' was considered as the GnuS sequence. At the same time, when using other GnuS sequences (5'-AATGATCTTAACCAAGTATA-3', 5'-TCGGTTGGGGAGCCTAT-3' and 5'-TGGCTAATCCCCTCGGATA-3'), i.e. while maintaining the length of the probe from 15 to 20 nucleic acids and the content of cytosine and guanine of at least 20%, similar sensitivity, selectivity, rapidity and reliability of the analysis were provided.

As a system for detecting mutations C228T and C250T in the promoter of the hTERT gene, a construct consisting of a deoxyoligonucleotide was used, which ensures the immobilization of the probe on the sequence of silver nanoparticles (Gnus), to which the sequence with the R6G tag is associated, complementary to the analyte sequence (Fig. 11) . The sequences A-228 and A-250 containing a fragment of the hTERT gene promoter with single nucleotide substitutions С228Т and С250Т, respectively, were analytes.

From stock aqueous solutions of oligonucleotides GnuS-C228T-R6G, Gnus-C250T-R6G, A-C250T and A-C228T with concentrations of 1 mM, working aqueous solutions were prepared for each sample with concentrations of 10 ^-8 - 10 ^-14 M. In order to register individual SERS spectra for the GnuS-C228T-R6G and Gnus-C250T-R6G oligonucleotides were applied to the surface of silver nanoparticles in a volume of 10 μL, and the SERS spectra were recorded after the substrate had completely dried at room temperature. To record the spectra of the systems GnuS-C228T-R6G + A-C228T and Gnus-C250T-R6G + A-C250T (Table 1), the components were pre-mixed until the final concentrations of the solutions were 10 ^-8 - 10 ^-14 M. After mixing, they were kept at T = 22°C approx. 10 min, then the mixture was heated at T = 95°C for 5 min, after which the mixture was left to cool to T = 22°C for approx. 60 min. Then, the resulting solution was applied to the surface of silver nanoparticles in a volume of 10 µl, and the SERS spectra were recorded after the substrate had completely dried at room temperature.

Obtained SERS spectra for individual solutions of the GnuS-C250T-R6G and GnuS-C228T-R6G probes applied in a volume of 10 μl with a dispenser on the planar surface of silver nanoparticles at four different concentrations, as well as the spectra of probe + analyte systems: GnuS-C250T-R6G + A250 and GnuS-C228T-R6G + A228 demonstrated the ability to selectively detect mutations C250T and C228T (Fig. 12 - 15). Before applying the probe + analyte system, the mixture was preliminarily heated at 94°C for 5 min, and then gradually cooled to room temperature.

In the case of SERS spectra obtained separately for the GnuS-C250T-R6G and GnuS-C228T-R6G probes, characteristic peaks are either absent or their intensity is at the background level. In the case of using the complete developed indicator systems GnuS-C250T-R6G + A250 and GnuS-C228T-R6G + A250, intense peaks were observed that are characteristic of the SERS-label R6G at 1380, 1470 and 1590 cm- ¹ . The SERS spectra for the proposed GnuS-C250Т-R6G + A250 and GnuS-C228Т-R6G + A250 detection systems had the same position of the characteristic peaks: 1383, 1470, and 1590 cm ^-1 , regardless of the concentration of mutations in the analyzed sample.

In the absence of the analyzed molecule, the free probe formed a hairpin (the GnuS anchor was not involved in the hairpin formation), and the label was at a close distance from the surface. Depending on how close this distance is and whether the label is located in the hot spot, the signal could both appear and fade ["Method for detecting and determining DNA with a given sequence by giant Raman spectroscopy" RF Patent No. 2723160 dated 06/09/2020 .]. When an analyte was introduced into the system, it interacted with the complementary sequence of the probe, the hairpin opened, as a result of which a change in the intensity of the SERS signal was observed compared to the situation when the label was in the hairpin. Thus, when the hairpin opened, the covalent bond with the label was preserved, and the DNA structure changed, which led to a change in the relative position of the label and the surface. In the absence of the analyzed molecule, the probe formed a complex secondary structure (hairpins, intramolecular dimers, as well as internal G-quadruplexes) (Fig. 16). The 5' end of the probe, which is responsible for immobilization on the surface of the nanoparticle, was involved in the formation of this secondary structure. When attempting to hybridize the detected damaged DNA sequences with a sequence-inappropriate labeled oligonucleotide: GnuS-C250Т-R6G with A228 and GnuS-C228Т-R6G with A250, the analytical signal could not be registered by SERS spectroscopy. This fact additionally demonstrated that without effective immobilization and the formation of structured aggregates, it was impossible to obtain a significant increase in the signal; the complete absence or low intensity of signals for other DNA fragments indicated that the method was selective enough for analysis.

Only in the presence of C228T and C250T mutant DNA fragments, the probe interacted with the analyte to form a duplex; and the 5'-end of the probe involved in the formation of the secondary structure was released, and the {probe + analyte} system was effectively immobilized on the surface of the nanoparticles, which leads to the observed increase in signal intensity. The created system made it possible to detect C228T and C250T mutations in the promoter of the hTERT gene using SERS spectroscopy; conclusions about the quantitative content of the detected fragments were provided by the analyte-dependent enhancement of the SERS signal. In the case of application of GnuS-C228T-R6G to the surface of the probe in the presence of the C250 mutation, no interaction occurred and no analytical signal was observed in the SERS spectra, as in the case of GnuS-C250T-R6G in the presence of the C228 mutation, which confirmed the cross-selectivity of the developed method. It is important that when using oligonucleotides labeled with a GKR tag that differs from the proposed length, it was not possible to achieve the required sensitivity for detecting mutant DNA fragments, which was shown for oligonucleotides with RnuS and PnuS probes (Table 2). According to the lengths calculated in accordance with the chemical structures, the distance from the nanostructured surface to the SERS label was 4 nm for GnuS, 5 nm for RnuS, and 3 nm for PnuS. The use of dye-labels with the Gnus sequence from a number of rhodamine, cyanine, xanthene and diazo dyes, methylene blue and crystal violet ensured the achievement of the required level of sensitivity (Table 2). Sample preparation of all samples (up to the amount that needs to be analyzed), which consisted in hybridization of the DNA fragment to be determined with labeled oligonucleotides, took about 60 min. At the same time, no additional manipulations, except for turning the thermostat on/off, are required from the laboratory assistant at this moment, so it is advisable to carry out this procedure for several samples at the same time. The total analysis time took no more than 1 min with a sufficient spectrum accumulation time of 10 s.

The reproducibility of analytical signals was less than 5%. Received software for mutant DNA fragments - 10 ^-14 M, which is approx. 6,000 damaged fragments in 1 µl of the analyzed sample. The achieved software allows diagnosing bladder cancer starting from stage I (at the initial stages of the tumor process: stage T1 or more superficial tumors), when the tumor is non-invasive. In the case of using the label-dye R6G, the conclusion about the qualitative composition and quantitative content was carried out by the presence of characteristic bands: 1383, 1470 and 1590 cm ^-1 on the SERS spectra and the intensity of the received signals according to the calibration dependences, respectively.

Table 1. Determined nucleic acid sequences, sequences of GKR-labeled oligonucleotides and their short designations

Table 2. Metrological characteristics (with the number of parallel measurements n = 5 and confidence probability P = 0.95) of determination of mutant DNA sequences with covalently SERS-labeled oligonucleotides of complementary sequences of various lengths (Parameters of recording SERS spectra: excitation wavelength - 514 nm, laser power - 10%, extinction time - 10 s)

Calibration equation
I = (a±Δa)lgc + (b±Δb) ^* DOK, pM PO, fM r s _r (at s _n ) ^** Determination of the mutant DNA fragment: A-C228T labeled R6G GnuS-pr-C228T I = (1.3 ± 0.2)×10 ⁴ lgc + (1.6 ± 0.1)×10 ³ 0.003 - 10 one 0.990 0.05 RnuS-pr-С228Т I = (2.9 ± 0.3)×10 ¹ logc + (7.6 ± 0.6) 1 - 100 300 0.969 0.09 PnuS-pr-С228Т I = (4.0 ± 0.3)×10 ¹ logc + (4.4 ± 0.2) 10 - 500 3000 0.981 0.15 Determination of the mutant DNA fragment: A-C228T labeled with Cy5 GnuS-pr-C228T I = (6.0 ± 0.5)×10 ³ logc + (3.2 ± 0.2)×10 ³ 0.005 - 10 1.5 0.989 0.05 Determination of the mutant DNA fragment: A-C250T labeled R6G GnuS-pr-C250T I = (4.9 ± 0.6)×10 ⁴ lgc + (1.8 ± 0.3)×10 ³ 0.003 - 10 one 0.990 0.04 RnuS-pr-C250T I = (3.9 ± 0.4)×10 ¹ logc + (6.6 ± 0.3) 1 - 1000 300 0.974 0.10 PnuS-pr-C250T I = (5.9 ± 0.3)×10 ¹ logc + (1.3 ± 0.1) 20 - 100 600 0.980 0.13 Determination of the mutant DNA fragment: A-C250T labeled crystal violet GnuS-pr-C250T I = (5.2 ± 0.5)×10 ⁴ lgc + (2.4 ± 0.4)×10 ³ 0.003 - 10 one 0.988 0.05

^* - type of dependence of the intensity of the analytical signal at the characteristic Raman shift on the concentration of the determined DNA (mol/l);

^** - standard relative deviation for measurements at the minimum concentration (c _n ) from the range of determined contents.

Example 2 Validation of C228T and C250T Mutation Results by SERS Spectroscopy Using the ccPCR Method

Since the procedures for clinical diagnosis of diseases must be performed according to certain standards, it is necessary to conduct a preliminary assessment of diagnostic methods: determine their analytical sensitivity and other characteristics. Determining the capabilities of the method itself on clinical specimens or on cell lines does not give fully reliable results, since tumor samples are inherently heterogeneous, and cell lines do not contain 100% of the mutant allelic fraction. Therefore, in order to introduce ccPCR or the proposed technique based on SERS spectroscopy into clinical practice, it is necessary to create a unified standard analytical system for comparing and validating the results obtained by various laboratory methods in various laboratory conditions.

Model reference constructs were obtained as preclinical trials closest to clinical trials by cloning fragments of the human genome and introducing detectable mutations: C228T and C250T.

Cloning. Four plasmids were obtained containing all the necessary inserts:

1. wild type (pUC19_235_wt),

2. with the C228T mutation (pUC19_235_C228T),

3. with the C250T mutation (pUC19_235_C250T),

4. simultaneously with both mutations С228Т and С250Т (pUC19_235_С228Т+С250Т).

To obtain wild-type inserts, Human Genomic DNA 11691112001 Roche was used as a template for PCR, and for inserts with the C228T mutation at position -146, DNA of the HepG2 cell line (70% mutant DNA content) was used, for inserts with the C250T mutation at position -126, DNA of the cell line A375. (Fig. 1). The cloned inserts were GC-rich fragments (77% GC for the 235 bp fragment), so their amplification was difficult due to the complex secondary structure (presumably the formation of G-quadruplexes). To amplify the wild-type fragment with human genomic DNA, 7-deaza-2'-deoxyguanosine-5'-triphosphate (7-deaza-dGTP) was added to the reaction mixture to a final concentration of 200 μM (Fig. 2, A). The PCR product of the correct length was purified by gel electrophoresis separation and agarose gel isolation.

Polymerase chain reaction (PCR). To amplify the wild-type fragments and the fragment with the C228T mutation, the following PCR mixture was composed:

2x Maxima Hot Start PCR Master mix 25 µl Matrix DNA (genomic) 1 mcg

TERT_235_fw_KpnI 0.5 µM

TERT_235_rev_HindIII 0.5 µM

7-deaza-dGTP 0.05 µM

H ₂ O up to a total volume of 50 µl

Mix thoroughly and place in a Bio Rad T100 PCR Thermal Cycler. Amplification parameters: 95°C for 4 min - 1 cycle; 95°C - 30 s, 58°C - 20 s, 72°C - 30 s - 39 cycles; 72°C for 5 min - 1 cycle.

Amplification of fragment c of the genome of cell lines HepG2 and A375 was carried out using Q5 DNA polymerase (New England Biolabs). The composition of the reaction mixtures:

5x Q5 GC buffer 10 µl dNTP blend 0.2 mM each TERT_235_fw_KpnI 0.5 µM TERT_235_rev_HindIII 0.5 µM Matrix DNA (A375 or HepG2) 0.2 µg Q5 polymerase (2u/µl) 0.5 µl 5x Q5 GC enhancer 10 µl H ₂ O up to a total volume of 50 µl

Amplification parameters: 98°C for 30 s - 1 cycle; 98°C - 7 s, 68°C - 15 s, 72°C - 10 s - 35 cycles; 72°C for 2 min - 1 cycle.

In the case of using HepG2 and A375 cell lines as templates for amplification of the cloned DNA fragment, the approach with the addition of 7-deaza-dGTP was not successful and it was not possible to obtain a quantitative PCR product even in the case of two-stage PCR. To solve this problem, Q5 polymerase, whose accuracy is more than 100 times higher than that of c Taq polymerase, was used in a GC-rich template amplification buffer with the addition of an enhancer. Typically, GC enhancers include compounds such as DMSO and glycerol, which contribute to the disruption of a complex secondary structure and increase the efficiency of amplification (Fig. 2, B).

The resulting PCR products were digested with restriction endonucleases HindIII and KpnI and ligated at the sticky ends with the plasmid pUC19 (Fig. 3) linearized with the same restriction endonucleases. The resulting ligation mixture was transformed into competent E. coli JM109 cells and seeded on a Petri dish with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) and isopropyl-beta-galactopyranoside (IPTG) for white - blue selection. The next day, night cultures were sown from the white colonies. A plasmid was isolated from overnight cultures, and after restriction analysis with PvuII restriction endonuclease confirmed the presence of the insert, the insert was sequenced by the Sanger method (FIG. 4).

Separation of DNA fragments in agarose gel, isolation from the gel. After PCR, the obtained PCR products were purified in 1.2% agarose gel. To prepare an agarose gel, 1.0 g of low-melting agarose was added to 100 ml of a 1xTBE solution. The bands were visualized by adding 2.5 μl of the fluorescent dye SybrGreen I (10000x) to the agarose. The voltage was ~100 - 150 V. The separation of DNA fragments was controlled using a camera with an ultraviolet lamp.

DNA isolation from the agarose gel was performed using the Cleanup mini reagent kit from Evrogen. The desired band, visualized by SybrGreen I, was cut from the gel and placed in a test tube. A 3-fold volume of "Binding Buffer" was added to the gel (assuming that 100 μg of gel has a volume of 100 μl), incubated at 55°C until complete dissolution of the gel. The resulting solution was transferred to a DNA binding column, and the column was placed in a collection tube. The collection tube with the column was centrifuged at maximum speed in a tabletop centrifuge for 30 s (all further centrifugations were performed at maximum speed). The slip was poured out, 700 µl of wash solution was added to the column and centrifuged for 30 s. The slip was discarded and the column was centrifuged for 1 min. After that, the column was placed in a new tube, 30 µl of elution buffer was added, and centrifuged for 30 s. The resulting DNA was stored at -20°C.

Cooking vectors and inserts. Before ligation, the vector and inserts were treated with FastDigest KpnI and FastDigest HindIII restriction endonucleases, Thermo Scientific.

10x Fast Digest buffer 2 µl

plasmid DNA/PCR fragment 1 mcg/200ng restrictase KpnI 1 µl restriction enzyme HindIII 1 µl H ₂ O up to a total volume of 20 µl

All necessary reaction components were mixed and incubated for 1 h at 37°C. After that, the enzymes were inactivated by heating at 80°C for 10 min. After that, the restriction mixture with plasmid DNA was applied to an agarose gel and the desired fragment was isolated from the gel as described above.

Ligation. Ligation was performed using T4 DNA ligase (Thermo Scientific). To 10-fold T4 DNA ligase buffer was added 50 - 100 ng of linearized plasmid and insert in a molar ratio with the plasmid 5:1, T4 DNA ligase to a concentration of 0.05 units/µl, the required amount of water. The ligation mixture was left overnight at +4°C.

Obtaining competent E. coli cells and their transformation. 250 ml of LB medium was inoculated with a fresh cell colony and the culture was grown at 18°C with moderate stirring to A600 ~0.4. Cells were cooled in ice and harvested by centrifugation at 5000 rpm for 10 min at 4°C in a JA-14 rotor (Beckman). After that, the cells were washed twice with TB buffer. To do this, the cell pellet was resuspended in 30 ml of chilled TB buffer, incubated with gentle stirring in ice for 10 min, and centrifuged at 5000 rpm for 10 min at 4°C. After the last centrifugation, the cells were resuspended in TB buffer, DMSO was added to 3.5% and then to a final concentration of 7% at 10 min intervals at 0°C with gentle agitation. Aliquots of the cell suspension, 100 µl each, were frozen in liquid nitrogen and stored at -80°C.

For transformation, the suspension of competent cells was thawed on ice, 20–50 ng of the plasmid or 5–10 µl of the ligation mixture were added, and kept on ice for 20 min. The mixture was placed in a thermostat heated to 42°C, kept for 40 - 50 s, then 300 μl of LB medium was added and incubated for 1 h at 37°C. Half of the cells were seeded on a Petri dish with LB solid medium containing ampicillin. Incubated at 37°C for 16 hours Selection of colonies with an insert was carried out using blue-white selection.

Isolation of plasmid DNA. Plasmids from the overnight culture were isolated using the Evrogen Plasmid Miniprep reagent kit from E. coli. To do this, 2–5 ml of overnight culture was grown from one colony, the cells were precipitated for 2 min at 8000g in a table top microcentrifuge. The medium was taken, the cells were resuspended in 250 µl of "Resuspending Solution", 250 µl of "Lysing Solution" was added and gently mixed until the lysate became clear. 350 µl of "Neutralizing solution" was added, mixed, incubated for 1 min. Debris was removed by centrifugation at a maximum speed of 10 min in a tabletop microcentrifuge (further centrifugations were performed at maximum speed). The supernatant was transferred to the column in a collection tube and centrifuged for 30 s. The filtrate was removed from the collection tube. Two washings of 300 µl of the “Washing solution” were performed with column centrifugation for 30 s. After the last wash, the empty column was centrifuged for 1 min. After that, the column was transferred to a new test tube, 50 μl of the "Elution solution" was added, incubated at room temperature for 1 min, and centrifuged for 1 min. The resulting plasmids were stored at -20°C.

Restrictive analysis. To confirm the presence of the insert, restrictive analysis of plasmids was carried out using FastDigest PvuII restrictase, Thermo Scientific. For restriction, 200 ng of plasmid, 2 µl of 10× FastDigest Green buffer, and 5 units of enzyme activity per 20 µl reaction. Incubated at 37°C for 1 h. The products were analyzed in 1.2 wt.% agarose gel.

Site directed mutagenesis. For site-directed mutagenesis, PCR amplification of the resulting plasmid pUC19_235_C228T was performed using Pfu DNA Polymerase and DreamTaq DNA Polymerase, Thermo Scientific.

In a test tube with a capacity of 0.2 ml, the following PCR mixture was composed:

10X Pfu buffer with MgSO ₄ 2.5 µl dNTP blend 0.2 mM each primer 1 20 pmol primer 2 20 pmol plasmid DNA 50 ng polymerase Taq:Pfu (5:1) (2.5 u/µl) 1 µl DMSO 100% 1.25 µl H ₂ O up to a total volume of 25 µl

Amplification parameters: 95°C for 30 s - 1 cycle; 95°C - 50 s, 65°C - 50 s, 68°C - 4min - 18 cycles; 68°C for 7 min - 1 cycle.

After PCR, 0.5 μl of DpnI (10 U/μl, Thermo Scientific) was added to the reaction mixture and incubated at 37°C for 16 h. The enzyme was inactivated at 80°C for 20 min. Next, competent E. coli cells were transformed with 10 μl of the reaction mixture and seeded on a Petri dish. White colonies were subcultured into liquid medium, plasmids were isolated from the cells, and the presence of the insert was checked using Sanger sequencing.

Determination of the primary structure of plasmid DNA. Nucleotide sequences during the creation and use of genetically engineered constructs were determined using an automatic sequencer ABI prism 3100-Avant genetic Analyzer (4-capillary).

PCR in real time. Real-time PCR was performed using Maxima hot start PCR Master mix, Thermo Scientific. 2xPCR Master mix primers were added to a concentration of 0.9 μM, 5 - 50 ng of the template, 7-deaza-dGTP, fluorescent probes to a concentration of 250 nM and adjusted to the desired volume. The annealing temperature and amplification parameters varied depending on the experiment, the elongation time was calculated under the condition that Taq polymerase synthesizes DNA at a rate of 1000 nucleotides per minute.

Digital drip PCR. For staging ccPCR, the following reaction mixture was prepared, with a volume of 22 µl:

2x ddPCR Supermix (no dUTP) 11 µl sample 1 250 nM sample 2 250 nM primer 1 900 nM primer 2 900 nM template DNA (plasmids) variable quantity

7-deaza-dGTP 2 µM

H ₂ O up to a total volume of 22 µl

PCR amplification for the detection of C228T and C250T mutations occurred separately, amplification parameters: 95°C for 10 min; 40 cycles 94°C - 30 s, temperature change rate 2.5°C/s, 54°C for C228T (64°C for C250T) for 30 s; 98°C - 10 min, then storage at 12°C after completion of amplification. Before registration of the fluorescent signal, the plate with the PCR product was stored for no more than a week at a temperature of +4°C.

Droplets were generated using an automatic generator QX200, Biorad. The fluorescence intensity in each drop was measured using a QX200 Droplet reader, Biorad. The results were analyzed using QuantaSoftTM Analysis Pro 1.0.596 software, Biorad.

According to the concept of mutation detection (Fig. 5), 2 pairs of fluorescent probes were used to detect each of the mutations: one pair of probes labeled with rhodamine 6G (R6G) (Fig. 6) to the wild-type sense and template strand (TERT_250_wt_prb1, TERT_250_wt_prb2 for wild-type at position S250T, TERT_228_wt_prb1, TERT_228_wt_prb2 wild type at position S228T), the second pair of probes labeled with fluorescein (FAM) to the sense and the template strand of the mutant-type (TERT_250_mut_prb1 and TERT_250_mut_prb2 on S250T mutation and TERT_228_mut_prb1 and TERT_228_mut_prb2 on S228T mutation). At the beginning of the PCR, the temperature was increased in order for the DNA double helix to denature. At this point, the signal from the fluorescent dye at the 5' end of the probe was quenched by the non-fluorescent quencher located at the 3' end of the probe. In the next step, the temperature was lowered, allowing the primers and probes to form duplexes with the target sequence. Taq polymerase synthesized complementary DNA on the denatured strand. When the polymerase reached the probe, it cleaved the probe with its 5' → 3' exonuclease activity, separating the fluorophore from the quencher. With each PCR cycle, more and more fluorophore molecules were released, which led to an increase in the fluorescence intensity proportional to the number of molecules of the synthesized amplicon. The recommended amplicon size of 60-200 bp was used. (65 bp for C228T and 92 bp for C250T), a region for amplification with 40-60% content of GC-pairs was chosen and regions with a complex secondary structure were avoided. Since the content of GC-pairs in the resulting amplicons is 77–80% (Fig. 7), GC-enhancers were added to the reaction mixture for the efficient operation of polymerases, which disrupt the secondary structure and promote normal separation of DNA strands. For these purposes, we used 7-deaza-dGTP, which can be included in the synthesized chain by DNA polymerases. Since there is no nitrogen atom in the seventh position of the cycle in 7-deaza-dGTP, the inclusion of 7-deaza-dGTP in DNA prevented the formation of G-quadruplexes in GC-rich regions. Figure 8 shows the result of the analysis of a system consisting of plasmid pUC19_235_wt wild type in the amount of 5000 copies per 20 μl of the reaction mixture, and plasmid pUC19_235_C228T in the amount of 1250 copies per 20 μl of the reaction mixture. The analysis of the experimental results was carried out using the QuantaSoftTM analysis pro software. The cut-off value for channel 1 (C228T mutant probe) was 4000. For channel 2 (wild-type probe C228T), the cut-off value was 2500. The area with blue dots represents the droplets with mutant-type plasmids, the area with green dots is the droplets with plasmids wild-type, the area with orange dots - drops containing plasmids of both types (Fig. 8).

When optimizing the C228T mutation detection system, consisting of pUC19_235_C228T and pUC19_235_wt plasmids, the maximum resolution between clusters was achieved at a temperature of 54°C. At this temperature, at 100% content in the system of only wild-type plasmids in the amount of 2500 positive drops, there are no false positive results (drops) in the FAM channel. Thus, the selected conditions provide 100% detection selectivity provided that the analyzed mixture is not contaminated with mutant-type sequences. Next, we analyzed the systems containing the plasmid with the C228T mutation against the background of an excess of the wild-type plasmid, the concentration of which is constant and amounts to 5000 copies per 20 μl of the reaction mixture. (Fig. 9). Adjusting for specificity and control, the sensitivity was 2%.

The selected conditions (temperature regime, 7-deaza-dGTP concentration, fluorescent TaqMan probes and primers) for digital droplet PCR made it possible to achieve selective detection of the hTERT gene promoter sequence with C228T and C250T mutations, which was necessary to validate the results obtained by SERS spectroscopy. However, SERS spectroscopy makes it possible to achieve lower detection limits, and most importantly, the use of this method does not require preliminary enzymatic amplification of the target.

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SEQUENCE LIST

<110> Federal state budgetary educational institution

higher professional education "Moscow State

University named after M.V. Lomonosov "(MSU)

<120> Kit for diagnosing bladder cancer using C228T mutations

and C250T in the promoter of the HTERT gene and method for its implementation

<160> SEQ ID NO:1

<210> 1

<211> 19

<212> GnuS

<213> Artificial sequence

<400> 1

1 TGCCTTTTGG GGACGGATA

<160> SEQ ID NO:2

<210> 2

<211> 20

<212> GnuS

<213> Artificial sequence

<400> 2

1 AATGATCTTA ACCAAGTATA

<160> SEQ ID NO:3

<210> 3

<211> 17

<212> GnuS

<213> Artificial sequence

<400> 3

1 TCGGTTGGGG AGCCTAT

<160> SEQ ID NO:4

<210> 3

<211> 19

<212> GnuS

<213> Artificial sequence

<400> 4

1 TGGCTAATCC CCTCGGATA

<---

Claims (18)

1. Kit for the diagnosis of bladder cancer using C228T and C250T mutations in the hTERT gene promoter using giant Raman spectroscopy (GRS), including a solid-phase planar sensor made of a dielectric chemically inert material and a coating based on noble metal nanoparticles, 1 thickness -10 µm; and solutions labeled with a dye with a detection limit of not more than 1 μM of oligonucleotides GnuS-C228T-tag and GnuS-C250T-tag, where Gnus is a sequence consisting of 15-20 amino acids, of which at least 20% are cytosine (C) and guanine ( G). 2. The set according to claim 1, characterized in that Gnus is the nucleotide sequence of SEQ ID no. 1 - no. 4. 3. Set according to claim 1, characterized in that the coating has a nanostructure with a roughness of at least 2 nm. 4. The kit according to claim 1, characterized in that the dye with a detection limit of not more than 1 μm is a rhodamine, cyanine, xanthene and diazo dye, methylene blue, crystal violet. 5. The set according to claim 1, characterized in that the noble metal nanoparticles are made of silver, platinum, gold. 6. A kit according to claim 2, characterized in that the nanoparticles are 20-90 nm in size. 7. The kit according to claim 1, characterized in that the dielectric chemically inert material for coating with nanoparticles is silicate glass. 8. The kit according to claim 1, characterized in that the concentration of the GnuS-C228T-tag or GnuS-C250T-tag of the oligonucleotide in solution is from 10 ^-6 to 10 ^-10 M. 9. A method for diagnosing bladder cancer using the kit according to claim 1, including taking a biological sample, one part of which is mixed with a GnuS-C228T-tag oligonucleotide solution, and the other part with a GnuS-C250T-tag solution, their hybridization is carried out, followed by determination mutations of DNA C228T and C250T, by applying the resulting mixtures to the surface of the sensor, irradiation with a monochromatic laser beam with a wavelength of 485-785 nm, with the registration of a Raman signal, while the qualitative and quantitative content of DNA mutations C228T and C250T is judged by the position and intensity of the bands characteristic of the dye label. 10. The method according to claim 9, characterized in that the patient's urine is used as a biological sample. 11. The method according to claim 9, characterized in that the amount of SERS-labeled oligonucleotide is used in a volume ratio from 1:5 to 5:1 to the analyzed sample containing the C228T and C250T DNA mutations. 12. The method according to claim 9, characterized in that pre-hybridization of nucleic acid sequences with a dye-labeled solution is carried out. 13. The method according to claim 12, characterized in that hybridization is carried out by mixing the analyte and the dye-labeled solution, heating them and keeping them for 5-10 minutes at a temperature of 95 ° C, followed by cooling to room temperature. 14. The method according to claim 9, characterized in that the analyzed biological samples are used, mixed with a solution of the oligonucleotide GnuS-C228T-tag or GnuS-C250T-tag by dropping them onto the surface of the nanoparticles. 15. The method according to claim 9, characterized in that samples with a volume of 10 μl are used with a concentration of C228T and C250T DNA mutations of 10 ^-15 - 10 ^-2 M. 16. The method according to claim 9, characterized in that monochromatic laser radiation with a power not exceeding 200 mW is used for up to 10-60 s. 17. The method according to claim 10, characterized in that the presence and content of C228T and C250T DNA mutations in the sample is judged by the position and intensity of the bands at 1385, 1470 and 1590 cm ^-1 with an allowable error of not more than 10 cm ^-1 . 18. The method according to claim 9, characterized in that calibration curves are pre-built to determine the quantitative content of mutations.

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