METHODS OF DETECTION OF TARGET RNAs IN A SAMPLE AND USES THEREOF

Abstract

The present invention is directed to a rapid method of detection of oligonucleotide sequences allowing rapid, sensitive (low femtomolar) and specific (single nucleotide resolution) detection of cancer-associated nucleic acids and especially mutated RNA / DNA from blood, plasma, and other bodily fluids or tissues.

Description

[0001] METHODS OF DETECTION OF TARGET RNAs IN A SAMPLE AND USES

[0002] THEREOF

[0003] Field of the Invention

[0004] The present invention relates to the detection of target nucleic acids in a biological sample.

[0005] Background of the Invention

[0006] Cancer is one of the main leading causes of mortality worldwide and remains a major public health challenge (Mehlen et al., 2006, Nat Rev Cancer, 6 (6), 449-458. DOI: 10.1038 / nrcl886). Many of the screening methods applied for the early detection of cancer are either ineffective, expensive, or highly invasive, and thus only performed on a minority of people, or only after the appearance of symptoms. Tissue biopsy remains an essential technique that is invaluable to confirm diagnosis and determine the nature of the cancer, however it has intrinsic limitations: it is an invasive procedure that is applicable only when the lesion is visible (clinically through imaging methods), is challenging to repeat, it provides only a limited number of specimens for further genetic testing, and it may pose a risk for the patient as some tumors are difficult to access due to their anatomical location (Perakis et al., 2017, BMC Med, 15 (1), 75. DOI: 10.1186 / S12916-017-0840-6; Tanaka et al, 2017, Nat Biomed Eng 2017, 1 (10), 796-806. DOI: 10.1038 / s41551-017-0139-0). The discovery of tumor-derived biomarkers in plasma and other body fluids of cancer patients has emerged as a new approach known as liquid biopsy (Lone et al., 2022, Mol Cancer, 21 (1), 79. DOI: 10.1186 / sl2943-022-01543-7).

[0007] Liquid biopsy is being considered as a minimally invasive tool that may be rapidly used for cancer detection at an early stage Wan et al., 2017, Nat Rev Cancer, 17 (4), 223-238. DOI: 10.1038 / nrc.2017.7).

[0008] It may also be used to validate the efficiency of a cancer treatment by taking multiple samples in the following weeks and months for the monitoring of cancer patients for relapse.

[0009] Liquid biopsy obtained from peripheral blood encompasses different tumoral components including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), extracellular vesicles (EVs), mRNAs and micro-RNA (miRNA) (Siravegna etal., 2017, Nat Rev Clin Oncol, 14 (9), 531-548. DOI: 10.1038 / nrclinonc.2017.14).

[0010] These elements can be isolated for the identification of various tumor-specific genomic aberrations including point mutations, copy number variations, structural rearrangements, or epigenetic (methylation) patterns (Palacin-Aliana et al, 2021 Biomedicines, 9 (8). DOI: 10.3390 biomedicines9080906.) The level of circulating nucleic acids (i.e. ctDNAs, mRNAs, IncRNAs and miRNAs) were found to be elevated in a broad spectrum of cancer types relative to healthy individuals and the expression can be further altered during the progression of the particular cancer type (Kosaka etal., 210, Cancer Sci, 101 (10), 2087-2092. DOI: 10.1111 / j.l349-7006.2010.01650.x).

[0011] Moreover, the cancer specific mutations (e.g. KRAS mutation) that were detected in the patient' s blood were found to be identical in the patient’s tumor, thereby confirming that the mutant DNA fragments in the plasma were of tumor origin and reflects therefore tumor genetics (Sorenson et al. , 1994, Cancer Epidemiol Biomarkers Prev 1994, 3 (1), 67-71).

[0012] After the approval of the first liquid biopsy test developed by Roche (EGFR mutation test) in 2016 (Kwapisz et al., 2017, Ann Transl Med 2017, 5 (3), 46. DOI: 10.21037 / atm.2017.01.32) the United States Food and Drug Administration (FDA) has granted approval for diagnostic tests and many companies started the development of liquid biopsy tests for the early detection, prognosis, and post-treatment monitoring of cancer (e.g. Qiagen therascreen kits, Galleri MCED test) (Vallee et al., 2014, Clin Chim Acta, 429, 8-11. DOI: 10.1016 / j.cca.2013.11.014; Duffy et al., 2023, Cr it Rev Clin Lab Sci, 1-13. DOI: 10.1080 / 10408363.2023.2275150).

[0013] Considering the fact that the concentration of circulating nucleic acids in the bloodstream could be in the range of low femtomolar (fM) concentrations, sensitive and specific detection methods are urgently needed for precise cancer diagnostics (Shin et al., 2023, Biosens Bioelectron, 242, 115694. DOI: 10.1016 / j.bios.2023.115694).

[0014] Common detection techniques involve complex, expensive and time-consuming approaches such as next-generation sequencing (NGS), digital PCR (dPCR) or microarray hybridization. Although these sensing techniques enable high-throughput detection of multiple nucleic acids, they can suffer from low sensitivity and low specificity, especially when detecting highly homologous sequences that can differ by a single nucleotide due to cross-hybridization, and reliable detection of mutations below 1% could be challenging due to amplification and sequencing errors (Song et al., 2022, Nat Biomed Eng., 6 (3), 232-245. DOI: 10.1038 / s41551- 021-00837-3).

[0015] In this regard, DNA nanotechnology approaches offer promising tools for the detection of cancer biomarkers, including CTCs (Rafiee, 2020, ChemMedChem, 15 (8), 661-666. DOI: 10.1002 / cmdc.201900697), cell free RNA and DNA (Domljanovic et al., 2022, Nanoscale, 14 (41), 15432-15441. DOI: 10.1039 / d2nr03985k; Kocabey et al., 2023, Biosens Bioelectro, 224, 115053. DOI: 10.1016 / j.bios.2022.115053) or cellular membrane proteins (Sun et al., 2022. Sci Adv, 8 (48), eadd!106. DOI: 10.1126 / sciadv.addl 106). Existing approaches for the detection of proximal proteins using Proximity -PER (US 2021 / 147902) and protein detection via PER conjugated to antibodies (US 2020 / 362398) are primarily performed on fixed cells, involving labor-intensive procedures and requiring complex bioconjugation techniques for DNA-protein coupling. Methods using beads (US 5591841) require extra steps to separate and isolate DNA complexes from the beads prior to detection. Recently, a variety of signal amplification strategies have been developed to enhance the sensitivity of cancer biomarker detection including deoxyribozymes (DNAzyme) (McConnell, et al., 2021, Chem Soc Rev, 50 (16), 8954-8994. DOI: 10.1039 / dlcs00240f), catalyzed hairpin assembly (CHA) (He et al, 2024, ACS Nano, 18 (6), 5017-5028. DOI: 10.1021 / acsnano.3cl 1147), rolling circle amplification (RCA) (Li et al, 2023, Biosens Bioelectron 2023, 231, 115273. DOI: 10.1016 / j.bios.2023.115273) and hybridization chain reaction (HCR) (Lazaro et al., 2022, ACS Sens, 7 (3), 758-765. DOI:

[0016] 10.1021 / acssensors.lc02220). However, many of these strategies mainly rely on an on-going reaction and they are incompatible with a ready-to-use design with higher controllability (Huang et al., 2023, ACS Sens, 8 (3), 1308-1317. DOI: 10.1021 / acssensors.2c02819).

[0017] Moreover, the in situ enzymatic reaction could be hard to control or tune for individual targets. By contrast, primer exchange reaction (PER) (WO 2018 / 132392) is a signal and nucleic acid amplification technology that enables the autonomous synthesis and assembly of single strand DNA molecules. It is based on a catalytic hairpin reaction in the presence of polymerase with exonuclease activity and branch migration, which allows controlled extension of a short primer sequence in an iterative manner through binding to the complementary hairpin strand (Saka et al., 2019, Nat Biotechnol, 37 (9), 1080-1090. DOI: 10.1038 / s41587-019-0207-y).

[0018] On its own, PER has moderate sensitivity and efficiency and is not easily amenable to high throughput.

[0019] Summary of the invention

[0020] The present invention relates to the unexpected finding of a rapid method of detection of oligonucleotide sequences allowing rapid, sensitive (low femtomolar (fM) range) and specific (single nucleotide resolution) detection of cancer-associated nucleic acids and especially mutated RNA / DNA from blood, plasma, and other bodily fluids. The detection is highly sensitive with a limit of detection down to 27 fM without pre-amplification.

[0021] The method of the invention allowed to successfully detect target RNA in cellular extracts of known genes such as actin and vimentin, as well miR-21, an oncogenic microRNA expressed in many types of cancer. The results demonstrated the detection of actin and vimentin RNA fragments in the range of 0.3 pM to 0.45 pM, which is an expected range. The miR-21 (low pM range) was easily detected in two breast cancer cell lines (MCF-7 and MDA-MB-231). Importantly, the method made possible to identify oncogenes vs the corresponding protooncogene through gene mutations in three cancer cell lines containing known oncogenes (P53, KRAS and PI3K), giving a significant signal shift.

[0022] The results showed that the fluorescence intensities were significantly higher in all conditions when the fragmented RNAs of target regions were incubated with mutant probes.

[0023] The technology is also applicable to direct biological samples since with DNA extracted from the plasma of 8 patients, it was possible to detect a mutation in one of the most common oncogenes, the gene P53, and to differentiate samples from healthy donor versus breast cancer patient by detecting the mutation named P53-R280K.

[0024] The technology is also applicable to the detection of circulating tumor DNA (ctDNA) carrying known mutations that affect patient’s response to therapy and prognosis, present at very low concentrations in plasma among DNA of wild-type sequence derived from normal cells.

[0025] It is advantageous to provide a method for the direct detection of target RNAs from cell extracts without pre-amplification.

[0026] It is advantageous to provide a method for the direct detection of single nucleotide mutations in target RNAs notably for early cancer detection.

[0027] It is advantageous to provide a method of detection of target RNAs in a sample that allows sequential sequence targeting and detection within the same system.

[0028] It is advantageous to provide a method of detection of target RNAs in a sample that allows to adjust specifically the design of complementary sequences for the detection of specific mutations and prepare the target RNA material by a simple pre-processing step accordingly.

[0029] It is advantageous to provide a method of detection of target RNAs in a biological sample that can be completed in less than 7 hours.

[0030] It is advantageous to provide a method of detection of various type of target oligonucleotides (mRNAs, miRNAs and ctDNAs) present in small quantities in a biological sample.

[0031] It is advantageous to provide a method of detection of various type of target oligonucleotides wherein said target oligonucleotides, DNA concatemers and beads can be mixed sequentially to generate a signal that can be directly detected by flow cytometry without the need of repeated purification or isolation procedures. An object of this invention is to provide a method of detection of target RNAs in a sample, said method comprising contacting a complex formed by a microbead and at least one hook sequence, wherein said at least one hook sequence is a hybridizing sequence complementary to only a part of the sequence of the target RNA to form a hooked target DNA / RNA complex at the surface of the microbead wherein the target RNA is hybridized to the said at least one hook sequence; contacting said formed hooked target DNA / RNA complex to a composite elongated single strand DNA primer, to form an imaging target complex wherein the unhybridized part of the formed hooked target DNA / RNA complex forms a duplex with the x domain of said composite elongated single strand DNA primer, wherein said composite elongated single strand DNA primer sequence comprises i) a x domain: a sequence hybridizing complementary to the part of the sequence of the target RNA which is not complementary to the hook sequence and ii) a plurality of p domains formed after a plurality of PER cycles; subjecting said formed imaging target complex to a plurality of fluorophore labelled imager strands, wherein each p domain hybridizes with a fluorophore labelled imager strand; detecting a fluorescence signal emitted by the so formed fluorescent complex.

[0032] Another object of this invention is to provide a use of a method of the invention for the detection of different targets (multiplexed detection) in the same sample.

[0033] Another object of the invention is to provide a kit for the detection of target RNAs in a sample by a method according to the invention.

[0034] Objects of this invention have been achieved by providing a method according to claim 1 and a kit according to claim 15.

[0035] Description of the figures

[0036] Figure 1 provides a schematic representation of the formation of a fluorescent complex 9 by a method of the invention for the detection of a target RNAs 4 in a sample through the measurement of the fluorescence signal emitted by said fluorescent complex resulting from the hybridization of the plurality of p domains from the elongated single strand DNA primer 6 formed after a plurality of PER cycles with a plurality of fluorophore labelled imager strands 8 (81, 82, 83), wherein said elongated single strand DNA primer 6 is itself hybridized through its x domain to the unhybridized part 4b of the hooked target DNA complex 5 formed by the hybridization of a hooking complex 3 via its hook sequence 2a which is complementary to only a part 4b of the sequence of the target RNA 4. The hooking complex 3 is formed by the immobilization of a hook sequence 2 on the microbead surface through the formation of strept(avidin) / biotin complex.

[0037] Figure 2 provides a schematic representation of the steps of the formation of an elongated single strand DNA primer 6 by PER (PER concatemer) starting from a bridge primer which comprises a hybridizing sequence complementary to the part 4b of the sequence of the target RNA 4 which is not complementary to the hook sequence 2 and a p domain which binds to molecules in a hairpin shape (p1), initiating the PER reaction. Said steps comprise the binding of the bridge primer to the p* domain of the respective hairpin oligonucleotide (1), the isothermal extension (2) in the presence of strand-displacing DNA polymerase until encountering with the stopper base-pair, branch migration (3), spontaneous separation between the extended primer and hairpin leading to their dissociation (4), the extended primer (xpp) being able to re-joins a next cycle for further elongation (5), finally leading to an elongated single strand DNA primer 6 formed after a plurality of PER cycles which can be used in the method of the invention as elongated single strand DNA primer.

[0038] Figure 3 provides the detection of KRAS DNA oligonucleotide at the concentrations of 1 fM to 100 pM as described in Example 2, using a method of the invention A) linear signal amplification and B) branched signal amplification approaches. At least 20’000 events were recorded to detect fluorescence signals and calculate MFIs using Cytek Aurora flow cytometer and C) comparing the method using linear, branched and iterative signal amplification at 10 nM target oligonucleotide incubated on the beads for all conditions. MACSQuant Analyzer flow cytometer was used to detect fluorescence signals and calculate MFIs.

[0039] Figure 4 illustrates the detection of actin and vimentin mRNAs and miR-21 from cancer cell extracts using PER-based branched signal amplification as described in Example 2. Synthetic single strand RNA oligonucleotides were used at the concentrations of 1 pM to 10 nM to calculate the detection sensitivity. At least 30’000 events were recorded to detect fluorescence and calculate MFIs using Cytek Aurora flow cytometer. IVT cut: in vitro transcribed and cleaved by RNase H, IVT uncut: in vitro transcribed but not cleaved by RNase H.

[0040] Figure 5 illustrates the detection of single mutations (KRAS-G12S, P53-R280K and PIK3CA- E545K) from cancer cell extracts using PER-based linear signal amplification (A) and of P53- R280K mutation after extraction of ctDNAs from the plasma of breast cancer patients and healthy donors (B) as described in Example 3. At least 27x104events were recorded to calculate MFIs using MACSQuant flow cytometer.

[0041] Figure 6 illustrates the multicolor detection of KRAS G12S mutation in the background of KRAS wild-type DNA fragment. AF-647 labelled imager strands were used for G12S probe and AF-488 labelled imager strands were used for WT probe as described in Example 3. At least 29x104events were recorded to detect fluorescence and calculate MFIs using MACSQuant flow cytometer. Statistical analysis was performed by an unpaired t-test (****p < 0.0001).

[0042] Figure 7 shows the detection of multiple target RNA fragments (EGFR R858, EGFR L861 and BRAF V600) by the method of the invention as described in Example 4. A: by multiplexed detection of different target RNA fragments in a mixture (one pot) of different sizes of beads using two fluorophores; B: by separate detection of those fragments using two fluorophores.

[0043] Detailed description

[0044] The expression “biological sample” includes any biological sample comprising nucleotide material of interest containing target RNAs such as blood, plasma, and other bodily fluids such as saliva, urine, tears, breast milk, exudates.

[0045] Referring to the figures, in particular to Fig. 1A is provided a method of detection of target RNAs 4 in a sample, said method comprising contacting a hooking complex 3 formed by a microbead 1 and a hook sequence 2, to said target RNA sequence 4 to form a hooked target DNA / RNA complex 5 wherein the target RNA 4 is hybridized to the hook sequence, wherein said hook sequence 2 is a hybridizing sequence complementary to only a part 4b of the sequence of the target RNA 4; contacting said formed hooked target DNA / RNA complex 5 to a composite elongated single strand DNA primer 6, to form an imaging target complex 7 wherein the unhybridized part 4b of the hooked target DNA / RNA complex 5 forms a duplex with the x domain of said composite elongated single strand DNA primer 6, wherein said composite elongated single strand DNA primer 6’s sequence comprises i) a x domain: a hybridizing sequence complementary to the part 4b of the sequence of the target RNA which is not complementary to the hook sequence 2 and ii) a plurality of p domains formed after a plurality of PER cycles; subjecting said formed imaging target complex 7 to a plurality of fluorophore labelled imager strands 8 (81, 82, 83) to form a fluorescent complex 9, wherein each of the plurality of p domains of the elongated single strand DNA primer 6 hybridizes with a fluorophore labelled imager strand 8; detecting a fluorescence signal emitted by said formed fluorescent complex 9.

[0046] According to a particular aspect, when contacting a plurality of hooking complex 3 with a plurality of target RNA sequences 4, the detected fluorescence signal will be amplified by the method and will be proportional to the amount of target RNAs in said sample.

[0047] According to a particular aspect, the target RNAs are sequences from about 20 to about 30 nt.

[0048] According to a particular aspect, the target RNAs can be directly used after extraction from a biological sample by standard techniques, e.g. as described herein.

[0049] According to another particular aspect, the target RNAs, in particular for RNAs longer than 70 nt, those can be subjected to a targeted cutting to obtain shorter fragments, typically from 20 to 30 nt.

[0050] According to another particular aspect, a targeting cutting can be conducted by using guide oligonucleotides. Typically, guide oligonucleotides can be around 20 nt and the distance between two guide oligonucleotides are around 20 to 30 nt.

[0051] According to another particular aspect, the target RNAs can be used without pre-amplification and after a preliminary pre-amplification step when the concentrations of target RNAs are lower than 27 fM or when target RNAs need to be transcribed from other source of nucleic acids such ctDNAs.

[0052] According to another particular aspect, the target RNAs can be generated through a preamplification step that includes DNA pre-amplification (e.g., from ctDNA or genomic DNA or reverse transcribed RNA), followed by in vitro transcription and RNAse H mediated cleavage.

[0053] According to a particular aspect, the fluorescence signal emitted by said formed fluorescent complex is measured by fluorescence spectroscopy under an excitation wavelength specific to said imager strands.

[0054] According to another particular aspect, the composite elongated single strand DNA primer is a linear primer for a direct amplification through hybridization with a fluorophore labelled imager strand with the plurality of p domains of the elongated single strand DNA primer. According to another particular aspect, the hooking sequence or single-stranded DNA primers are designed to be complementary and specific to a mutant target RNAs, typically spanning about 9 to 11 nt with mismatch melting temperatures (Tm) below 25°C.

[0055] According to another particular aspect, the composite elongated single strand DNA primer hybridized with the hooked target DNA / RNA complex is then further hybridized with a second composite elongated single strand DNA primer (second concatemer) which is branched and therefore presenting further possible hybridization sites for the plurality of fluorophore labelled imager strands for a further amplification of the fluorescence signal.

[0056] According to another particular aspect, the method can be used for multiplexed RNAs detection using different fluorophores at different wavelengths. Typical fluorophores are provided in the Example section to illustrate those.

[0057] According to a particular aspect, each step of the method can be performed in 30 min (max) and washing steps (about 5 min) are carried out between each step. The whole detection method can be completed in 3-7 h.

[0058] Method of preparation of hooking complexes formed by a microbead and a hook sequence According to a particular aspect, a hooking complex according to the invention suitable for a method of the invention is a complex formed by a microbead and a hook sequence wherein said hook sequence is a hybridizing sequence complementary to only a part of the sequence of the target RNA.

[0059] According to a particular embodiment, the hook sequence has a length from about 10 to 20 nt (e.g. 15 nt).

[0060] The microbead advantageously serves as a solid support for the sequential assembly of the components targeting the target sequence, the generation of a detectable signal and its detection.

[0061] According to a particular embodiment, microbeads have any diameter, typically ranging from 2 to about 30 pm.

[0062] According to a particular embodiment, the hooking complex comprises strept(avidin) coated microbeads onto which surface a biotinylated oligonucleotide hook sequence is immobilized through the formation of strept(avidin) / biotin complex, wherein said oligonucleotide hook sequence is a hybridizing sequence complementary to only a part of the sequence of the target RNA. According to a particular embodiment, microbeads are polystyrene beads.

[0063] According to a particular embodiment, the hooking complex is achieved through the formation of a complex between the surface of the said bead and a carrier of the hook sequence. Typically, as exemplified in Figure 1A, first step, the microbeads 1 used to form hooking complex 3 are stept(avidin) lb coated microbeads la are interacted with a biotinylated 2a oligonucleotide sequence 2b to form a hook sequence 2, wherein said oligonucleotide sequence 2b is a hybridizing sequence complementary to only a part 4a of the sequence of the target RNA 4 to form a hooking complex 3 such that the target RNA sequence can bind partially to the hook sequence 2, once immobilized on the microbeads surface through the formation of stept(avidin) / biotin complex as depicted on Fig. 1A.

[0064] The hooking complex advantageously provides a defined and stable support for immobilizing a composite elongated single strand DNA primer comprising a hybridizing sequence complementary to the part of the sequence of the target RNA which is not complementary to the hook sequence.

[0065] Method of preparation of composite elongated single strand DNA primer

[0066] According to a particular aspect, composites elongated single strand DNA primers suitable for a method according to the invention comprise: i) a x domain: a hybridizing sequence complementary to the part of the sequence of the target RNA which is not complementary to the hook sequence and ii) a plurality of p domains formed after PER cycles.

[0067] Referring to the figures, in particular to Fig.2, the elongated single strand DNA primers are prepared from a ‘bridge primer’ which is elongated to form a concatemer (single strand DNA comprised of multiple copies of itself) by primer exchange reaction (PER) as described in WO 2018 / 132392. The ‘bridge primer’ comprises a x domain: a hybridizing sequence complementary to the part of the sequence of the target RNA which is not complementary to the hook sequence and a p domain which binds to molecules in a hairpin shape (p1), initiating the PER reaction.

[0068] As illustrated in Fig. 2, to assemble the elongated single strand DNA primer (PER concatemer) which recognizes the target oligonucleotide sequence, the bridge primer first binds (1) to the p* domain of the respective hairpin oligonucleotide and extended (2) isothermally in the presence of strand-displacing DNA polymerase until encountering with the stopper base-pair. The stopper is made of a G-C pair and the on-going polymerization halts at this point due to lack of dGTP in the solution, which induces the dissociation of the newly synthesized sequence (xpp) from the hairpin through the branch migration (3). This leads to spontaneous separation between the extended primer and hairpin (4). After this separation, the extended primer (xpp) re-joins a next cycle for further elongation (5). Finally, a long (e.g. 300-600 nt) ssDNA concatemer 6 with repeated p domains (e.g. 30 to 60) formed after many PER cycles (e.g. 30 to 60) which can be used in the method of the invention as elongated single strand DNA primer. Those repeated p domains provide multiple target segments for fluorophore labelled imager strands leading to fluorophore signal amplification.

[0069] According to a further particular aspect, once the hooked target DNA complex wherein the target RNA is hybridized to the hook sequence immobilized on the microbeads is contacted with the composite elongated single strand DNA primer as described herein, an imaging target complex is formed wherein the unhybridized part of the formed hooked target DNA complex forms a duplex with the x domain of said composite elongated single strand DNA primer. The repeated p domains of the elongated single strand DNA primer provide regions available for hybridization with complementary fluorophore labelled imager strands.

[0070] Detecting fluorescence signal from the formed imaging target complex

[0071] According to a further particular aspect, the imaging target complex is then contacted with a plurality of fluorophore labelled imager strands, each of the plurality of p domains hybridizes with a fluorophore labelled imager strand and said imager strand emits a fluorescence signal under an excitation wavelength specific to said imager strands, which, when hybridized to the corresponding p domain is proportional to the amount of target RNAs in said sample.

[0072] According to a further particular aspect, the fluorophore labelled imager strands amount is from about 10 to 20-fold the amount of imaging target complex.

[0073] For this purpose, the composite elongated single strand DNA primer of the imaging target complex is hybridized to fluorophore labelled imager strands directly for linear amplification or through to secondary concatemers and then to fluorescent imager strands for branched signal amplification as illustrated under Figure 3C.

[0074] The image strands generate high fluorescence signal detectable by fluorescence under an excitation wavelength specific to said imager strands. The fluorescence can be measured by fluorescence spectrophotometer or preferably by flow cytometry.

[0075] According to a further particular aspect, wherein a plurality of hooking complexes is contacted with a plurality of the different target RNA sequences, the so-formed plurality of hooked target DNA complexes ais contacted with a plurality of composite elongated single strand DNA primers specific for each different target RNA sequences and a plurality of fluorophore labelled imager strands is used wherein different fluorophores at different wavelength for the multiplexed detection of said different target RNA sequences within the same sample.

[0076] According to a further particular aspect, for the multiple detection of more than one target RNA, different composite elongated single strand DNA primers are contacted to the same beads. For example, for a 2-color detection, two different PER concatemers derived from 2 different primers for 2 different targets (e.g. WT and mutant or 2 different mutants for example) are prepared and those are detectable by the use of two different fluorophore labelled imagers (IS- 488 and IS-647). Both PER concatemers are added in 1 :1 ratio and the fluorophore labelled imagers are added in 1 : 1 ratio as well. The same could be extended to more than two targets to be detected using the same number of different fluorophores (e.g. IS-488, IS-532, IS-560 and IS-647) and 4 unique PER concatemers with different sequences targeting the different targets.

[0077] According to a further particular aspect, beads of different diameters can be used (e.g. of 2 pm, 4 pm and 10 pm) to carry out multiplexed detection of different fragments in a mixture of different sizes of beads using two fluorophores. Multiplexed detection can be based on the combined use of different size of beads and also different fluorophores on each bead. For example, a total 6 different targets can be detected using 3 different sizes of beads with 2 colors. The multiplexing can be further enhanced by increasing the range of bead sizes.

[0078] This allows for applications such as multiplexing (having several cancer biomarkers tested at once), high throughput analyses.

[0079] Kits according to the invention

[0080] According to another aspect of the invention, is provided a kit for the detection of target RNAs 4 in a sample, said kit comprising:

[0081] 1) Biotinylated hook sequences wherein said hook sequences are hybridizing sequences complementary to only a part of the sequence of a target RNA;

[0082] 2) One or more buffered solutions of composites elongated single strand DNA primers said primers comprising: i) a x domain: a hybridizing sequence complementary to the part of the sequence of the target RNA which is not complementary to the hook sequence; and ii) a plurality of p domains formed after a plurality of PER cycles; 3) Fluorophore labelled imager oligonucleotides (IS-647 labelled oligonucleotides) wherein said fluorophore labelled imager oligonucleotides (fluorophore labelled imager strand) comprise a hybridizing sequence complementary to each of the p domains of the composites elongated single strand DNA primers and

[0083] 4) Optionally guide oligonucleotides for the targeting cutting of a target RNA.

[0084] According to another aspect of the invention, the buffered solutions are prepared in ThermoPol® Reaction Buffer as described herein.

[0085] According to another aspect of the invention, a kit of the invention may further comprise one or more of strept(avidin) coated beads and RNAse free water.

[0086] According to another aspect of the invention, the kit comprises different fluorophores at different wavelength for the multiplexed detection of different target RNA sequences within the same sample.

[0087] According to another aspect of the invention, the above kit is suitable for use in a method according to the invention.

[0088] Examples illustrating the invention will be described hereinafter in a more detailed manner and by reference to the embodiments represented in the Figures.

[0089] EXAMPLES

[0090] Materials:

[0091] All unmodified and biotin modified DNA oligonucleotides were ordered from LubioScience- Switzerland IDT (Zurich, Switzerland). Alexa-488 and Alexa-647 modified imager DNA strands (HPLC purified) were purchased from Eurofins MWG Operon (Ebersberg, Germany). Synthetic miRNAs and RNA oligonucleotides were purchased from Microsynth AG (Balgach, Switzerland). The detailed sequences of the oligonucleotides are given in Table SI. Streptavidin coated polystyrene beads (6-6.9 pm diameter) were obtained from Spherotech, Illinois, USA. Bst DNA polymerase (M0275L, 8000 U / mL), MgSO4(100 mM), ThermoPol® reaction buffer, deoxynucleotide (dNTP) solution mix (N0447S), RNase H (M0297S), HiScribe T7 High Yield RNA Synthesis Kit (E2040S) and Monarch® RNA Cleanup Kit (T2050L, 500 pg) were purchased from New England Biolabs (NEB) (Ipswich, MA, USA). NucleoSpin® Gel and PCR Clean-up kit (740609.50), cfDNA isolation kit (cfDNA XS, 740900.50) and RNA isolation kit (NucleoSpin® RNA plus) were purchased from Macherey- Nagel. High Pure miRNA isolation kit was purchased from Roche. Taq DNA polymerase (cat: 10342053), SYBR safe DNA gel stain (lOx, cat: S33102), GeneRuler DNA ladder mix (cat: SM0331), RiboRuler RNA ladders (cat: SM1821 and SM1831) and all cell culture reagents were ordered from Thermo Fisher Scientific (Basel, Switzerland).

[0092] Table 1 - List of oligonucleotide sequences In reference to Figure 1 : x: the first part of any primer for linear PER that is complementary to the target DNA or RNA sequence (e.g. KRAS-G12-Linear-Primer: ACCAGCTCCA); p: extended region by PER & the repetitive segments in the linear PER concatemer (CCAATAATA); p*: complementary region for p that is found in the Hairpin-25 and Imager- 647 (TATTATTGG); T' s in bold represent the spacers.

[0093] Example 1: Preparation of the material for the method of the invention

[0094] The preparation of the target RNAs, hooking complex and composite elongated single strand DNA primer suitable for use in the method of the invention is described below. a) Isolation of RNAs from human cells and cfDNAs from human plasma

[0095] Human cancer cell lines (MCF-7, MDA-MB-231 and A549) were cultured at 37°C, 5% CO2 and 95% humidity in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with Glutamax, 10% fetal bovine serum (FBS), and 1% Penicillin and Streptomycin. mRNA isolation from these cell lines was performed using the NucleoSpin® RNA plus RNA isolation kit (106cells). Extraction was done as described in the kit protocol. To isolate miRNAs from cells, the column protocol for the isolation of the small RNA was applied as described in the High Pure miRNA isolation kit. To detect circulating DNAs from human blood samples, 240 pL of plasma was used for each sample (8 samples from breast cancer patients and 3 samples from healthy donors). MiRNAs were extracted using NucleoSpin miRNA Plasma kit for circulating miRNA. Previously collected plasma samples were used in this study (Cattin et al., 2021, Breast Cancer Res 23(1), 64. DOI: 10.1186 / sl3058-021-01441-8). b) Providing a hooking complex formed by a microbead and a hook sequence:

[0096] 20 pL of streptavidin coated polystyrene beads (6-6.9 pm diameter, 0.5% w / v) were transferred to V-bottom 96-well microplate (Thermo Fisher Scientific (Basel, Switzerland)) and completed up to 100 pL with lx PBS. The solution was centrifuged at 2000 rpm (Awel MF48-R) for 5 min and the supernatant removed completely. Then, the plate was placed on ice and the beads were incubated sequentially with biotinylated oligonucleotide (100 nM) for 30 min and target RNA containing solution for 30 min. c) Providing a composite elongated single strand DNA primer according to the invention: Composite elongated single strand DNA primers (PER concatemers) were prepared based on a previous method with slight modification (Saka et al., 2019, Nat Biotechnol, 37 (9), 1080-1090. DOI: 10.1038 / s41587-019-0207-y). Typically, 100 pL reaction mixtures was prepared in lx ThermoPol reaction buffer (diluted from lOx stock) with final concentrations of 10 mM MgSCU, 400 U / mL of Bst LF polymerase, 600 pM each of dATP, dCTP, and dTTP, 100 nM of Clean G hairpin and 0.15 pM of hairpin for target oligonucleotide. The bridge primer is designed in a way that a part of it named as “x“ binds to the target RNA (by considering the melting temperature and GC content the length is varying between 9-12 nt) and the other part of it named as “p” is the 9 nt region that is elongated by primer exchange reaction.

[0097] After addition of water to 99 pL and the reaction mixture was incubated for 15 min at 37°C, followed by the addition of 1 pL of 100 pM primer (Xp) to obtain 1 pM final concentration, and the reaction was incubated for another 1-3 h at 37 °C. The reaction was terminated by heating to 80°C for 20 min to deactivate the polymerase.

[0098] Independently assembled secondary concatemers

[0099] Secondary concatemers were prepared as the primary concatemer, the only difference being in the sequence of the used primer: called as a bridge primer, which is not target / dependent. In this case, the primary concatemers were prepared using the target specific primers, which are elongated in the presence of another hairpin (Hairpin-29, Table 1).

[0100] The generation of PER concatemers and hybridization of imager strands to those were verified by gel analysis as follows:

[0101] The PER solution was incubated for 3 h at 37°C. After heat inactivation of polymerase for 20 min, samples were mixed with 6x loading dye and run in 1.5 % agarose containing lx SYBR safe at 80 V for 40 min. 20 pL of unpurified solution out of 100 pL PER assembly was loaded into the gel in the presence or absence of imager strand (IS-647). 100 pmol primer and 100 pmol hairpin were loaded as controls. The length of the concatemers was 300-600 nt.

[0102] Example 2: Applying the method of the invention to the detection of target sequences

[0103] In order to test whether a method according to the invention may achieve fast and ultrasensitive detection of cancer-associated nucleic acids including mRNAs, miRNAs and ctDNAs that carry cancer driver mutations, the method of the invention was implemented as follows:

[0104] Streptavidin-coated beads with a diameter of 6 pm and a hooking complex were prepared as described above hooking complex was then incubated with 20 pL of a primary PER solution obtained as described above for 30 min, 20 pL of secondary PER solution for 30 min (for branched amplification) and imager strand (1 pM) for 30 min in 100 pL of lx PBS. Between each step, the beads were rinsed with 100 pL of lx PBS and centrifuged at 2000 rpm for 5 min to wash unbound molecules away. After incubation for the respective times, the beads were analyzed using Cytek Aurora flow cytometer (Figures 3 and 4) or MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec) (Figures 5 and 6). All flow cytometers were calibrated using specific QC beads and protocol, and instrument setting were standardized using the obtained gain and minimal aspiration speed. For Cytek Aurora, unmixing was done using proper reference and unstained controls with autofluorescence extraction. Data was analyzed with FlowJo Software (vlO, FlowJo LLC). Mean fluorescence intensity (MFI) for all samples were background corrected by subtracting the MFI values from the appropriate controls.

[0105] Determination of the detection sensitivity

[0106] A synthetic single stranded DNA fragment of human KRAS proto-oncogene was used as target oligonucleotide for the detection with a linear and branched fluorescence signal amplification approaches.

[0107] Different flow cytometers that are both conventional and full spectrum technology (some of them are commonly used in the clinics, e.g. BD FACS Canto II) (Cytek Aurora, BD FACS Canto II, BD LSR Fortessa and MACSQuant Analyzer) were used to compare the detection sensitivities based on their internal parameters (e.g. laser power). Target oligonucleotide concentrations in the range from 100 nM to 100 fM. Among these systems, the highest sensitivity was achieved using the full spectrum flow cytometer Cytek Aurora in which 100 fM of oligonucleotide could be detected with linear fluorescence amplification, whereas the detection limit using MACSQuant flow cytometer was reached at 10 pM. The detection sensitivities obtained by using both BD instruments (Canto II and LSR Fortessa) were similar with a detection limit of 1 pM and less sensitive compared to Cytek Aurora.

[0108] Therefore, Cytek Aurora flow cytometer was used for the sensitivity experiments. To calculate the detection sensitivity using linear signal amplification method, the beads were incubated in the buffer solutions containing different concentrations of target oligonucleotides ranging from 100 pM to 1 fM as shown in Figure 3A and those were incubated with linear elongated single strand DNA primers of the invention prepared as described above and finally incubated with fluorophore labelled imager strands of the invention as described herein. Flow cytometry analysis with a linear signal amplification demonstrated that the binding of KRAS oligonucleotide is maximal, with a mean fluorescence intensity (MFI) of 3.95 x 104when 100 pM of oligonucleotide was added to the beads and the binding of KRAS oligonucleotide was gradually decreased down to the concentration of 1 fM, with an MFI of 7.24 x 104. The MFI without target oligonucleotide was measured as 7.45 x 104. The theoretical limit of detection (LoD) of target oligonucleotides was defined as the mean fluorescence intensity plus three standard deviations (3 c) of the blank sample. By fitting all the measured data points (100 pM to 1 fM) to an asymmetric five-parameter curve, the LoD of KRAS oligonucleotide was determined as 94 fM.

[0109] In order to provide further signal enhancement, a sequential amplification strategy was used where after the first primary concatemers were added and a washing and a centrifugation step were carried out, secondary concatemers are added to the mixture and finally the fluorophore labelled imager strands were added and the mixture was analyzed by flow cytometry after washing and centrifugation steps. (Primary and secondary concatemers were prepared in separate PER reactions using different primers and hairpins). The secondary concatemers were hybridized and branched off the primary concatemers so that more binding sites for fluorescent imager strands are generated (Figure 3B). Compared to linear signal amplification, the fluorescence intensity generated by branched signal amplification (Figure 3B) was increased up to 3.17-fold at the concentration of 100 pM target oligonucleotide (MFI: 1.25 x 105) and 3.57-fold at the concentration of 100 fM of target oligonucleotide (MFI: 3.43 x 104). Using the same formula generated from the asymmetric curve, the LoD of KRAS oligonucleotide using branched signal amplification was determined as 27 fM. With respect to unamplified signal, the linear PER assembly method yielded 75-fold signal amplification and branched PER assembly method yielded 284-folds signal amplification (Figure 3C). The amplification levels were comparable with previous studies where PER-based signal amplification was applied for in situ hybridization on fixed samples (Saka et al., 2019, Nat Biotechnol, 37 (9), 1080-1090. DOI: 10.1038 / s41587-019-0207-y).

[0110] Detection of Target RNAs from Cell Extracts

[0111] Three different target RNAs: 1) actin mRNA, the housekeeping gene encoding actin cytoskeleton protein, 2) vimentin mRNA, a marker of epithelial to mesenchymal transition (EMT), and 3) miR-21, an oncogenic miRNA highly expressed in several cancer types. Initially, to estimate the detection sensitivity of the method of the invention for RNA targets, the beads were incubated separately with the synthetic RNA fragments of the corresponding target RNAs (actin-RF: 30 nt, vimentin-RF: 32 nt and miR-21 : 22 nt, the sequences are given in Table 1 above) using different concentrations of target RNAs ranging from 10 nM to 1 pM (Figure 4A). All of target RNAs were detectable at the concentration of 1 pM (MFIactin: 19.9xl03, MFIvimentin: 23xl03, MFImiR2i: 29.3xl03). Then, to expand the detection capability cell-derived mRNAs and miRNAs were extracted from the human breast cancer cell lines MCF-7 and MDA-MB-231 as described in Example 1.

[0112] First, for the detection of target mRNAs, RT-PCR and T7 transcription were performed to amplify and generate large quantities of the actin and vimentin mRNAs. Agarose gel analysis revealed the successful production of transcripts in vitro with desired lengths (actin mRNA: 633 nt and vimentin mRNA: 718 nt). When long target mRNA strands were directly incubated the beads (Figure 4, IVT uncut), the obtained signal was lower than expected even though the initial mRNA amount is high (MFIactin: 59xl03and MFIvimentin: 23.4xl04). This is most likely due to the secondary structure formation of transcribed mRNAs masking the target sequence thereby interfering with the efficient capture of the target RNA. To avoid this problem, a targeted cutting of target mRNA using ribonuclease (RNase) H was carried out as described above. The guide oligonucleotides (ssDNA, 20 nt) (Table 1: Actin guide 1 & 2, Vimentin guide 1&2) were designed to bind upstream and downstream on the specific (capture) regions of the target mRNAs. Then, RNase H was used to digest the RNA sequence in DNA: RNA hybrids thereby inducing the release of the target RNA fragment in the middle. The bands visualized in the agarose gel confirmed the production of the respective RNA fragments of actin and vimentin. After cutting with RNase H, the signal obtained from the beads incubated with fragmented target RNAs increased by an order of magnitude. The mean fluorescence intensity of actin fragment was 9.62-folds higher than fluorescence intensity of the uncut actin mRNA (MFIactin-cut: 5.72xl05) and the mean fluorescence intensity of vimentin fragment was 17.4-folds higher than the signal obtained from the uncut mRNA (MFIvimentin-cut: 4.07xl06). With these experiments, it is demonstrated that targeted cutting of long mRNA strands into shorter fragments further increased the detection capacity of the PER-based signal amplification approach (Figure 4).

[0113] After the successful detection of in vitro transcribed and fragmented target RNAs, the direct detection of selected target RNAs from the cell extracts without any pre-amplification steps was carried out. For this purpose, extracted RNAs were mixed with respective guide oligonucleotides for actin and vimentin as described herein. After cutting with RNase H and incubating the solution containing fragmented RNAs with beads, branched signal amplification was applied. The results demonstrated the detection of actin and vimentin RNA fragments in the range of 0.3 pM to 0.45 pM. The concentration of actin was higher in both cell lines compared to vimentin (MDA-MB-231 : 0.45 pM, MCF-7: 0.37 pM). The concentration of vimentin is slightly higher in MDA-MB-231 cells (0.32 pM) compared to the MCF-7 cells (0.3 pM) (Figure 4). Then, for the detection of miR-21, the beads were incubated with a solution containing cell-extracted small RNA. The results demonstrated that the concentration of miR- 21 was high in both cancer cell lines (MDA-MB-231 : 48.2 pM and MCF-7: 40.3 pM).

[0114] Example 3: Applying the method of the invention to the single-nucleotide mutations from cell extracts and human plasma

[0115] To assess the specificity of the method of detection the invention, the ability to discriminate wild type sequences from single nucleotide mutations that occur frequently in cancer was investigated.

[0116] Plasma ctDNAs carrying these mutations originated from the cancer cells serve as significant and reliable cancer biomarkers in liquid biopsy but their concentrations in the bloodstream of cancer patients is extremely low and are mixed with ctDNA fragments of wild-type sequences derived from normal cells (Elazezy et al., 2018, Comput Struct Biotechnol J, 16, 370-378. DOI: 10.1016 / j.csbj.2018.10.002).

[0117] To determine the ctDNA detection capability of our sensing approach, a screening for the genetic lesions for the common mutations that are found in several cancer types by PCR and Sanger sequencing. Among these mutations, the Gly-12 codon (G12) in KRAS (GGT) is a site of multiple base changes observed in cancer was carried out (Zhang et al., 2022, Nat Chem Biol, 18 (11), 1177-1183. DOI: 10.1038 / s41589-022-01065-9).

[0118] The KRAS G12S is a prominent cancer driver mutation, accounting for 4.4 % of all KRAS mutations that has been observed, including in 2.5% of non-small-cell lung cancer (NSCLC) and 2.8% of colorectal adenocarcinoma (O'Bryan, 2019, Pharmacol Res, 139, 503-511. DOI: 10.1016 / j.phrs.2018.10.021; Consortium, A. P. G. AACR Project GENIE: Powering Precision Medicine through an International Consortium. Cancer Discov 2017, 7 (8), 818-831. DOI: 10.1158 / 2159-8290.CD-17-015P). Moreover, PIK3CA mutations (e.g. E542K, E545K) are common activating mutations in breast cancer (occurring in 20-30% of all cases) and are potent predictive markers for responses to PI3K inhibitors in Estrogen Receptor-Positive, HER2- Negative (ER+ / HER2 ) breast cancers (Shimoi et al., 2018, Cancer Sci, 109 (8), 2558-2566. DOI: 10.111 l / cas.13696) . In addition to this, P53 mutants (e.g., R248Q, R273H, and R280K) can be classified as contact mutants associated with breast cancer, particularly ER', that are involved in DNA binding without causing protein unfolding but inhibit its transcriptional activity (Gomes et al., 2018, IntJMol Sci, 19 (4). DOI: 10.3390 / ijmsl9041184

[0119] To screen for these genetic alterations, RNAs were isolated from lung (A549) and breast cancer cell lines (BT-474, MDA-MB-231 and MCF-7) and the transcripts including target mutations were amplified by RT-PCR. After sequencing of these regions (sequences in Table 2 below), it was observed that the KRAS G12S mutation is present only in A549 cells, whereas PIK3CA E545K and P53 R280K mutations are present in ER+ / HER2‘ and ER7HER2' breast cancer cells, MCF-7 and MDA-MB-231 respectively.

[0120] Table 2 - The sequences of amplified regions from cell extracted RNAs using the designed primers given in Table 1

[0121] Binding sites for guide oligonucleotides were depicted with underline and mutation codon was depicted in bold.

[0122] Then, to detect these mutations using the method of the invention, the amplified regions were first in vitro transcribed and incubated the purified RNA strands with respective guide oligonucleotides for RNase H cleavage (KRAS guide 1&2, P53 guide 1&2 and PIK3CA guide 1&2 in Table 1). After cleavage, the fragmented RNA strands carrying mutations (18-23 nt, sequences in Table 1) were separately incubated on the beads covered with respective biotinylated oligonucleotides (WT or mutant probes) to form a hooking complex according to the invention. The KRAS mutation was located in the region (10 nt) (4b) that hybridizes to the composite elongated single strand DNA primer (PER concatemer), whereas P53 and PIK3CA mutations were located in the regions (9 nt and 11 nt respectively) that hybridize to the biotinylated oligonucleotides (4a). The beads were further incubated with PER concatemers complementary to the target fragments non-hybridized to the hooking complex and the fluorescence intensities were recorded using MACSQuant flow cytometer. For the wild-type and mutant probes, two different biotinylated oligonucleotides were used for P53 and PIK3CA RNAs and two different PER concatemers were used for KRAS RNA. The results showed that the fluorescence intensities were significantly higher in all conditions when the fragmented RNAs of target regions were incubated with mutant probes (MFIKRAS-WT: 2.16, MFIKRAS-GOS: 18.7, MFIP53-WT: 3.84, MFIPS3-R28OK: 16.2, MFIPIK3CA-WT: 31.9, MFIPIK3CA-E545K: 65.5) (Figure 5A). The signal obtained from PPK3CA-WV is higher compared to the other wild-type signals, which is probably due to the presence of two different alleles (heterozygosity) for PIK3CA gene in MCF-7 cells (Table 2). Overall, it was demonstrated that the abovementioned cancerspecific mutations could be efficiently distinguished from the wild-type sequence using the method of the invention.

[0123] The detection specificity of the method of the invention being confirmed on cellular extracts, the method was then used to identify ctDNAs with target mutations from the plasma of breast cancer patients. For this, ctDNAs were extracted from the plasma of the 8 patients with early, non-metastatic, breast cancer and 3 age-matched healthy donors for a proof-of-concept experiment. First, the P53 gene was amplified for all samples and sequenced the amplicons using Sanger sequencing. After screening of all samples, a heterozygous R280K mutation was observed in the ctDNA extracted from one of the patient plasma samples (Pl), whereas none of the ctDNAs extracted from the healthy individuals carry the respective mutation. The sequencing results are given in Table 3.

[0124] Table 3 - Sequences of P53 gene region amplified from ctDNAs extracted from human plasma from healthy and patient plasma samples using the designed primers given in Table 1

[0125] Binding sites for guide oligonucleotides were depicted with underline and mutation codon was depicted in bold.

[0126] Then, to distinguish these mutations using the method of the invention, the respective RNA strands were produced from three ctDNA samples (Pl, P8 and H3) by in vitro transcription from the amplified regions and mixed the purified RNA strands with guide oligonucleotides for RNase H cleavage. After cleavage, PER-based linear signal amplification was applied by incubating fragmented RNAs on the beads and the fluorescence signal was recorded with MACSQuant flow cytometer according to the method of the invention. The results showed that one can specifically detect the heterozygous mutation located in the R280K codon (ARA) from the patient sample Pl using PER-based approach (Figure 5B). The mean fluorescence intensity of the beads treated with RNA fragments of the Pl sample was significantly higher (MFIpi: 115) than the beads treated with the RNA fragments of P8 and H3 (MFIps: 15.1 and MFIm: 10). Consistent with this finding, the intensity of the adenine signal in the heterozygous R280K codon (G / A) of the Pl amplicon was also higher than the adenine signal of the P8 and H3 amplicons according to the Sanger sequencing (Figure 5B). CtDNAs exist alongside a vast background of wild-type DNAs, which are frequently distinguished by single-nucleotide changes with a variant allele frequency as low as 0.01% (Shin etal., 2023, Biosens Bioelectron, 242, 115694. DOI: 10.1016 / j.bios.2023.115694).

[0127] To precisely detect and accurately quantify these single mutations, multicolor detection of the KRAS G12S mutation was performed in the background of KRAS wild-type DNA fragments. For this, the beads were first incubated with biotinylated oligonucleotides and then added the WT and mutant KRAS fragments in different ratios in lx PBS (WT:M; 1 :1, 10: 1, 100:1, 1000: 1 and only WT) by keeping the WT fragment concentration at 100 nM. Then, corresponding PER concatemer probes were added in equal amounts designed for WT and mutant fragments distinctively, where the WT probe hybridizes with Alexa-488 labelled imager strands and the mutant probe hybridizes with Alexa-647 labelled imager strands. Flow cytometry data demonstrated that the mean fluorescence intensity obtained from mutant strands were highest at 1 : 1 ratio (MFIGOS SO%: 391) and decreased linearly down to 1000: 1 ratio (MFIGOS IO%: 174, MFIGOS I%: 126, MFIGOS O.I%: 118) (Figure 6). When there is no mutant strand in the solution the MFI obtained from Alexa-647 channel was 114, showing that one can detect the mutant strand even at the 0.1% of the WT strand concentration. In comparison, the MFI of WT strand at 1 : 1 ratio was 993 and the fluorescence signal did not change dramatically in all the other conditions (MFIGOS IO%: 1171, MFIGOS I%: 1195, MFIGOS O.I%: 1188 and MFIWT: 1206). Overall, these results indicate that the method according to the invention can specifically detect the single mutations in target DNA and RNA strands and with the sensitivity of 0.1% of the concentration of the WT strands.

[0128] In summary, those data support that the method of the invention allows a rapid, ultrasensitive and highly specific detection of cancer-associated nucleic acids and cancer-specific mutations. It has been demonstrated to be precise and accurate detection of both synthetic and natural nucleic acids extracted from human cells and plasma from breast cancer patients.

[0129] The whole detection process can advantageously be completed in 3 h starting from biotinylation of the beads to the flow cytometry measurements and can take up to 7-8 h when preamplification, RNA synthesis and cleavage steps are required.

[0130] Example 4: Applying the method of the invention to multiplexed detection

[0131] For multiplexing, different size of beads were used in addition to use of two different fluorophores as shown in Figure 6. For this, each bead (with the diameter of 2 pm, 4 pm and 10 pm) was incubated with two different biotinylated oligonucleotides (Table 4) (2 pm: for EGFR R858 and T790, 4 pm: for EGFR L861 and PIK3CA R1047, 10 pm: for BRAF V600 and KRAS G12) at 1 : 1 ratio (100 nM each) in 100 pl of lx PBS. 30 min after incubation, the beads were washed with IxPBS and centrifuged, and then the beads were mixed in the same well in 100 pl of lx PBS solution containing target oligonucleotides (EGFR R858, EGFR L861 and BRAF V600, 25 nM each). Then, corresponding PER concatemers were added in equal amounts (5 pl each in 100 pl of lx PBS) designed for these targets distinctively, where the EGFR R858 probe hybridizes with Alexa-488 labelled imager strands and the EGFR L861 and BRAF V600 probes hybridize with Alexa-647 labelled imager strands. Finally, the imager strands were added (500 nM each) and analyzed the mixture by flow cytometry after washing and centrifugation steps as shown under Figure 7. The bead populations were gated using forward and side scattering by using the corresponding gates from the separate detection. Figures 7A shows it is possible to carry out multiplexed detection of different fragments in a mixture of different sizes of beads using two fluorophores with the same sensitivity as the separate detection of those fragments using two fluorophores (Figures 7B).

[0132] Table 4 - The sequences of oligonucleotides used for multiplexed detection

[0133] Example 5: Example of a kit according to the invention and uses there of

[0134] A kit for the detection of target RNAs in a sample may comprise at least the following components: 1) Biotinylated hook sequences wherein said hook sequences are hybridizing sequences complementary to only a part of the sequence of a target RNA;

[0135] 2) One or more buffered solutions of composites elongated single strand DNA primers said primers comprising: i) a x domain: a hybridizing sequence complementary to the part of the sequence of the target RNA which is not complementary to the hook sequence; and ii) a plurality of p domains formed after a plurality of PER cycles;

[0136] 3) Fluorophore labelled imager oligonucleotides wherein said fluorophore labelled imager oligonucleotides (fluorophore labelled imager strand) comprise a hybridizing sequence complementary to each of the p domains of the composites elongated single strand DNA primers and

[0137] 4) Optionally guide oligonucleotides for the targeted cutting of said target RNA. A kit of the invention may further comprise one or more of strept(avidin) coated beads and RNAse free water.

[0138] Alternatively, to one or more solutions of composites elongated single strand DNA primers, the kit may comprise the corresponding primers for PER and hairpin oligonucleotides (Clean G hairpin, Target hairpin) to prepare the composites elongated single strand DNA primers.

[0139] The kit can be used for the following steps: RNAse cleavage (Steps 1-3), PER (Steps 4-7) and detection of target RNA (8-14). Steps 1-3 and Steps 4-7 are independent reactions and can be done simultaneously.

[0140] Typical instructions of use for those respective steps are as follows:

[0141] 1) Mix the solution containing transcribed target RNA sample (5 pg) or cell / tissue / plasma isolated total RNA (20 pg) with 2 pL of guide oligonucleotides (1 pM each) in lx RNAse H reaction buffer (not provided in the kit) and complete up to 19,5 pL with RNase free water.

[0142] 2) Heat the solution to 70°C for 5 min for DNA: RNA hybridization. Then, add 0,5 pL of RNase H (not provided in the kit), mix and incubate for 2 h at 37°C to allow the enzyme to cut RNA in the DNA: RNA hybrid for the release of the target RNA fragment for detection.

[0143] 3) Inactivate RNase H by incubation at 65°C for 10 min.

[0144] 4) Prepare 100 pL of PER solution in lx ThermoPol reaction buffer (diluted from lOx stock) with final concentrations of 10 mM MgSO4, 400 U / mL of Bst LF polymerase, 600 pM each of dATP, dCTP, and dTTP, 100 nM of Clean G hairpin and 0.15 pM of hairpin for target oligonucleotide. (ThermoPol reaction buffer, MgSO4, Bst polymerase and deoxynucleotide triphosphates (dNTPs) are not provided in the kit)

[0145] 5) Add water to complete up to 99 pL and incubate the reaction mixture for 15 min at 37°C.

[0146] 6) Add 1 pL of 100 pM primer solution to obtain 1 pM final concentration and incubate the reaction for another 1-3 h at 37°C.

[0147] 7) Terminate the reaction by heating to 80°C for 20 min to deactivate the polymerase.

[0148] 8) Transfer 10 pL of streptavidin coated polystyrene beads to V-bottom 96-well microplate (not provided in the kit) and complete up to 100 pL with lx PBS.

[0149] 9) Centrifuge the plate at 2000 rpm for 5 min and remove the supernatant completely.

[0150] 10) Put the plate on ice and incubate the beads with biotinylated oligonucleotide (100 nM) for 30 min. 11) Wash the mixture by adding 100 pL of lx PBS and repeat step 9. Add target RNA containing solution (from step 3) in 100 pL of lx PBS and incubate for 30 min.

[0151] 12) Wash the mixture by adding 100 pL of lx PBS and repeat step 9. Add 10 pL of PER solution (from step 6) in 100 pL of lx PBS and incubate for 30 min. 13) Wash the mixture by adding 100 pL of lx PBS and repeat step 9. Add imager oligonucleotide (1 pM) in 100 pL of lx PBS and incubate for 30 min.

[0152] 14) Wash the mixture by adding 100 pL of lx PBS and repeat step 9. Analyze the beads using flow cytometer.

Claims

Claims:

1. A method for the detection of target RNAs in a sample, said method comprising:- contacting a complex formed by a microbead and at least one hook sequence, wherein said at least one hook sequence is a hybridizing sequence complementary to only a part of the sequence of the target RNA to form a hooked target DNA / RNA complex at the surface of the microbead wherein the target RNA is hybridized to the said at least one hook sequence;- contacting said formed hooked target DNA / RNA complex to a composite elongated single strand DNA primer, to form an imaging target complex wherein the unhybridized part of the formed hooked target DNA complex forms a duplex with the x domain of said composite elongated single strand DNA primer, wherein said composite elongated single strand DNA primer sequence comprises i) a x domain: a sequence hybridizing complementary to the part of the sequence of the target RNA which is not complementary to the hook sequence and ii) a plurality of p domains formed after a plurality of PER cycles;- subjecting said formed imaging target complex to a plurality of fluorophore labelled imager strands, wherein each p domain hybridizes with a fluorophore labelled imager strand;- detecting a fluorescence signal emitted by the so formed fluorescent complex.

2. A method according to the preceding claim, wherein the target RNAs are sequences from about 20 to about 30 nt.

3. A method according to the preceding claims, wherein the microbeads have a diameter ranging from 2 to about 30 pm.

4. A method according to any one of the preceding claims, wherein the hooking complex comprises strept(avidin) coated microbeads onto which surface a biotinylated oligonucleotide hook sequence is immobilized through the formation of strept(avidin) / biotin complex, wherein said oligonucleotide hook sequence is a hybridizing sequence complementary to only a part of the sequence of the target RNA.

5. A method according to any one of the preceding claims, wherein the microbeads are polystyrene beads.

6. A method according to any one of the preceding claims, wherein the composite elongated single strand DNA primer is a linear primer for a direct amplification through hybridization with a fluorophore labelled imager strand with the plurality of p domains of the elongated single strand DNA primer.

7. A method according to any one of the preceding claims 1 to 6, wherein the composite elongated single strand DNA primer, once hybridized with the hooked target DNA / RNA complex, is then further hybridized with a second the composite elongated single strand DNA primer (second concatemer) before subjecting said formed imaging target complex to a plurality of fluorophore labelled imager strands, wherein said second the composite elongated single strand DNA primer is branched and therefore presenting further possible hybridization sites for the plurality of fluorophore labelled imager strands for a further amplification of the fluorescence signal.

8. A method according to any one of the preceding claims 1 to 6, wherein the composite elongated single strand DNA primer is concatemer with 30 to 60 repeated p domains formed after 30-60 PER cycles.

9. A method according to any one of the preceding claims, wherein a washing steps (about 5 min) are carried out between each step.

10. A method according to any one of the preceding claims, wherein the target RNAs is directly used after extraction from a biological sample without pre-amplification.

11. A method according to any one of the preceding claims 1 to 9, wherein the target RNAs is obtained after a pre-amplification step comprising DNA pre-amplification, in vitro transcription and RNAse H mediated cleavage.

12. A method according to any one of the preceding claims, wherein a plurality of hooking complexes is contacted with a plurality of the same target RNA sequences, the detected fluorescence signal is to be proportional to the amount of target RNAs in said sample.

13. A method according to any one of the preceding claims, wherein a plurality of hooking complexes is contacted with a plurality of the different target RNA sequences, the so-formedplurality of hooked target DNA / RNA complexes is contacted with a plurality of composite elongated single strand DNA primers specific for each different target RNA sequences and a plurality of fluorophore labelled imager strands is used wherein different fluorophores at different wavelength for the multiplexed detection of said different target RNA sequences within the same sample.

14. A method according to claim 13, wherein a plurality of hooking complexes comprising beads of different diameters to carry out is used for the multiplexed detection of different fragments in a mixture of different sizes of beads using a plurality of fluorophores.

15. A kit for the detection of target RNAs in a sample, said kit comprising:- Biotinylated hook sequences wherein said hook sequences are hybridizing sequences complementary to only a part of the sequence of a target RNA;One or more buffered solutions of composites elongated single strand DNA primers said primers comprising: o a x domain: a hybridizing sequence complementary to the part of the sequence of the target RNA which is not complementary to the hook sequence; and o a plurality of p domains formed after PER cycles;- Fluorophore labelled imager oligonucleotides wherein said fluorophore labelled imager oligonucleotides (fluorophore labelled imager strand) comprise a hybridizing sequence complementary to each of the p domains of the composites elongated single strand DNA primers; andOptionally guide oligonucleotides for the targeted cutting of said target RNA.

Images