When the molecular beacon encounters its target sequence, the complementary base pairing between the loop and the target sequence is stronger than the base pairing in the stem. This causes the stem to unwind and the loop to hybridize with the target. As a result, the fluorophore and quencher are separated, allowing the fluorophore to emit fluorescence. The intensity of the fluorescence is directly proportional to the amount of target sequence present, making molecular beacons useful for quantitative assays.

The typical length of the nucleic acid probe, including those used as molecular beacons, may range from 5 to 50 nucleotides, from 8 to 45 nucleotides, from 10 to 40 nucleotides, from 15 to 30 nucleotides, from 18 to 25 nucleotides or about 20 nucleotides. This length is generally sufficient to provide specific and stable hybridization with the target sequence while avoiding non-specific binding.

The design of the probe may take into account the following factors:

- The sequence of the probe is carefully selected to be complementary to a unique region of the target nucleic acid, minimizing the likelihood of cross-reactivity with other sequences.
- The length of the probe also influences its melting temperature (Tm), which is the temperature at which half of the probe-target duplex dissociates.

For molecular beacons specifically, the length of the loop region (the part complementary to the target) typically falls within this range, while the stem region usually consists of 4 to 6 base pairs. The exact length and composition can vary based on the specific application and the nature of the target sequence.

In one embodiment the hybridization done in steps b3), c3) and d3), as well as steps cc3) and dd3), at an temperature (Ta) which is below Tm (Ta<Tm). In some embodiments Ta is a temperature from 0° C. to the melting temperature of both molecules. In some embodiments Ta is below Tm of at least 1° C., of at least 2° C., of at least 3° C., of at least 4° C., or of at least 5° C. Ta is in one embodiment at 0° C., in other embodiments above 0° C. (Ta>0° C.) in order to avoid freezing of the buffer medium. It depends on the freezing point of the buffer medium how low Ta may be selected. Thus, in one embodiment Ta is >freezing point of the buffer medium. In further embodiments Ta is 1° C. or more, 2° C. or more, 3° C. or more, 5° C. or more, 10° C. or more, or 15° C. or more. Thus, in one embodiment Ta may be selected from freezing point of buffer to Tm, from >freezing point of buffer to <Tm, 0° to Tm, from 0° C. to <Tm, from >0° C. to <Tm, from 0° C. to Tm−1° C., from 0° C. to Tm−2° C., from 0° C. to Tm−3° C., from 0° C. to Tm−4° C., from 0° C. to Tm−5° C., from >0° C. to Tm−1° C., from >0° C. to Tm−2° C., from >0° C. to Tm−3° C., from >0° C. to Tm−4° C., from >0° C. to Tm−5° C., from 1° C. to Tm−1° C., from 2° C. to Tm−2° C., from 3° C. to Tm−3° C., from 5° C. to Tm−4° C., or from 10° C. to Tm−5° C.

The term “freezing point” is defined herein as the temperature at which a liquid turns into a solid under normal atmospheric pressure. At this temperature, the molecules of the liquid slow down enough due to a decrease in thermal energy to form a solid crystalline structure. This phase transition is a characteristic property of the substance and varies depending on the nature of the liquid.

In a further embodiment a method is disclosed wherein instead to the described steps c) to e), steps cc) and dd) and ee) are performed, comprising:

- cc) repeating step b) in the presence of a sample molecule selected from a small molecule, peptide or protein, thereby measuring the energy E^Pwhich is the energy of the hybridized nucleic acid molecule in the presence of the sample molecule;
- dd) calculate ΔE according to the formula ΔE=E^K−E⁰; calculate ΔE′^Paccording to the formula ΔE′=E′^K−E′⁰;
- ee) calculate ΔE^Paccording to the formula ΔE^P=E^P−E^0P; calculate ΔE′^Paccording to the formula ΔE′^P=E′^P−E′^0P,
- wherein the difference between the values of ΔE and ΔE^Pand ΔE′ and ΔE′^Pindicates an interaction of the sample molecule with the duplex of the probe and the sample nucleic acid molecule.

Please note also FIG. 6 for further information.

As outlined above the method may also be used to detect the “interference” of a molecule, selected from a small molecule (i.e. a small organic molecule), peptide and/or protein on the nucleic acid duplex. In such a case the molecule is added to the buffer and the energy shifts with respect to the solutions without the presence of the molecule are detected and calculated.

In another embodiment of the disclosure the “interference of the molecule” (such as a sample small molecule (i.e. a small organic molecule), peptide and/or protein) with the nucleic acid is selected from the group consisting of bending of the nucleic acid, looping of the nucleic acid, altering accessibility and compaction of the nucleic acid, unwinding of the nucleic acid, supercoiling the nucleic acid, and modify the nucleic acid, as well as combinations thereof.

Further, the method may also be used to define “permissive” buffer medium conditions. Permissive buffer conditions are conditions in which the energy difference between 100% complete hybridization and mispairing hybridization is either nearly zero or the mispairing event is even energetically “favoured”. In those “permissive” buffer conditions therefore the mispairing event may either be statistically equal to a 100% complementary hybridization and/or may even be statistically more likely. These “permissive” buffer conditions may then be used for PCR or other nucleic acid amplification techniques in which the mispairing of the two nucleic acid sequences is preferred. Without being bound to this theory, the insight that there are buffer conditions in which nucleic acid mismatches are favoured also seems to explain why mismatched nucleic acids are more frequently amplified in some cells. This allows for the hypothesis that, at least in certain cancers and other diseases, where nucleic acid mismatches play a role, it might be explained by the presence of buffer conditions such cells in which the energetic advantage lies with incomplete hybridization rather than 100% complementary hybridization. In this sense, the disclosed method can also be adapted to measure the energetic conditions of a cell medium, and in the case of a “permissive” cell medium composition, to predict the likelihood of disease development. Thus, in one embodiment the disclosed methods may be used to predict the chances to develop a disease originating form nucleic acid mispairings, such as one selected from the group consisting of cancer, lynch syndrome (Hereditary Nonpolyposis Colorectal Cancer, HNPCC), xeroderma pigmentosum, mutations leading to genetic disorders, cystic fibrosis, sickle cell anaemia, neurodegenerative disorders such as Huntington's disease and certain forms of spinocerebellar ataxia, Fanconi anaemia, hereditary breast and ovarian cancer syndrome (such as caused by mutations in BRCA1 and BRCA2 genes), and ataxia-telangiectasia.

In one embodiment the buffer medium is water or any buffer allowing a hybridization of the nucleic acid molecules tested, i.e. which provide the necessary ionic strength and environment to facilitate the hybridization of nucleic acids to complementary strands.

In some embodiments the buffer medium may comprise:

- a) a salt selected from sodium chloride (NaCl), sodium citrate, and sodium phosphate, as well as any combination thereof;
- b) a detergent selected from the group consisting of sodium dodecyl sulfate (SDS), polyethylene glycol tert-octylphenyl ether (Triton X-100), nonylphenoxypoly(ethyleneoxy)ethanol (Nonidet P-40 (NP-40)), octylphenoxypolyethoxyethanol (Igepal CA-630), Polysorbate 20 (Tween 20), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), N-lauroylsarcosine sodium salt (Sarkosyl) and sodium deoxycholate (Deoxycholate), as well as any combination thereof;
- c) a blocking agent selected from Denhardt's solution (comprising Ficoll, polyvinylpyrrolidone, and bovine serum albumin), salmon sperm DNA and herring sperm DNA, as well as any combination thereof;
- d) a buffer such as Tris-HCl-buffer, HEPES-NaOH, saline-sodium citrate buffer, saline-sodium phosphate-EDTA buffer, as well as any combination thereof;
- e) a chelating agent such as EDTA; and has a pH from 7.0 to 8.0.

In one embodiment the buffer medium is selected from the group consisting of standard SSC (Saline-Sodium Citrate) buffer, Denhardt's solution, Church buffer, formamide hybridization buffer, DIG Easy Hyb, HEPES buffer, and lysis buffer, as well as combinations thereof.

SSC (Saline-Sodium Citrate) buffer is a widely used buffer solution in molecular biology, especially in nucleic acid hybridization techniques. It provides the ionic strength and pH conditions necessary for the hybridization of DNA or RNA to complementary strands. The standard formulation for SSC buffer is as follows:

- 3 M NaCl (Sodium Chloride)
- 0.3 M Sodium Citrate (Tri-sodium citrate dihydrate)
- pH adjusted to 7.0 with HCl

Denhardt's solution is a blocking agent used in molecular biology, particularly in nucleic acid hybridization techniques, to prevent non-specific binding of probes to the membrane or other surfaces. It helps to reduce background noise and increase the specificity of the hybridization signal. The standard formulation for Denhardt's solution is as follows:

- 2% (w/v) Ficoll 400
- 2% (w/v) Polyvinylpyrrolidone (PVP)
- 2% (w/v) Bovine Serum Albumin (BSA)

These components are dissolved in distilled water to create a 100× stock solution, which can be diluted to the desired working concentration.

Church buffer, also known as Church and Gilbert buffer, is a hybridization buffer used in molecular biology to enhance the sensitivity and specificity of nucleic acid hybridization reactions. It was developed by George M. Church and Wally Gilbert. This buffer provides an optimal environment for the hybridization of DNA or RNA probes to target sequences on membranes, reducing non-specific binding and background noise. The composition of Church buffer can vary slightly depending on the specific application, but the standard formulation typically includes:

- 1% Bovine Serum Albumin (BSA)
- 1 mM EDTA (Ethylenediaminetetraacetic acid)
- 0.5 M Phosphate Buffer (pH 7.2)
- 7% SDS (Sodium Dodecyl Sulfate)

Formamide hybridization buffer is a commonly used solution in molecular biology for nucleic acid hybridization, particularly in procedures like in situ hybridization, Southern blotting, and Northern blotting. The inclusion of formamide in the buffer helps to lower the melting temperature (Tm) of nucleic acid duplexes, allowing hybridization to occur at lower temperatures, which can be crucial for preserving the integrity of nucleic acids and improving hybridization specificity.

A typical formamide hybridization buffer might include:

- 50% Formamide
- 5× SSC (Saline-Sodium Citrate) Buffer
- 0.1% SDS (Sodium Dodecyl Sulfate)
- 0.02% Polyvinylpyrrolidone (PVP)
- 0.02% Ficoll
- 0.02% Bovine Serum Albumin (BSA)
- Denhardt's Solution (sometimes included)
- 100 μg/mL denatured, sheared salmon sperm DNA (or other blocking DNA)

DIG Easy Hyb is a hybridization buffer designed for use in molecular biology applications, particularly in hybridization techniques that employ DIG (digoxigenin)-labeled probes. It is optimized to enhance the specificity and sensitivity of hybridization reactions, facilitating the detection of nucleic acids in various assays such as Southern blotting, Northern blotting, and in situ hybridization. While the exact composition of DIG Easy Hyb is proprietary, it typically includes components that promote efficient hybridization and stability of the DIG-labeled probes, such as:

- Blocking Agents: To reduce non-specific binding.
- Salts: To maintain ionic strength and stabilize nucleic acid duplexes.
- Detergents: To reduce background noise.
- Buffering Agents: To maintain optimal pH.

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer is a zwitterionic buffering agent commonly used in biological and biochemical research due to its excellent ability to maintain a stable pH over a wide range of temperatures. In the context of nucleic acid hybridization, HEPES buffer is sometimes used to provide optimal conditions for the hybridization process, ensuring that the pH remains stable during the reaction. A typical HEPES hybridization buffer may include:

- HEPES: 10-50 mM (depending on the specific application)
- NaCl: To maintain ionic strength, typically around 0.1-1 M
- EDTA: 1-5 mM, to chelate divalent cations and prevent degradation of nucleic acids
- Detergents: Such as SDS (0.1-1%) or other non-ionic detergents, to reduce background noise
- Denhardt's Solution: Often included to block non-specific binding sites
- Formamide: 0-50%, to lower the melting temperature (Tm) of nucleic acid duplexes, facilitating hybridization at lower temperatures
- Blocking Agents: Such as sheared salmon sperm DNA or BSA, to prevent non-specific probe binding.

As lysis buffer any buffer may be used, in particular in cases in which the nucleic acid needs to be separated from other biologic material, such as in case of a cell or tissue-sample. In one embodiment the lysis buffer may comprise 50 mM Tris-HCl, 22 mM EDTA and 1.2% Triton X-100, and the pH is 7.0-7.5. In another embodiment the lysis buffer may comprise 150 mM NaCl, 10 mM Tris-HCl (pH 7.4) and 0.25% Triton X-100.

In one embodiment a simple Tris-HCL-Buffer may be used comprising 50 mM Tris-HCl (pH 7.4) and 100 mM HEPES-NaOH (pH 8.5).

In another embodiment of the disclosure the nucleic acid of the probe, the control nucleic acid and/or the sample nucleic acid is selected independently from each other from the group consisting of DNA (Deoxyribonucleic Acid), RNA (Ribonucleic Acid), PNA (Peptide Nucleic Acid), XNA (Xeno Nucleic Acid), LNA (Locked Nucleic Acid), morpholino oligomers, 2′O-Methyl-RNA, phosphorothioate DNA or RNA, guanosine-rich oligonucleotides, and D-amino acid-based nucleic acids, as well as combinations thereof.

In another embodiment of the disclosure the fluorescence-label is selected from the group consisting of fluorescent dyes such as DAPI, ethidium bromide, SYBR Green, Alexa Fluor dyes, and CyDyes, Quantum Dots and lanthanide-based labels, as well as combinations thereof.

In another embodiment of the disclosure the probe is excited by light in the wavelength corresponding to the absorption wavelength of the fluorescence-label.

In another embodiment of the disclosure the chaotropic agent is selected from guanidinium chloride (GuHCl), guanidinium thiocyanate (GuSCN), phenol, formamide, urea, potassium ions (K⁺), lithium ions (Li⁺), and magnesium ions (Mg²⁺), as well as any combination thereof;

In another embodiment of the disclosure the excitation light is provided by light-source selected from the group consisting of a mercury arc lamp, a xenon arc lamp, an LED, a LASER, a metal halide lamp, a tungsten-halogen lamp, and a deuterium lamp, as well as combinations thereof.

In another embodiment of the disclosure the mispairing is a pairing selected from the group consisting of A-A, A-C, C-A, A-G, G-A, T-T, T-C, C-T, T-G, G-T, C-C, G-G, U-U, A-U, U-A, as well as combinations thereof, wherein A is adenosine, C is cytosine, T is tyrosine, G is guanine, and U is uracil.

In another embodiment of the disclosure the sample nucleic acid molecule may be derived from a cell-lysate, a biological sample such as a tissue sample, or a body fluid selected from the group consisting of blood, urine, saliva, sweat, lymph fluid, cerebrospinal fluid (CSF), gastric juice, pleural fluid, peritoneal fluid, synovial fluid (joint fluid), and sputum (phlegm), as well as combinations thereof.

In another embodiment of the disclosure the sample nucleic acid molecule is a nucleic target for use in the detection of a nucleic acid mispairing in diagnostics, research, or a therapeutic application in a biological and/or medical field.

In one aspect the present disclosure relates to a detection device as described in EP20169908.9 which is incorporated by reference hereinunder. In short, the detection device is characterized by a light source for the excitation light, and a microprocessor that is arranged for automatically receiving data from the detector. The detection device may be further characterized by comprising a beam splitter with high pass optical filter, or a dichroic mirror between the sample and the detector which differentiates the excitation energy from an emission energy to be measured by the detector. The detection device may be further characterized in that the optical element is an optical fibre. The detection device may be further characterized by comprising at least one focusing element between the light source and the sample which delivers the excitation light onto the sample. The detection device may be further characterized by comprising at least one beam collimating element between the sample and the optical element which collects the emitted light (emission energy) to the optical element.

In particular, the present disclosure concerns a method for detecting a target nucleic acid in a sample, comprising the following steps:

- Bringing the sample in contact with a probe, that is a nucleic acid which can hybridize with the target, wherein, optionally, the probe may be labelled with a fluorescent molecule;
- b. Irradiating the probe with exciting energy, preferably excitation light,
- c. Detecting the emission energy from the sample, preferably emission light,
- d. Calculating ΔE from the energy-difference from the sample in comparison to a standard which is the probe-target-pair of same length and chemical composition, but 100% complementary to each other allowing a perfect hybridization.

In another aspect the present disclosure pertains to a sensor vessel as described in detail in PCT/EP2022/083446 which is included by reference herein. In short, the vessel is characterized in comprising of at least a reaction chamber, a ferrule, a light conducting element, such as optical fibre (glass or plastic) with a light conducting core element and a sheath encapsulating the light conducting core element, wherein the ferrule comprises a detection buffer and a probe.

The sensor vessel combines several technical solutions:

- Safe collection and storage of biological sample (in at least one embodiment being pathogenic);
- Facilitation of a lysis of the biological sample and facilitation of the binding of the nucleic acid probe with the target nucleic acid molecule within the liquid;
- Facilitation a light conduction from the optical detection device to the complex of the a least two hybridized nucleic acid molecules (e.g. the probe and the target), thereby producing as little as possible “light pollution” in form of reflection, absorption and scattering.

In one embodiment, the sensor vessel is a “single use” or “disposable” device, which poses specific requirements regarding cost-effective production without jeopardizing the optical quality of each device.

In order to perform the disclosed method, a number of energy-levels need to be detected.

Generally, the energy difference ΔE corresponds to energy-difference of the only probe emission and the emission light of the probe-target duplex and corresponds to the hybridization energy of the at least two hybridized nucleic acid molecules; wherein a mispairing of the at least two hybridized nucleic acid molecules is characterized by a decrease or increase of energy-difference as compared to a 100% complementary control. By then adding a chaotropic substance the energy-levels may be further increased and decreased allowing for a quantitative analysis of how many base mispairings have occurred. The addition of the chaotropic substance also reduces any other artefacts which may be measured.

This finding is surprising because prior art literature suggests that mispairings should always decrease stability energy of the hybridized complex, |ΔE^H|>|ΔE^HM|. However, it seems that at least in certain buffer conditions, this rule does not apply, and the overall energy of the hybridization complex may be actually reduced.

The theoretical background is explained for example in the review-article (Petruska J, Sowers LC, Goodman MF. Comparison of nucleotide interactions in water, proteins, and vacuum: model for DNA polymerase fidelity. Proceedings of the National Academy of Sciences. 1986 March; 83(6): 1559-62.)

In one embodiment the sensor vessel is configured to be used with the detection device such as described in detail in DE 10 2019 132 525.0.

In some embodiments, in particular in case of biological samples comprising intact cells, a buffer may be chosen which breaks the cells open (“lysis function”) for use in the further detection reaction in order to analyze the nucleic acids within the cell, respectively, the biological sample. The biological sample may be a blood sample, a saliva sample and/or a tissue sample, or any other human body fluid or tissue sample, and therefore the cells therein need to be broken open in order to analyze the nucleic acid within. In one embodiment between at least 50% and up to 100% of the cells in a sample are “broken open”, in a preferred embodiment between 60% and 99%, in a further preferred embodiment between 70 and 85%.

Another role of the buffer is to promote the detection reaction itself and not interfere with the detection (“detection function”), for example the buffer must promote hybridization of target molecules with the sensor probes (for example a hybridization and/or binding of the sensor probe with the target molecule) and allow the analysis to take place. For the detection function, it is important, that the buffer solution is “optically clear” at the wavelength, where the biomarker molecules are active, e.g. between 300 nm-1100 nm, preferably between 500 nm-700 nm.

Another role of the buffer-solution is to facilitate the binding of the biomarker with the sensor-probe.

The detection buffer of the disclosure comprises:

- a) (optional) a salt selected from sodium chloride (NaCl), sodium citrate, and sodium phosphate, as well as any combination thereof;
- b) a detergent selected from the group consisting of sodium dodecyl sulfate (SDS), polyethylene glycol tert-octylphenyl ether (Triton X-100), nonylphenoxypoly(ethyleneoxy)ethanol (Nonidet P-40 (NP-40)), octylphenoxypolyethoxyethanol (Igepal CA-630), Polysorbate 20 (Tween 20), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), N-lauroylsarcosine sodium salt (Sarkosyl) and sodium deoxycholate (Deoxycholate), as well as any combination thereof;
- c) (optional) a blocking agent selected from Denhardt's solution (comprising Ficoll, polyvinylpyrrolidone, and bovine serum albumin), salmon sperm DNA and herring sperm DNA, as well as any combination thereof;
- d) a buffer such as Tris-HCl-buffer, HEPES-NaOH, saline-sodium citrate buffer, saline-sodium phosphate-EDTA buffer, as well as any combination thereof;
- e) a chelating agent such as EDTA;
- f) a chaotropic agent selected from guanidinium chloride (GuHCl), guanidinium thiocyanate (GuSCN), phenol, formamide, urea, potassium ions (K^′), lithium ions (Li⁺), and magnesium ions (Mg²⁺), as well as any combination thereof;
- g) and has a pH from 7.0 to 8.0.

The concentration of the probe (e.g. the molecular probe, MB) depends on the size of the probe, the lifetime and signal intensity of the fluorochrome and the properties of the exciting light (e.g. wavelength and signal intensity of the LASER and loss of energy in the optical pathway). In some embodiments the concentration of the sensor probe was chosen to be between 0.5 μM and 20 μM, preferably between 1 μM and 10 μM, preferably between 2 μM and 5 μM, in one embodiment about 1 M in case of a LASER with 0.25 mW of nominal power, fibre diameter of 200 μm and NA (0.22).

As fluorochrome one may use any of the following dyes: Methoxycoumarin, DyLight®, Alexa Fluor®, Brilliant Violet 421™, HiLyte Fluor™, DyLight®, Alexa Fluor®, Aminocoumarin (AMCA), BD Horizon™, Pacific Blue™, EviTag™ quantum dots-Lake Placid Blue, AMCyan, BD Horizon™, Cy2®, Chromeo™, DyLight®, Alexa Fluor®, FAM, Fluorescein Iso-thiocyanate (FITC), EviTag™ quantum dots-Adirondack Green, Chromeo™, HiLyte Fluor™, Alexa Fluor (405, 488, 514, 532, 546, 555, 568, 633, 647, 660, 680, 700, 750, 790), EviTag™, quantum dots-Catskill Green, Pacific Orange™, HEX, EviTag™ quantum dots-Hops Yellow, Cy3®, 5-TAMRA, Phycoerythrin (PE), Tetramethyl Rhodamine Isothiocyanate (TRITC), EviTag™ quantum dots-Birch Yellow, Cy3.5®, Rhodamine Red-X 570 590, PE-Dy-omics®, EviTag™ quantum dots-Fort Orange, ROX, Red 613, Texas Red®, PE-Texas Red®, EviTag™ quantum dots-Maple-Red Orange, Allophycocyanin (APC), Quantum Red, Cy5®, PE-Cy5®, SureLight® P1, PE-Dyomics®, Peridinin Chlorophyll (PerCP), IRDye® 700DX, PE-Cy5.5®, APC-Cy5.5®, TruRed, APC-Cy7®, Cy7R, PE-Dyomics®, DyLight®, PE-Cy7®, IRDye® 800RS, DAPI, Hoechst 33258, Hoechst 33342, SYTOX Blue, YOYO-1, SYTOX Green, TOTO-1, TO-PRO-1, Mithramycin, SYTOX Orange, Chromomycin A3, CyTRAK Orange™, Ethidium Bromide, Propidium iodide (PI), DRAQ5™ and DRAQ7™, and/or combinations thereof.

In one embodiment system comprising the nucleic acids and the buffer medium of the present disclosure is adapted to a LASER which emits light at a wave length of at least one of the wave lengths selected from 325, 360, 405, 407, 488, 514, 532, 543, 568, 595, 633, 635 and/or 647 nm. The nominal power of the LASER may be between 1 mW and 3 W, preferably 5 mW to 1 W, in some embodiments about 10 mW, 25 mW, 50 mW, 100 mW, 200 mW, 300 mW, and up to 1 W, 2 W, 3 W. Higher nominal power may lead to heating of the probe, lower nominal power usually does not generate enough signal. In general, the number of photons (and related with this the emitted optical power) which reach the carrier element should be between 2×10¹⁵and 5×10¹⁵photons/s, for example about 3.2×10¹⁵photons/s. Of course, this may be further adapted to the concentration of immobilized probes and optical activity of the used fluorochrome. In one embodiment a 532 nm LASER (diode LASER) with nominal power applied to the sample of about 0.25 mW is used.

In one embodiment the probe is a molecular beacon (i.e. nucleic acid such as for example an aptamer or RNA-probe, MB) and/or a target-binding molecule (i.e. an antigen-binding molecule, such as an antibody, fab-fragment, anticalin, or the like). In another embodiment the probe is a linear nucleic acid.

In one embodiment the method according to the disclosure preferably a peak wavelength of the detected light is defined that is specific to a hybrid of the target and the probe, and if the emitted light has the peak wavelength, it is concluded that the target is in the sample. This method is based on the finding that each target-probe hybrid has a unique wavelength of emitted light, and allows for identifying any known target, in a patient's sample.

In particular, the peak wavelength λ_emmay be calculated from h*c/λ_em=h*c/λ_ref−E₀, wherein λ_refis a reference emission of the fluorescent dye without hybridization, h is the Planck constant, c is the speed of light and E₀is the hybridization energy of the target-probe hybrid, as known from Santalucia J Jr, Proc. Natl. Acad. Sci. USA 95 (1998) and calculated in terms of Nearest-Neighbor (NN) model, where change of the energy is 0.0243 eV. In some embodiments the change of the energy is between 0.01-0.05 eV, preferably between 0.02-0.03 eV, however, in some embodiment the energy may be up to 0.1 eV or even up to 0.2 eV.

In this respect it is important to note that the energy change of the peak wavelength upon hybridization and/or binding is measured (depicted as a shift between energy levels, generally called “ΔE”).

This measurement is not to be confused with prior art methods based for example on fluorescence excitation and/or FRET. The measurement of the present disclosure rather relies on the reduction of total free energy of the complex as compared to the single molecules, which is reduced after hybridization and/or binding.

In another embodiment the measurement allows even the differentiation between single nucleotide polymorphisms (SNPs), such as for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mispairings as long as the overall molecule still hybridizes. This allows the differentiation between different mutants or strains, e.g. different viral mutants, cancer types, and/or MRSA-strains.

In case of larger numbers of target macromolecules, such as in nano-molar-range or, preferably, micro-molar-range or, preferably, millimolar-range, as it may be the case of biomarkers found for example in blood samples, a quantitative measurement offers beneficial accuracy/precision in comparison with diagnostic methods used now (such as selective electrodes, titration methods etc.).

In one aspect the disclosure pertains to labelling a biomarker with the sensor-probe. Such a biomarker may be a nucleic acid contained in a patient's sample. According to the present disclosure in case of nucleic acids the probe is a molecular beacon. The detection buffer supports the lysis of the biological sample and the hybridization process. It is not necessary to extract the target nucleic acid (RNA or DNA) in advance since the method is very sensitive and is able to detect less than 20, preferably less than 10 copies per test. However, depending on the sample obtained, extraction of the target nucleic acid by generally known methods and/or commercially available extraction kits may be applied. In addition, when a double-stranded nucleic acid (DNA or RNA) has to be detected a strand separation can be made in advance or simultaneously with the hybridization. This strand separation may be conducted e.g. chemically could be performed simultaneously, or with UV-light or by heat treatment to be made in advance.

Detecting single nucleotide polymorphisms (SNPs) or nucleic acid mispairings under in vivo conditions opens up several significant applications in biomedical research and clinical practice.

Thus, the present disclosure encompasses the following uses and applications:

- Disease Diagnosis and Personalized Medicine
  - Genetic Disorders: Identifying SNPs associated with genetic diseases allows for early diagnosis and personalized treatment plans.
  - Cancer: Detecting SNPs linked to cancer susceptibility or drug resistance helps in designing targeted therapies.
  - Infectious Diseases: Identifying genetic variations in pathogens helps in tracking outbreaks and selecting appropriate treatments.
- Pharmacogenomics
  - Drug Response: SNPs influencing drug metabolism or efficacy can guide personalized drug selection and dosage adjustments.
  - Adverse Drug Reactions: Predicting susceptibility to adverse drug reactions based on genetic variations improves patient safety.
- Population Genetics and Evolutionary Studies
  - Studying SNP frequencies in populations provides insights into genetic diversity, migration patterns, and evolutionary relationships.
  - Identifying genetic adaptations to environmental pressures helps in understanding human evolution and adaptation.
- Forensic Analysis
  - Using SNPs for individual identification and forensic DNA profiling enhances accuracy and reliability in criminal investigations and paternity testing.
- Monitoring Treatment Efficacy
  - Monitoring changes in SNP profiles during treatment helps in assessing treatment response and predicting outcomes.
- Non-Invasive Prenatal Testing (NIPT)
  - Detecting fetal SNPs from maternal blood allows for non-invasive prenatal screening for genetic disorders.
- Environmental Monitoring and Agriculture
  - Assessing genetic diversity and health of wildlife populations or agricultural crops using SNP analysis contributes to conservation and breeding programs.

In summary, the ability to detect SNPs and nucleic acid mispairings in vivo has transformative implications across medicine, research, and beyond, enabling more precise diagnostics, treatments, and understanding of genetic diversity and disease mechanisms.

In another aspect of the disclosure the method can be applied to analyse the influence of other macromolecules such as peptides, proteins, carbohydrates or lipids on nucleic acid duplexes in a patient. The inventive method may be used both for predictive a well as diagnostic purposes.

In case of larger numbers of target macromolecules, such as in nano-molar-range or, preferably, micro-molar-range or, preferably, millimolar-range, as it may be the case of biomarkers found for example in blood samples, a quantitative measurement becomes especially feasible.

As used herein the term “optically clear” refers to a material which shows a transmissibility of the light at least in the detection wavelength of 500 to 700 nm of at least 95% or more, preferably of 98% or more, most preferably of 99% or more, such as more than 99.9%, more than 99.95%, more than 99.99%. Optically clear also implies that the material does not show any fluorescent effects neither in itself, nor on the probe alone or bound to the target molecule (i.e. the biomarker). Furthermore, “optically clear” materials preferably do not absorb and/or scatter the light.

As used herein the term “optically inactive” refers to a material which shows weak (a few %, i.e. 0.1%, 0.5%, 1%, less than 5%, less than 2.5%) to no transmissibility of the light at least in the in the detection wavelength of 500 to 700 nm . For example, a transmission of 0.01% or less, preferably of 0.001% or less, most preferably of 0.0001% and even better less than the detection limit. Furthermore, the term “optically inactive” as used herein refers to a material which shows no other optical effects such as luminescence or phosphorescence upon excitation with light. According to the present disclosure “optically inactive” material is used to avoid any “light contamination”, e.g. any light which comes from sources apart from the sensor-probe.

Definitions

As used herein, “nucleic acid” or “polynucleic acid” refers to the order or sequence of nucleotides along a strand of nucleic acids. The nucleic acid sequence may be single-stranded or double-stranded or contain portions of both double-stranded and single-stranded sequences. The nucleic acid sequence may be composed of DNA, both genomic and cDNA, RNA or DNA/RNA hybrid.

As used herein, “macromolecule” refers to any large biomolecule including nucleic acids, peptides, proteins, carbohydrates or lipids.

As used herein, “peptide” refers to short chains of between two and fifty amino acids, linked by peptide bonds. Chains of fewer than ten or fifteen amino acids may also be called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.

As used herein, “protein” refers to large biomolecules that are comprised of one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity.

As used herein, “carbohydrate” refers to a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen-oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula C_m(H₂O)_n(where _mmay or may not be different from _n). However, not all carbohydrates conform to this precise stoichiometric definition (e.g., uronic acids, deoxy-sugars such as fucose), nor are all chemicals that do conform to this definition automatically classified as carbohydrates (e.g. formaldehyde). It is used herein primarily as a synonym of saccharide, a group that includes sugars, starch, and cellulose.

As used herein, “lipid” refers to a macro biomolecule that is soluble in nonpolar solvents. Non-polar solvents are typically hydrocarbons used to dissolve other naturally occurring hydrocarbon lipid molecules that do not (or do not easily) dissolve in water, including fatty acids, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, and phospholipids. As such the term encompasses fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols and saccharolipids.

As used herein the term “sensor-probe” is used herein for any molecule which is able to specifically bind to a target molecule, i.e. via hybridization and/or other binding modes, such as for example via van-der-Waals-bonds, covalent bonds and/or hydrogen bonds. Thus, the term “sensor-probe” without further definition encompasses “molecular beacons”, or “(molecular beacon) probes”, as well as “target-binding molecules” (such as for example antibodies).

As used herein, the terms “target/probe molecule complex” or “complex” are used in a general manner for any group of two or more associated molecules. For example, such an association may be the hybridization of the molecular beacon and the target nucleic acid and/or the binding of the target-binding molecule and the target macromolecule. The complex is excited by light and the emission is detected in the analysing device.

As used herein, “molecular beacons” are oligonucleotides that can report the presence of specific nucleic acids in homogenous solutions. Molecular beacons are hairpin-shaped molecules with a fluorochrome. This is a novel nonradioactive method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the molecular beacon-target hybrids from an excess of the molecular beacons.

A typical molecular beacon is 25-40 nucleotides long. The middle nucleotides are complementary to the target DNA or RNA and do not base pair with one another, while the five to seven nucleotides at each terminus are complementary to each other rather than to the target DNA.

A typical molecular beacon structure can be divided in 4 parts: 1) loop, an 18-30 base pair region of the molecular beacon that is complementary to the target sequence; 2) stem formed by the attachment to both termini of the loop of two short (5 to 7 nucleotide residues) oligonucleotides that are complementary to each other; 3) 5′ fluorescent dye at the 5′ end of the molecular beacon, a fluorescent dye is covalently attached; 4) 3′ quencher (non-fluorescent) dye that is covalently attached to the 3′ end of the molecular beacon. When the beacon is in closed loop shape, the quencher resides in proximity to the fluorescent dye, which results in quenching the fluorescent emission of the latter. If the nucleic acid to be detected is complementary to the strand in the loop, the event of hybridization occurs. The duplex formed between the nucleic acid and the loop is more stable than that of the stem because the former duplex involves more base pairs. This causes the separation of the stem and hence of the fluorescent dye and the quencher. Once the fluorescent dye is no longer next to the quencher, illumination of the hybrid with light results in the fluorescent emission. The presence of the emission reports that the event of hybridization has occurred and hence the target nucleic acid sequence is present in the test sample. The fluorescent dye of the molecular beacon may be any suitable fluorescent dye, for example ABI dyes (e.g. FAM™, HEX™, TET™, JOE™, ROX™, CAL Fluor™ Red 610), cyanine dyes (e.g. Yakima Yellow or ATTO) or molecular dyes (e.g. ALEXA-fluor or BODIPY dyes). The quencher of the molecular beacon may be any suitable quencher, for example TAM, BHQ1, BHQ2, DAB, Eclip, BBQ650. Molecular beacons are synthetic oligonucleotides whose preparation is well documented in the literature (Tyagi S; Kramer FR, (1996). “Molecular beacons: probes that fluoresce upon hybridization”. Nat. Biotechnol. 14 (3): 303-308). Further, the molecular beacon design is within the skill of a molecular biologist and there are many bioinformatics tools for this purpose available (e.g. Beacon Designer™). In addition, molecular beacons are commercially available for many target sequences (e.g. Eurofins Genomics, Ebersberg, Germany; Integrated DNA Technologies, Inc., Coralville, Iowa, USA).

In the context of this disclosure, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside and/or nucleotide bases. Hybridization takes place under stringent or non-stringent conditions. As used herein, “stringent” refers to the conditions, i.e. temperature, buffer composition or ionic strength under which hybridization between polynucleotides occurs. These conditions depend mainly on the composition and complexity of the target nucleic acid and length of the molecular beacon probe.

For the hybridization temperature conditions the “T_m” (melting temperature) of the nucleic acids has to be considered. “T_m” means under specified conditions the temperature at which half of the nucleic acid sequences are disassociated and half are associated. Generally, suitable hybridization conditions may be easily determined by a person skilled in the art.

“Complementary” as used herein, refers to the capacity for pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the target DNA or RNA are considered to be complementary to each other at that position. For example, the sequence 5′-A-C-T-3′ is complementary to the sequence 3′-T-G-A-5′.

Complementarity may be partial, in which only some of the nucleotides match according to base pairing, or complete, where all the nucleotides match according to base pairing. For purposes of the present disclosure “substantially complementary” refers to 90% or greater identity over the length of the target base pair region. The complementarity can also be 45, 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementary, or any amount below or in between these amounts. In other words, the oligonucleotide and the target DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can build a hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the molecular beacon probe and the DNA or RNA target. It is understood in the art that the molecular beacon probe does not need to be 100% complementary to that of its target nucleic acid to be specifically hybridizable. “Oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent inter-nucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.

As used herein, “target-binding molecule” refers to a molecule, preferably a protein, which is able to specifically bind to a target macromolecule (such as a peptide, protein, carbohydrate or lipid) in a sample. Such a target-binding molecule may be selected from the group comprising antibody, antibody-fragment, Fab-fragment, anticalin protein, DARPin, nanoCLAMP, affilin, affimer, affitin, alphabody, avimer, Kunitz domain peptide, monobody, intrabody, micro-antibody, single-chain variable fragment (scFv), and combinations thereof. Target-binding molecules typically bind to unique surface patterns on the target macromolecule, sometimes referred to as “epitope”.

As used herein, “specifically binding” refers to the binding of the target-binding molecule to the target macromolecule (such as a peptide, protein, carbohydrate or lipid) in a sample with a high specificity. The fewer ligands a protein can bind besides the desired target, the greater is its specificity. Specificity describes the strength of binding between a given protein and ligand. This relationship can be described by a dissociation constant (K_D), which characterizes the balance between bound and unbound states for the protein-ligand system. K_Dis the equilibrium dissociation constant, a calculated ratio of K_off/K_on, between the target-binding molecule and its target. The association constant (K_on) is used to characterise how quickly the target-binding molecule binds to its target. The dissociation constant (K_off) is used to measure how quickly a target-binding molecule dissociates from its target. The specificity of the target-binding molecule is at least micromolar (μM, K_Dbetween 10⁻⁴to 10⁻⁶), more preferably nanomolar (nM, K_Dbetween 10⁻⁷to 10⁻⁹), even more preferably picomolar (pM, K_Dbetween 10⁻¹⁰to 10⁻¹²), and most preferably femtomolar (fM, K_Dbetween 10⁻¹³to 10⁻¹⁵).

“Subject” or “patient” as used herein refers to a living organism, such as a mammalian individual, including murines, cattle, simians and humans. Preferably, the patient is a human.

“Biological sample” or just “sample” as used herein refers to a biological sample encompassing liquid and solid samples. Liquid samples encompass saliva, sputum, sweat, urine, nasal secretion, bronchoalveolar lavage fluid, laryngo-pharyngeal scrape test, vaginal secretion, blood liquids (e.g. serum, plasma) and cerebrospinal fluid (CSF). Solid samples encompass tissue samples such as tissue cultures or biopsy specimen. A preferred patient's sample is sputum. The patient's sample will be collected with the disposable pipette as described below.

In a particular preferred embodiment of the present disclosure, the method is used for detecting a biomarker (including pathogens) in a patient's sample.

“Biomarker” as used herein, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. As such the disclosure pertains to labeling a biomarker such as a macromolecule (such as nucleic acids, peptides, proteins, carbohydrates or lipids) contained in a patient's sample (e.g. blood-sample, liquor-sample and/or saliva sample comprising the target macromolecule) with a sensor-probe. For simplification the definition of “biomarker” within this application encompasses also pathogens.

“Pathogens” as used herein, is any organism that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ. Typically, the term is used to describe an infectious microorganism or agent, such as a virus, bacterium, protozoan, prion, viroid, or fungus.

As used herein, “nucleic acid” or “polynucleic acid” refers to the order or sequence of nucleotides along a strand of nucleic acids. The nucleic acid sequence may be single-stranded or double-stranded or contain portions of both double-stranded and single-stranded sequences. The nucleic acid sequence may be composed of DNA, both genomic and cDNA, RNA or DNA/RNA hybrid.

The fluorescent dye attached (optionally covalently bound) to the probe may be any suitable fluorescent dye, for example ABI dyes (e.g. FAM™, HEX™, TET™, JOE™, ROX™, CAL Fluor™ Red 610), cyanine dyes (e.g. Yakima Yellow or ATTO) or molecular dyes (e.g. ALEXA-fluor or BODIPY dyes). The quencher of the probe may be any suitable quencher, for example TAM, BHQ1, BHQ2, DAB, Eclip, BBQ650.

In the context of this disclosure, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. Hybridization takes place under stringent or non-stringent conditions.

As used herein, “stringent” refers to the conditions, i.e. temperature, buffer composition or ionic strength under which hybridization between polynucleotides occurs. These conditions depend mainly on the composition and complexity of the target nucleic acid and length of the probe.

For the hybridization temperature conditions the “Tm” (melting temperature) of the nucleic acids has to be considered. “Tm” means under specified conditions the temperature at which half of the nucleic acid sequences are disassociated and half are associated. Generally, suitable hybridization conditions may be easily determined by a person skilled in the art.

“Complementary” as used herein, refers to the capacity for pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the target DNA or RNA are considered to be complementary to each other at that position.

For example, the sequence 5′-A-C-T-3′ is complementary to the sequence 3′-T-G-A-5′. Complementarity may be partial, in which only some of the nucleotides match according to base pairing, or complete, where all the nucleotides match according to base pairing. For purposes of the present disclosure “mispairing” refers to a situation where at least two bases are not complementary to each other, e.g. where the base-pair is C-A, A-C, T-G, G-T, U-A or A-U.

The term “substantially complementary” refers to 90% or greater identity over the length of the target base pair region. The complementarity can also be 45, 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementary, or any amount below or in between these amounts. In other words, the oligonucleotide and the target DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can build a hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the probe and the DNA or RNA target. It is understood in the art that the probe does not need to be 100% complementary to that of its target nucleic acid to be specifically hybridizable.

“Oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.

“Subject” or “patient” as used herein refers to a mammalian individual, including murines, cattle, simians and humans. Preferably, the patient is a human.

“Sample” as used herein refers to a biological sample encompassing liquid and solid samples. Liquid samples encompass saliva, sputum, sweat, urine, nasal secretion, bronchoalveolar lavage fluid, laryngo-pharyngeal scrape test, vaginal secretion, blood liquids (e.g. serum, plasma) and cerebrospinal fluid (CSF). Solid samples encompass tissue samples such as tissue cultures or biopsy specimen. A preferred patient's sample is sputum. The patient's sample will be collected with a disposable device as described hereinunder.

In a particular preferred embodiment of the present disclosure, the method is used for detecting a virus in a patient's sample. “Virus” or “viral” means any single-or double-stranded DNA or RNA virus. In particular, Human Immunodeficiency Virus (HIV), Zika, MERS-Corona, SARS-COV-1, Sars-COV-2, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Lyphotropic Virus-type 1-4 (HTLV-1-4), Epstein-Barr Virus (EBV), Human Papilloma Virus (HPV), Influenza A, B or C, Non-Influenza Respiratory Viruses (NIRVs, e.g. respiratory syncytial virus, parainfluenza virus, rhinovirus, metapneumovirus), norovirus, Ebola virus, Reo-virus, Herpes virus (simplex 1, 2+B, 5, 6, 7, 8), Pox virus, FSME virus, Dengue virus, Rubi-virus, Varizella zoster virus and Cytomegalievirus.

In a method according to the disclosure, the target nucleic acid may in one embodiment be a virus RNA. This method provides for screening large numbers of patients for infection, in an extremely reduced time scale, compared to the state of the art. The virus may in particular be a coronavirus, further in particular SARS-COV-2. The method is thus applicable in a recent world-wide pandemic situation. In a method according to the disclosure, the excitation light may in particular be a laser beam. This method allows for limiting the excitation light to the excitation wavelength of the fluorescent dye, and thus for avoiding side effects induced by other wavelengths.

Types of nucleic acids disclosed hereinunder:

- Genomic DNA: Detecting specific genes, mutations, or overall genome integrity.
- Mitochondrial DNA (mtDNA): Used for studying maternal lineage, certain diseases, and forensic analysis.
- Plasmid DNA: Common in bacterial studies and biotechnology applications.
- Viral DNA: Detection of DNA viruses like herpesvirus, human papillomavirus (HPV), and adenovirus.
- mRNA (messenger RNA): Reflects gene expression levels; used in gene expression studies and diagnostics.
- rRNA (ribosomal RNA): Important for detecting and quantifying ribosomes; used in microbial identification and taxonomy.
- tRNA (transfer RNA): Less common in diagnostics but crucial for understanding translation processes.
- miRNA (microRNA): Small non-coding RNA molecules involved in gene regulation; used in cancer and other disease diagnostics.
- siRNA (small interfering RNA): Used in gene silencing studies and therapeutic applications.
- Viral RNA: Detection of RNA viruses like influenza, HIV, SARS-COV-2 (the virus responsible for COVID-19), hepatitis C virus (HCV), and Zika virus.
- lncRNA (long non-coding RNA): Involved in gene regulation; studied in various diseases including cancer.
- snRNA (small nuclear RNA): Involved in RNA splicing; studied in certain genetic disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—shows the energy measurements according to steps a) and b) which is the energy-shift upon hybridization of the probe with a 100% complementary control.

FIG. 2—shows the energy measurements according to step c) which is the energy-shift upon hybridization of the probe with a sample nucleic acid, which for example has one mismatch.

FIG. 3—shows the energy measurements according to step d) which is the repetition of the measurement b) in the same buffer but comprising a chaotropic agent.

FIG. 4—shows the different energy-differences which can used in order to qualitatively define that in the sample nucleic acid exists a mismatch as compared to the control.

FIG. 5