Fingerprint analysis of single molecule events

Abstract

The present disclosure relates to analysis of molecular events observed by irradiating a sample with primary electromagnetic radiation and detecting secondary electromagnetic radiation emitted by the sample caused by the primary electromagnetic radiation, and wherein at least one characteristic of the primary electromagnetic radiation is altered several times during a time period of the event.

Description

Technical Field

[0001] The present disclosure relates to analysis of molecular events observed by irradiating a sample with primary electromagnetic radiation and detecting secondary electromagnetic radiation emitted by the sample as a result of the primary electromagnetic radiation, wherein the sample is irradiated with the primary electromagnetic radiation and wherein at least one characteristic of the primary electromagnetic radiation is altered a plurality of times over a time period of the event. Background Art

[0002] The sequencing of the human genome or the genomes of other organisms and the determination and comparison of individual sequence variants require sequencing methods that are firstly rapid and secondly can be employed routinely and cost-effectively.

[0003] The high demand of cost-effective sequencing has promoted the development of high-throughput sequencing technology that makes sequencing process parallelization thereby concurrently produce multiple sequences.The example of these sequencing technologies is large-scale parallel signature sequencing (LynxTherapeutics), polymerase chain reaction sequencing (Life Technologies), 454 pyrophosphate sequencing (RocheDiagnostics), Illumina sequencing (SolexaInc.), connection sequencing (Life Technologies), ion current semiconductor sequencing (Life Technologies) or DNA nano ball sequencing (Complete Genomics).These technologies allow the rapid analysis of the consensus sequence in nucleic acid colony.But the mutation existing in the minority sequence in the nucleic acid colony to be analyzed (for example, in a minority cell genome) will not be detected because they are covered by most of other sequences existing in colony.

[0004] In order to address these problems, several different formats of single molecule sequencing processes have been developed. Several of these processes belong to the field of fluorescence spectroscopy (FCS) and relate to the detection and analysis of single molecules by fluorescence. Typically, nucleic acid polymerases and / or nucleic acid degrading enzymes and fluorescently labeled nucleic acids and / or nucleotide building blocks are used to determine the sequence of a single nucleic acid molecule based on the time-dependent variation of fluorescence when the nucleotide building blocks are incorporated into the nucleic acid molecule or cut off from the nucleic acid molecule. For example, single molecule sequencing processes and equipment suitable for performing such processes are described in co-owned applications WO 2002 / 097406, WO 2003 / 052137, WO 2006 / 013110, WO 2013 / 131888, WO 2015 / 104245, WO 2017 / 001407 and WO 2018 / 104301.

[0005] In known methods for analyzing single molecule events, a sample comprising the components of the single molecule event is irradiated with electromagnetic radiation that is constant over time. However, the fluorescence detected will change over time as the single molecule event proceeds. In exemplary embodiments in which a single DNA or RNA polymerase molecule incorporates fluorescently labeled nucleotides, the detected signal will change in real time as the labeled nucleotides are incorporated. If each of the four bases A, T, C, and G of a DNA molecule, or A, U, C, and G of an RNA molecule, is attached with a moiety that has unique fluorescent properties for each base / nucleotide, it is in principle possible to detect the incorporated base by detecting its unique fluorescent signature (see US 7,056,661 B2, the contents of which are incorporated herein by reference).

[0006] In practice, the emission wavelength distributions of the detected fluorescently labeled nucleotides will have some overlap, which makes it impossible, even in theory, to provide a 100% accurate determination of all incorporated nucleotides. Similar overlaps are found with respect to other parameters such as fluorescence lifetime, circular dichroism, and combinations thereof.

[0007] Besides DNA and / or RNA sequencing, there are a variety of additional applications where high-throughput single-molecule analysis is in high demand.

[0008] Thus, one of the several objects of the present disclosure is to provide improved means, e.g., spectroscopic methods such as fluorescence emission wavelength measurements, fluorescence lifetime measurements, circular dichroism measurements, absorbance measurements and / or combinations thereof, to distinguish those overlapping fluorescence emission spectra of labeled components participating in single molecule events. Summary of the Invention

[0009] In a first aspect, the present disclosure relates to a method for analyzing single molecule events, the method comprising:

[0010] - emitting primary electromagnetic radiation from a radiation source toward a sample undergoing a single molecule event, wherein the primary radiation causes emission of secondary electromagnetic radiation from components of the single molecule event, and wherein the secondary electromagnetic radiation is distinguishable from the primary electromagnetic radiation; and

[0011] - detecting said secondary electromagnetic radiation,

[0012] wherein the method further comprises modifying at least one characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event, thereby emitting primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one further characteristic towards the sample during the time period, wherein the first characteristic is different from the at least one further characteristic, and

[0013] The method further comprises modifying a characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event.

[0014] In certain embodiments, the single molecule event comprises sequence analysis of a single nucleic acid molecule. In other embodiments, the single molecule event comprises analysis of a single molecule receptor-ligand interaction, or analysis of a single molecule hybridization event.

[0015] In a preferred embodiment, at least one characteristic of the primary electromagnetic radiation is controlled and optionally adjusted based on the detected secondary electromagnetic radiation emitted from the sample of the single molecule event.

[0016] wherein, in particular, the controlling and optional adjusting is performed by a detector adapted to detect secondary electromagnetic radiation emitted from the sample of the single molecule event, and

[0017] Therein, more particularly, the control and optional adjustment is based on dynamic processing of previous measurements of secondary radiation emitted from the sample of single molecule events.

[0018] Additional aspects relate to an apparatus for analyzing single molecule events, comprising:

[0019] - means for providing at least one sample suitable for single molecule events;

[0020] a radiation source adapted to emit primary electromagnetic radiation towards the sample, wherein the primary radiation causes emission of secondary electromagnetic radiation from components of the single molecule event, and wherein the secondary electromagnetic radiation is distinguishable from the primary electromagnetic radiation; and

[0021] - means for detecting said secondary electromagnetic radiation,

[0022] The radiation source is adapted to modify at least one characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event, thereby emitting primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one further characteristic to the sample during the time period, wherein the first characteristic is different from the at least one further characteristic.

[0023] In certain embodiments, the apparatus is suitable for sequence analysis of single nucleic acid molecules.

[0024] In a preferred embodiment, the device further comprises

[0025] - means for controlling and optionally adjusting the primary electromagnetic radiation in dependence on detected secondary electromagnetic radiation emitted from the sample of single molecule events,

[0026] wherein, in particular, the components comprise a detector adapted to detect secondary electromagnetic radiation emitted from the sample of the single molecule event, and

[0027] Therein, more particularly, the components are adapted to control and optionally adjust the primary electromagnetic radiation by dynamic processing based on previous measurements of secondary radiation emitted from a sample of single molecule events.

[0028] A further aspect relates to the use of the above method or the above device for providing higher precision in the analysis of single molecule events.

[0029] In certain embodiments, the use is to provide improved discrimination between differently incorporated nucleobases during a polymerase reaction.

[0030] Items in the instruction manual

[0031] 1. A method for analyzing single molecule events, comprising:

[0032] - emitting primary electromagnetic radiation from a radiation source toward a sample undergoing a single molecule event, wherein the primary radiation causes emission of secondary electromagnetic radiation from components of the single molecule event, and wherein the secondary electromagnetic radiation is distinguishable from the primary electromagnetic radiation; and

[0033] - detecting said secondary electromagnetic radiation,

[0034] The method further comprises modifying at least one characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event, thereby emitting primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one other characteristic to the sample during the time period, wherein the first characteristic is different from the at least one other characteristic.

[0035] 2. The method of claim 1 , wherein the secondary electromagnetic radiation is detected separately for the first characteristic and the at least one further characteristic of the primary electromagnetic radiation.

[0036] 3. The method of item 2, wherein a combined signal of the secondary electromagnetic radiation is provided and analyzed for the first characteristic and the at least one further characteristic of the primary electromagnetic radiation.

[0037] 4. The method of claim 3, wherein the combined signal is a fingerprint of n different characteristics from the primary electromagnetic radiation, wherein n is in particular 2, 3 or 4.

[0038] 5. The method of any of the preceding items, wherein during the time period of the single molecule event, at least one characteristic of the primary electromagnetic radiation changes between at least 2 different states.

[0039] 6. The method of claim 5, wherein during the time period of the single molecule event, at least one characteristic of the primary electromagnetic radiation changes between approximately 2-10 different states.

[0040] 7. The method of any of items 5-6, wherein the spacing of one or more individual states of the primary electromagnetic radiation is selectively altered, eg, increased, compared to other individual states.

[0041] 8. The method of any of the preceding items, wherein the radiation source comprises at least one laser, in particular a plurality of lasers having different emission wavelengths.

[0042] 9. The method of any of the preceding items, wherein a plurality of individual samples are provided, for example, at least 10, at least 100, at least 1000 or at least 10000 individual samples.

[0043] 10. The method of item 9, wherein the primary electromagnetic radiation from the radiation source is split into a plurality of separate radiation beams, for example by a diffractive optical element.

[0044] 11. The method of item 10, wherein separate radiation beams are emitted into individual samples or groups of individual samples.

[0045] 12. The method of any of the preceding items, wherein the at least one sample comprises at least one sample site located on a support.

[0046] 13. The method of item 12, wherein the support comprises a substrate and a plurality of sample points on a surface of the support, wherein the sample points are spatially separated from each other.

[0047] 14. The method of item 12 or 13, wherein the components of the single molecule event are immobilized to the at least one sample site.

[0048] 15. The method of claim 14, wherein the immobilized component comprises a biological moiety, e.g., a biomolecule.

[0049] 16. The method of item 15, wherein the immobilized reaction component comprises a nucleic acid polymerase, in particular a DNA or RNA polymerase.

[0050] 17. The method of any of the preceding items, wherein the at least one sample comprises at least one luminescent component of a single molecule event, in particular at least one fluorescent component, e.g., a reactant, a reaction intermediate and / or a reaction product, wherein the at least one luminescent component emits the secondary electromagnetic radiation.

[0051] 18. The method of item 17, wherein at least one luminescent component is a compound comprising a luminescent group (eg, a luminescent labeling group), in particular a compound comprising a fluorescent group (eg, a fluorescent labeling group).

[0052] 19. The method of item 17 or 18, wherein the sample comprises a plurality of different luminescent components, wherein at least some of the luminescent components have distinguishable and partially overlapping emission spectra of secondary electromagnetic radiation.

[0053] 20. The method of any one of items 17-19, wherein the sample comprises a plurality of different luminescent components, in particular fluorescent components, wherein at least some of the luminescent components, in particular fluorescent components, have distinguishable and partially overlapping luminescent emission spectra, in particular distinguishable and partially overlapping fluorescence emission spectra.

[0054] 21. The method according to any one of items 17 to 20, wherein the at least one luminescent component is present in the sample in free form.

[0055] 22. The method of any of the preceding items, comprising detecting multiple single molecule events in different samples separately, in particular comprising detecting multiple single molecule events in different samples separately and in parallel.

[0056] 23. The method of any of the preceding items, comprising detecting multiple subsequent single molecule events in one sample, in particular comprising detecting multiple subsequent single molecule events in different samples separately and in parallel.

[0057] 24. The method of item 23, wherein the multiple single molecule events include subsequent nucleic acid extension and / or degradation steps for single molecule nucleic acid sequence analysis.

[0058] 25. The method of claim 24, wherein the single-molecule nucleic acid sequence analysis comprises multiple steps of nucleic acid elongation, wherein luminescent nucleotide building blocks (e.g., luminescent nucleoside polyphosphates, e.g., comprising 3 to 15 phosphate groups) are incorporated into nucleic acid molecules in the presence of a nucleic acid polymerase (e.g., a DNA or RNA polymerase).

[0059] 26. The method of item 25, comprising detecting secondary electromagnetic radiation generated when the luminescent nucleotide building block (e.g., the luminescent nucleoside polyphosphate) is incorporated into the nucleic acid molecule.

[0060] 27. The method of any of the preceding items, comprising modifying a characteristic of the primary electromagnetic radiation several times during the time period of the single molecule event.

[0061] 28. The method of any of the preceding items, comprising modifying a characteristic of the primary electromagnetic radiation during each time period of several subsequent single molecule events.

[0062] 29. The method of any of the preceding items, wherein the altering comprises temporarily shutting off the primary electromagnetic radiation during the time period of the single molecule event.

[0063] 30. The method of any of the preceding items, comprising modifying a characteristic of the primary electromagnetic radiation, the characteristic being selected from the group consisting of:

[0064] -wavelength,

[0065] -amplitude,

[0066] - Pulsating operation,

[0067] - polarization, and

[0068] - A combination of two or more of the above characteristics.

[0069] 31. The method of any of the preceding items, comprising modifying the wavelength of the primary electromagnetic radiation during the unimolecular reaction.

[0070] 32. The method of any of the preceding items, comprising modifying a characteristic of the primary electromagnetic radiation within a time interval in the range of about 50 ns to about 10 s, about 1 μs to about 500 ms, or about 10 μs to about 100 ms.

[0071] 33. The method of any of the preceding items, wherein at least one characteristic of the primary electromagnetic radiation is controlled and optionally adjusted based on detected secondary electromagnetic radiation emitted from the sample of single molecule events.

[0072] 34. The method of item 33, wherein the controlling and optionally the adjusting are performed by a detector adapted to detect secondary electromagnetic radiation emitted from the sample of the single molecule event.

[0073] 35. The method of item 33 or 34, wherein the controlling and optional adjusting is based on dynamic processing of previous measurements of the secondary radiation.

[0074] 36. An apparatus for analyzing single molecule events, comprising:

[0075] - means for providing at least one sample suitable for single molecule events;

[0076] a radiation source adapted to emit primary electromagnetic radiation towards the sample, wherein the primary radiation causes emission of secondary electromagnetic radiation from components of the single molecule event, and wherein the secondary electromagnetic radiation is distinguishable from the primary electromagnetic radiation; and

[0077] - means for detecting said secondary electromagnetic radiation,

[0078] The radiation source is adapted to modify at least one characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event, thereby emitting primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one further characteristic to the sample during the time period, wherein the first characteristic is different from the at least one further characteristic.

[0079] 37. The apparatus of item 36, suitable for performing the method of any one of items 1 to 35.

[0080] 38. The apparatus of item 36 or 37, adapted to perform single molecule nucleic acid sequence analysis.

[0081] 39. Use of the method of any one of items 1 to 35 or the apparatus of any one of items 36 to 38 for providing increased precision in the analysis of single molecule events.

[0082] 40. Use of item 39 for providing improved discrimination between differently incorporated nucleobases during a polymerase reaction. DETAILED DESCRIPTION

[0083] The present disclosure provides methods and apparatus for analyzing single molecule events, wherein the single molecule event is associated with the emission of electromagnetic radiation from a sample (e.g., a sample spot on a support comprising a component of the single molecule event). In certain embodiments, the single molecule event encompasses a reaction of a biological moiety associated with the emission of characteristic electromagnetic radiation.

[0084] The present disclosure addresses the need for increased precision in the analysis of single molecule events observed by irradiating a sample with primary electromagnetic radiation and simultaneously detecting secondary electromagnetic radiation emitted by the sample resulting from the irradiation.

[0085] In particular, the present disclosure provides apparatus and processes that improve the accuracy of the determination of each kinetic step of a single molecule event by allowing the source of electromagnetic radiation irradiating the sample to be varied over time during observation of any individual kinetic step of the single molecule reaction.

[0086] To this end, at least one characteristic of the primary electromagnetic radiation is modified during the time period of the single-molecule event to be analyzed. Accordingly, during the time period of the single-molecule event, primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one additional characteristic are emitted to the sample of the single-molecule event. The first characteristic of the primary electromagnetic radiation differs from the at least one additional characteristic. The characteristic can be selected, for example, from wavelength, amplitude, pulsation, polarization, and a combination of two or more of the aforementioned characteristics. Thus, the first wavelength (or any other characteristic or combination of characteristics) of the primary electromagnetic radiation differs from the at least one additional wavelength (or any other characteristic or combination of characteristics).

[0087] According to certain embodiments, secondary electromagnetic radiation emitted by a component associated with a single-molecule event is detected for a first characteristic and at least one additional characteristic of the primary electromagnetic radiation. In certain embodiments, a combined signal of the secondary electromagnetic radiation is provided. This combined signal is based on the secondary electromagnetic radiation signal caused by the first characteristic of the primary electromagnetic radiation and the secondary electromagnetic radiation signal caused by the at least one additional characteristic of the primary electromagnetic radiation. By analyzing this combined signal, the accuracy of detection is significantly improved.

[0088] In a particular embodiment, the combined signal is a fingerprint of n different characteristics from said primary electromagnetic radiation, wherein n is in particular 2, 3 or 4. The combined signal may be depicted as an n-dimensional graph or plot, wherein n is an integer of at least 2.

[0089] In an exemplary embodiment, a single-molecule DNA sequencing process is performed. Specifically, a polymerization reaction catalyzed by a DNA or RNA polymerase occurs, wherein DNA or RNA molecules bond to individual polymerase molecules and nucleotide building blocks are incorporated one by one, thereby extending the DNA or RNA molecule. During the time period during which each nucleotide is incorporated into the DNA or RNA chain, at least one characteristic of the radiation source that irradiates the sample with electromagnetic radiation is altered one or more times.

[0090] In certain embodiments, the characteristics of the primary electromagnetic radiation are altered several times, eg, 2, 3, 4, 5 or more times, eg, up to 100 times, during the time period of the single molecule event.

[0091] When analyzing multiple single-molecule events, the characteristics of the primary electromagnetic radiation can be modified during several consecutive single-molecule events. In those embodiments, the modification scheme can be the same for each of the multiple consecutive single-molecule events, or can be different between the multiple consecutive single-molecule events. In some embodiments, the modification can be to temporarily turn off the primary electromagnetic radiation during the time period of the single-molecule event, that is, to illuminate the sample with the primary electromagnetic radiation only during certain intervals during the time period of the single-molecule event.

[0092] In some embodiments, the characteristic of the primary electromagnetic radiation to be modified is selected from the group consisting of:

[0093] -wavelength,

[0094] -amplitude,

[0095] - Pulsating operation,

[0096] - polarization, and

[0097] - A combination of two or more of the above characteristics.

[0098] In certain embodiments, the wavelength of the primary electromagnetic radiation is altered at least once during the time period of the single molecule event.

[0099] According to the present disclosure, during the time period of the single molecule event, at least one characteristic of the primary electromagnetic radiation changes between at least two different states (e.g., between at least two different wavelengths or combinations of wavelengths). In certain embodiments, during the time period of the single molecule event, the characteristic of the primary electromagnetic radiation changes between approximately 2-10 different states (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 different states).

[0100] According to the present disclosure, at least one characteristic of the primary electromagnetic radiation is altered at least once during the time period of the single-molecule event. In some embodiments, the at least one characteristic of the primary electromagnetic radiation is altered at a time interval in the range of about 50 ns to about 10 s, about 1 μs to about 500 ms, or about 10 μs to about 100 ms.

[0101] In some embodiments, at least one characteristic of the primary electromagnetic radiation is controlled and optionally adjusted based on detected secondary electromagnetic radiation emitted from a sample of single-molecule events. In some embodiments, the control and optional adjustment are performed by a detector adapted to detect the secondary electromagnetic radiation emitted from a sample of single-molecule events. In certain embodiments, the control and optional adjustment are based on dynamic processing of previous measurements of the secondary radiation. In certain embodiments, the control and optional adjustment are based on real-time analysis of the detected secondary electromagnetic radiation. This control and optional adjustment can provide a feedback mechanism for optimizing a strategy for modifying at least one characteristic of the primary electromagnetic radiation.

[0102] In certain embodiments, the detector (optionally in combination with appropriate hardware and / or software) provides instructions for altering the primary radiation source based on the type of molecular event currently being analyzed, e.g., as evidenced by secondary radiation measured previously during analysis of the event.

[0103] In certain embodiments, the detector (optionally in combination with appropriate hardware and / or software) provides instructions for altering the primary radiation source based on general conditions (e.g., temperature, type and concentration of reagents, etc.), for example, as confirmed by secondary radiation measured during analysis of one or several prior events.

[0104] In a specific embodiment, the detector (optionally in combination with appropriate hardware and / or software) can indicate that the primary radiation source has become completely inactive for a predetermined interval during the time period of the single molecule event. This instruction can be provided based on an analysis of the initially measured secondary radiation.

[0105] In other embodiments, the change in the primary electromagnetic radiation is deterministic, without the need for a detector to analyze the secondary electromagnetic radiation in real time.

[0106] In certain embodiments, during single-molecule nucleic acid synthesis, the wavelength of the primary electromagnetic radiation is altered several times during each incorporation event. Each A, C, T / U, and G nucleotide building block is labeled differently, for example, by chemical attachment to a different fluorescent moiety, each having an emission spectrum with a unique maximum, but wherein each emission spectrum has a distribution around its maximum such that the distributions overlap. In this case, a radiation source capable of emitting multiple different wavelengths can be provided, for example, a collection of lasers with designated, unique output wavelengths. Thus, the wavelength of the primary electromagnetic radiation emitted by the radiation source can be altered between several different states during the duration of a single-molecule event (e.g., incorporation of a nucleotide building block into an elongated nucleic acid molecule). For example, in a first state, a first laser is turned on while the remaining lasers are turned off; in a second state, a second laser is turned on while the remaining lasers are turned off, and so on. This alternation between different states is a sequential process that is sufficiently rapid that several rounds of excitation of each laser can be performed during each single event, for example, several times during each incorporation event. Thus, it is possible to record, via a detector, the signal generated by the secondary radiation when each laser is activated several times during an incorporation event. The detectors are synchronized with the lasers so they know which laser is active at which time. This in turn allows for a specific response curve to be provided for each laser wavelength.

[0107] These specific response curves for each event can be combined to obtain a multi-dimensional response plot or map for each event. Accordingly, the lack of precision associated with overlapping emission profiles of individually labeled components can be overcome by providing a combined signal of the collected emissions recorded for each laser during one incorporation event. In general, such a combined signal can be presented as an n-dimensional plot or map or "fingerprint." In an exemplary but non-limiting embodiment, where three different lasers (n=3) are used, the following can be provided: Figure 3 According to this fingerprint, there is no longer any overlap between the emission spectra of the labeled nucleotide building blocks, and each labeled nucleotide can be clearly detected within a specific space in the 3D plot.

[0108] In further exemplary embodiments, the number of lasers may be increased from n=3 to a larger number, such as 4, 5, or even more, to further separate the different signals within the n-dimensional fingerprint.

[0109] The corresponding fingerprint can also be provided by changing other characteristics of the primary electromagnetic radiation or a combination of several characteristics.

[0110] Furthermore, the intervals between one or more individual states of the primary electromagnetic radiation can be selectively varied, e.g., increased in comparison to other individual states. For example, depending on the emission characteristics of the luminescent component, the length of one or more active periods for a selected wavelength of the primary electromagnetic radiation can be varied, e.g., increased in comparison to active periods for other wavelengths.

[0111] In some embodiments, the introduction of periods of zero primary electromagnetic radiation between active periods may be used, eg, different activation periods.

[0112] In some embodiments, the individual states of the primary electromagnetic radiation may comprise a combination of different wavelengths and their relative amplitudes. In some embodiments, the individual states of the primary electromagnetic radiation may comprise a combination of different wavelengths and polarizations.

[0113] A sample for a single molecule event typically includes a sample spot on a support. The support may include a substrate and a plurality of sample spots spatially separated from one another on a surface of the support. In certain embodiments, the surface of the support is formed by the substrate and the sample spots. In certain embodiments, the substrate forms a continuous region in which the sample spots are distributed. The individual sample spots on the surface of the support are surrounded by a substrate that is different from the sample spots, for example, in terms of material and / or surface. Typically, the substrate is adapted to inhibit and / or block adhesion of biomolecules (such as polypeptides), while the sample spots are adapted to allow adhesion of desired biomolecules.

[0114] A component, e.g., a biological moiety, that participates in a single-molecule event is immobilized on the sample site. In certain embodiments, the component comprises a biomolecule, particularly a single biomolecule. The term "single biomolecule" encompasses a single molecular entity, such as a polypeptide, or a complex composed of multiple individual units, e.g., individual molecular entities, where the individual units together form a functional biological moiety.

[0115] In some embodiments, the support is a substantially planar support, i.e., does not include protrusions or depressions of about 1000 nm or more or about 100 nm or more. In other embodiments, the support is a structured support, for example, including depressions (such as may have a diameter of about 5×10 -24 Increased to about 1×10 -15 The support may be a well having a volume of 100 liters or a support having a column height of about 1 nm to 500 nm. In principle, the support may have any design as long as it can form a reaction space that enables a single molecule event to occur at the at least two sample sites having single biomolecules immobilized thereon.

[0116] In some embodiments, the substrate is an optically transparent material, i.e., a material that is substantially transparent to electromagnetic radiation (e.g., radiation in the visible range and / or radiation in the near infrared range). In some embodiments, the substrate comprises a material having an absolute refractive index of at least 1.01 (e.g., from about 1.5 to about 3 in the visible range, or from about 1.5 to about 4 in the near infrared range). In other embodiments, the substrate is an optically opaque material, e.g., a metal or semi-metal (such as silicon).

[0117] In certain embodiments, the substrate comprises a non-conductive material. Specific examples are glass, quartz, plastic, metal oxide-based materials (e.g., silicon dioxide-based materials such as glass, silicon dioxide, or quartz), or composite materials comprising such materials. In other embodiments, the substrate comprises a conductive material, for example, an optically transparent material (such as indium tin oxide).

[0118] Typically, the substrate has a thickness of about 10 μm to about 5 mm, particularly about 20 μm to about 2 mm.

[0119] The surface of the support includes a plurality of sample sites that are spatially separated from each other by the substrate surface. The sample sites are suitable for attachment of biomolecules. In some embodiments, the support includes a plurality of sample sites, for example, at least 10, at least 100, at least 1,000, at least 10,000, or at least 100,000 sample sites. In some embodiments, the support may include up to 10 6 or 10 9 or even more sample points.

[0120] In some embodiments, the sample site comprises or consists of at least one conductive material, such as a metal, including a single metal or a combination of metals, such as an alloy or mixture of multiple different metals. For example, metals capable of attaching to a sulfur-containing moiety (e.g., in the form of a thiol or disulfide) or a metal capable of attaching to a chelating moiety (e.g., a polyhistidine tag) are suitable. In some embodiments, the metal has a positive electrochemical potential. Specific examples of suitable metals include, but are not limited to, Au, Cu, Ni, Pt, Pd, Rh, Ir, Os, Ru, and any combination thereof comprising at least two of the metals.

[0121] In certain embodiments, the sample site comprises or consists of at least one metal oxide (including a single metal oxide or a combination of metal oxides). For example, metal oxides capable of attaching to a phosphorus-containing moiety (e.g., in the form of a phosphonic acid or phosphonate) or a chelating moiety (e.g., a polyhistidine tag) are suitable. Specific examples of suitable metal oxides include TiO2 and NiO.

[0122] In further embodiments, the sample site comprises or consists of at least one non-conductive material.

[0123] The sample spots can be prepared by vapor deposition of a metal, which is evaporated on a support covered by a grid mask, which can be produced by electron beam lithography or an equivalent technique. The size of the holes in the grid mask can correspond to the size of the sample spots on the surface of the support. Alternatively, the sample spots on the support can be prepared by site-specific deposition of nanoparticles (e.g., having a size of 2-10 nm) by precise pipetting of the particles onto the support, in particular on a support with a flat surface.

[0124] In certain embodiments, the sample spot has a size suitable for attachment of a single biomolecule. In those embodiments, the sample spot has a diameter of about 1 nm to about 30 nm, particularly about 2 nm to about 20 nm.

[0125] In some embodiments, the sample point is a separate object on the surface of the substrate. In some embodiments, the sample point has a lower surface proximal to the substrate and an upper surface distal to the substrate, wherein the distance between the lower surface and the upper surface defines the height of the sample point. In certain embodiments, the height is approximately 50 μm to approximately 500 nm, particularly approximately 100 μm to approximately 20 nm, and more particularly approximately 500 μm to approximately 10 nm, for example, approximately 2 nm.

[0126] The sample sites on the support are suitable for attachment of biomolecules, for example, by covalent or non-covalent attachment. The biomolecules can be selected from polypeptides, nucleic acids, carbohydrates, and any combination thereof, for example, glycosylated polypeptides or ribonucleoproteins. In certain embodiments, the biomolecule is a complex composed of several individual units (e.g., several polypeptide units or several polypeptide and nucleic acid units).

[0127] In particular embodiments, the biomolecule is a nucleic acid polymerase, particularly a DNA polymerase or an RNA polymerase, or a nucleic acid polymerase complex, particularly a DNA or RNA polymerase complex comprising a nucleic acid polymerase and a nucleic acid molecule. In particular embodiments, the biomolecule is a DNA polymerase having a DNA bonding cleft, particularly a family A DNA polymerase, including but not limited to Klenow, Taq, or T7 DNA polymerases, or any genetically modified versions thereof, or a family B polymerase, including but not limited to therminator, Phi29, RB-69, or T4 DNA polymerases, or any genetically modified versions thereof. Reference is made herein to US 7,745,116 B2, the contents of which are incorporated herein by reference.

[0128] In further embodiments, the biomolecule is a nucleic acid degrading enzyme, in particular an exonuclease, or a nucleic acid degrading molecule complex, in particular a DNA or RNA degrading molecule complex comprising a nucleic acid degrading enzyme and a nucleic acid molecule.

[0129] In yet other embodiments, the biomolecule is a gene editing enzyme, in particular a Cas nuclease, such as Cas3, Cas9, Cas10 or Cas12 nuclease or any genetically modified version thereof, e.g., a Cas nickase, or a gene editing complex comprising a gene editing enzyme and a nucleic acid molecule (e.g., a guide RNA and / or a target nucleic acid).

[0130] The single-molecule event to be detected occurs in a sample that includes the desired components of the event (e.g., biomolecules and optionally small molecules). At least one of the components is a luminescent component that includes a luminescent group (i.e., a group that can emit secondary electromagnetic luminescent radiation in response to being irradiated with primary electromagnetic radiation). In certain embodiments, the luminescent component is a compound that carries a luminescent label group. In certain embodiments, the luminescent component is a compound that is itself luminescent.

[0131] In certain embodiments, the luminescent component of a single molecule event can be a reactant, a reaction intermediate, and / or a reaction product. In certain embodiments, the luminescent component is a fluorescent component, i.e., a component capable of emitting fluorescence in response to exposure to primary electromagnetic radiation. In certain embodiments, the fluorescent component is a compound that carries a fluorescently labeled group. In certain embodiments, the fluorescent component is a compound that itself is capable of emitting fluorescence.

[0132] In certain embodiments, a sample of single-molecule events includes a plurality of different luminescent components, wherein at least some of the luminescent components have partially overlapping emission spectra of secondary electromagnetic radiation. For example, a sample may include a plurality of different fluorescent components, wherein at least some of the fluorescent components have distinguishable and partially overlapping fluorescence emission spectra.

[0133] In certain embodiments, the fluorescent components have overlapping fluorescence wavelengths, fluorescence lifetimes, polarization characteristics (eg, circular dichroism), and any combination thereof.

[0134] In certain embodiments, the fluorescence lifetime is measured several times during each molecular event. Other spectroscopic methods, such as circular dichroism and absorption methods, are also encompassed by the present invention, wherein the absence of electromagnetic radiation in at least one state is observed during several spectroscopic measurements during a single molecule event. Combinations of several spectroscopic methods are also embodiments of the present invention.

[0135] The at least one luminescent component may be present in the sample in an immobilized form and / or in a free form. In certain embodiments, the at least one luminescent component is present in a free form.

[0136] The method of the present disclosure may include separately analyzing multiple single molecule events in different samples (particularly in different sample spots on a support). In certain embodiments, the method includes separately analyzing multiple single molecule events in different samples (particularly in different sample spots on a support) in parallel.

[0137] In certain embodiments, the methods of the present disclosure include analyzing multiple subsequent single molecule events in a sample, and in particular, include analyzing multiple subsequent single molecule events in different samples separately and in parallel. The term "multiple single molecule events" encompasses a series of consecutive single molecule events occurring at the same sample (e.g., at a sample point on a support). In certain embodiments, the sequence of consecutive single molecule events includes up to 10, up to 100, up to 1,000, up to 10,000, or even more individual single molecule events, for example, up to about 10,000,000 or 100,000,000 individual single molecule events. For example, the multiple single molecule events can include subsequent nucleic acid extension and / or degradation steps of a single molecule nucleic acid sequence analysis.

[0138] In certain embodiments, single-molecule nucleic acid sequence analysis includes multiple nucleic acid elongation steps in which luminescent nucleotide building blocks (e.g., luminescent-labeled nucleoside polyphosphates, e.g., comprising 3 to 15 phosphate groups) are incorporated into nucleic acid molecules in the presence of a nucleic acid polymerase (e.g., DNA or RNA polymerase). These embodiments may include detecting secondary electromagnetic radiation generated when the luminescent nucleotide building blocks (e.g., luminescent-labeled nucleoside polyphosphates) are incorporated into the nucleic acid molecules.

[0139] According to the present invention, a sample undergoing a single molecule event is irradiated with primary electromagnetic radiation, wherein during a time period of the single molecule event at least one characteristic of the primary electromagnetic radiation is altered.

[0140] The present disclosure also provides a device for analyzing single molecule events, comprising:

[0141] - means for providing at least one sample suitable for single molecule events,

[0142] a radiation source adapted to emit primary electromagnetic radiation towards the sample, wherein the primary radiation causes emission of secondary electromagnetic radiation from components of the single molecule event, and wherein the secondary electromagnetic radiation is distinguishable from the primary electromagnetic radiation, and

[0143] - means for detecting said secondary electromagnetic radiation,

[0144] The radiation source is adapted to modify at least one characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event, thereby emitting primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one further characteristic towards the sample during the time period, wherein the first characteristic is different from the at least one further characteristic.

[0145] In certain embodiments, the apparatus is adapted to perform the methods described above, for example, for performing single molecule nucleic acid sequence analysis.

[0146] The methods and apparatus of the present disclosure are useful for providing increased precision in the analysis of single molecule events. In certain embodiments, they are useful for providing improved discrimination between different incorporated nucleobases during a polymerase reaction.

[0147] Methods and apparatus for analyzing single molecule events are disclosed, for example, in WO 2002 / 097406, WO 2003 / 052137, WO 2006 / 013110, WO 2013 / 131888, WO 2015 / 104245, WO 2017 / 001407 and WO 2018 / 104301, the contents of which are incorporated herein by reference.

[0148] To analyze single molecule events, the biomolecules can be located at sample points on the support. There, they come into contact with a sample liquid containing free reactants. Thus, one or more reaction spaces are defined. In particular, at least 100, at least 1,000, or at least 10,000, or even more than 10,000, biomolecules can be analyzed on a single support (e.g., a single planar support). 6 molecules.

[0149] The nucleic acid molecule whose sequence is to be determined can be selected, for example, from a DNA molecule (such as a genomic DNA fragment, a cDNA molecule, a plasmid, etc.) or an RNA molecule (such as an mRNA molecule). The nucleic acid molecule can be derived from a genomic library or an expression library produced by a cell or organism (e.g., a eukaryotic or prokaryotic cell or organism). This allows for the sequencing of a plurality of different nucleic acid template molecules (e.g., at least 10, 100, 1000, or 10,000 and up to 100,000, 10,000, or ... 6 or 10 7 or even more different nucleic acid molecules) are sequenced in parallel.

[0150] The nucleic acid molecules to be sequenced can be linear or circular single-stranded nucleic acid molecules, for example, with the circular form of covalent linkage. In order to obtain the circular nucleic acid template, linear nucleic acid molecules can be carried out to cyclization process and alternatively chain separation process during sample preparation. Cyclization can be realized by the connection (ligation) carried out according to known scheme, for example, using DNA or RNA ligase. In certain embodiments, adapter and / or identifier molecule (that is, the nucleic acid molecules of known sequence) can be coupled to nucleic acid molecules.

[0151] Sequence determination may include nucleic acid extension and / or nucleic acid degradation.The sequencing process includes one or more sequencing cycles.

[0152] The nucleic acid synthetase molecule is capable of extending a primer annealed to a nucleic acid template molecule. Primer extension can be performed by the stepwise incorporation of individual nucleotide building blocks at the 3' end of the growing nucleic acid chain, thereby generating a nucleic acid molecule complementary to the sequence of the circular nucleic acid template. The nucleic acid synthetase is selected from a polymerase capable of template-specific nucleic acid polymerization, preferably a DNA polymerase and an RNA polymerase, for example, a natural or modified polymerase, including a thermostable DNA polymerase.

[0153] Specific examples of suitable DNA polymerases include Taq polymerase, exonuclease-deficient Taq polymerase, Escherichia coli DNA polymerase I, Klenow fragment, reverse transcriptase, Φ29-related polymerase (including wild-type Φ29 polymerase and derivatives of such polymerases, such as exonuclease-deficient ones), T7 DNA polymerase, T5 DNA polymerase, and RB69 polymerase, etc.

[0154] Nucleic acid degrading enzyme molecule can progressively cut individual nucleotide building blocks from nucleic acid molecules.Preferably use exonuclease, more preferably use the single-stranded nucleic acid exonuclease that degrades on 3'→5' direction or 5'→3' direction.The exonuclease that is particularly preferably used is 3'→5' exonuclease, such as Escherichia coli exonuclease I and Escherichia coli exonuclease III, and 5'→3' exonuclease, such as T7 exonuclease, Escherichia coli exonuclease II and Escherichia coli exonuclease VIII.In addition, the exonuclease activity of various polymerases can also be used, for example, Klenow fragment, Taq polymerase or T4 polymerase.

[0155] Nucleic acid synthetase molecule is for example contacted with a linear or circular nucleic acid template molecule (ssDNA or RNA molecule) and annealed to the nucleic acid template molecule or a primer molecule that can anneal to the nucleic acid template molecule. Primer molecule is preferably a single-stranded nucleic acid or a nucleic acid analog molecule with a free 3' end, and this free 3' end can be extended by the enzymatic reaction catalyzed by immobilized nucleic acid synthetase molecule. The length of primer molecule is selected to allow annealing to the template effectively under reaction conditions. Usually, the length of primer molecule is at least 8, at least 10, at least 12 or at least 15 nucleotides and for example up to 20, 25, 50 or 100 nucleotides, or even higher. In certain embodiments, primer resistance is digested by the nucleic acid degrading enzyme molecule, for example, by mixing stable nucleotide analog building blocks and / or links to degraded between the nucleotide building blocks. In other embodiments, the digestion sensitivity of primer to nucleic acid degrading enzyme molecule.

[0156] The sequence of primer is selected so that it is effectively annealed to template molecule under reaction conditions.For example, primer can be the universal degenerate primer that can statistically anneal to unknown nucleic acid sequence.In other embodiments, primer can be able to anneal to the known sequence part of nucleic acid template molecule.In this embodiment, known adapter and / or identifier sequence can be mixed in the nucleic acid template molecule.Primer can be unlabeled or comprise fluorescent marker group.

[0157] Additionally, the presence of a luminescent nucleotide building block is required, for example, a nucleotide building block carrying at least one fluorescent marker group. Preferably, each different nucleotide building block (A, G, C, T / U) comprises a different fluorescent marker group.

[0158] The fluorescent labeling group can be selected from known fluorescent labeling groups for labeling biopolymers (especially nucleic acids), such as, for example, fluorescein dyes, rhodamines, oxazines (e.g., Evoblue or GnothisBlue), phycoerythrin, Cy3, Cy5, IR dyes or their derivatives.

[0159] The nucleotide building blocks may carry (i) a fluorescent marker group that is retained with the building block when the building block is incorporated into the nucleic acid molecule during primer extension catalyzed by a nucleic acid synthetase molecule; and / or (ii) a fluorescent marker group that is cleaved from the building block when the building block is incorporated into the nucleic acid molecule during primer extension catalyzed by a nucleic acid synthetase molecule. The fluorescent marker group that is retained with the building block is preferably attached to an α-phosphate group, to a sugar group, and / or to a nucleobase group.

[0160] In a specific embodiment, the fluorescent marker group retained with the building block is for example attached to a core base via a linker, and the linker can have up to 15 carbon atoms, preferably a chain length of 10-12 carbon atoms, optionally including heteroatoms, for example, N, O or S atoms. The fluorescent marker group cut off when the building block is incorporated into nucleic acid molecules can be attached to a terminal phosphate group, for example, including but not limited to the terminal phosphate group of the polyphosphate building block of hexaphosphate, pentaphosphate, tetraphosphate or triphosphate building blocks, such as the gamma-phosphate group of triphosphate building blocks. In certain embodiments, the selected building block includes the fluorescent marker group retained after (i) incorporation and also includes the fluorescent marker group cut off during (ii) incorporation. In this case, the fluorescent group that can interact with each other (for example, by quenching and / or energy transfer) can be selected.

[0161] If the nucleic acid molecule to be sequenced is directly sequenced using a nucleic acid degrading enzyme molecule, the nucleic acid molecule will contain a fluorescent marker. On the other hand, if the nucleic acid molecule to be sequenced is used as a template in primer extension, the nucleic acid molecule may not contain a fluorescent marker.

[0162] The sequencing process can involve a step of generating a nucleic acid molecule incorporating nucleotide building blocks during primer extension catalyzed by a nucleic acid synthetase molecule, and / or a second step of cleaving individual nucleotide building blocks from the generated nucleic acid molecule during primer extension catalyzed by a nucleic acid degrading enzyme molecule. Depending on the type of fluorescent label, nucleic acid sequence determination can be performed during primer extension and / or degradation.

[0163] The sequence determination during primer extension involves the use of nucleotide building blocks that carry a fluorescent marker group. When the building block is incorporated into a nucleic acid molecule, the fluorescent marker group is cut off from the building block. In this case, the time-dependent fluorescence change caused by cutting off the fluorescent marker group from the nucleotide building block can be determined. The sequence determination during nucleic acid degradation involves the use of nucleotide building blocks that carry a fluorescent marker group. When it is incorporated into a nucleic acid molecule, the fluorescent marker group is retained with the building block. When the labeled nucleotide building block is released from the nucleic acid molecule, the individual nucleotide building blocks are gradually cut off from the nucleic acid molecule to cause the time-dependent change of fluorescence. In certain embodiments, it is also possible to perform sequence determination during extension and degradation, that is, when using nucleotide building blocks, the building block not only carries the fluorescent marker group that is retained with the building block when the building block is incorporated into a nucleic acid molecule, but also carries the fluorescent marker group cut off from the building block. In this embodiment, the two fluorescent groups can be the same or different.

[0164] In some embodiments, the method involves one or more cycles of nucleic acid synthesis and nucleic acid degradation to determine the base sequence of the nucleic acid template. Nucleic acid synthesis involves the extension of a primer annealed to the nucleic acid template molecule catalyzed by a nucleic acid synthetase molecule, wherein a nucleic acid molecule complementary to the sequence of the nucleic acid template is generated. In a next step, the generated nucleic acid molecule is degraded by a nucleic acid degrading enzyme molecule.

[0165] When the nucleotide building blocks are incorporated into the extended nucleic acid molecules, a time-dependent change in fluorescence can occur, which can be detected as indicated above. Preferably, the incorporation of the nucleotide building blocks into the extended nucleic acid molecules is associated with a detectable increase in fluorescence, preferably with an instantaneous increase in fluorescence. For example, a nucleotide building block can be used that carries a fluorescent marker group on the portion cut off in the molecule when the building block is incorporated into a primer (e.g., on a gamma-phosphate group).

[0166] When nucleotide building blocks are cleaved from a synthetic nucleic acid molecule, a time-dependent change in fluorescence can be determined due to the interaction of the fluorescent marker group incorporated into the nucleic acid chain with adjacent groups (e.g., with chemical groups of the nucleic acid, in particular nucleobases, such as, for example, G) or / and adjacent fluorescent marker groups, and these interactions lead to changes in fluorescence, in particular changes in fluorescence intensity, compared to the "isolated" form of the fluorescent marker group, which are caused by quenching and / or energy transfer processes. The removal of individual nucleotide building blocks by cleavage modifies the overall fluorescence, for example, the fluorescence intensity of the immobilized nucleic acid chain, and this change is a function of the removal of the individual nucleotide building blocks by cleavage, i.e., a function of time.

[0167] In certain embodiments, the association of labeled nucleotides with biomolecule complexes is detected by measuring the polarization of emitted photons. The polarization of excited-state photons changes due to rotational movement of the luminescent nucleotide labels and can be used to identify free, mobile, antibonding labeled nucleotides during the polymerization process.

[0168] This time-dependent change in fluorescence during elongation and / or degradation can be recorded for multiple nucleic acid molecules in parallel and correlated with the base sequence of the individual nucleic acid strands. Fluorescent labeling groups are preferably used that are at least partially quenched upon incorporation into a nucleic acid strand, such that the fluorescence intensity increases after the nucleotide building block containing the labeling group or the adjacent building block causing the quenching is removed by cleavage.

[0169] During the incorporation and / or removal of individual nucleotide building blocks, it is possible to measure the change in fluorescence intensity of the nucleic acid chain and / or the incorporated or removed nucleotide building block due to the quenching or energy transfer process. This change in fluorescence intensity over time depends on the base sequence of the nucleic acid chain being studied and can therefore be correlated with the sequence.

[0170] The complete sequence of a nucleic acid molecule can be determined by using a mixture of nucleotide building blocks, the building blocks being labeled at all four different bases, for example at A, G, C and T, or at a combination of two or three different bases. Where appropriate, it is possible to attach a "sequence identifier" (i.e., a labeled nucleic acid of known sequence) to the nucleic acid strand to be investigated, for example by enzymatically reacting with a ligase and / or a terminal transferase, so that at the start of sequencing a known fluorescence pattern is first obtained, followed by a fluorescence pattern corresponding to the unknown sequence to be investigated.

[0171] Detection comprises irradiating primary electromagnetic radiation from a radiation source into the support, preferably by means of a laser or another suitable light source, so as to cause excitation of the fluorescent marker group. In certain embodiments, the radiation source comprises a plurality of different lasers emitting radiation at different wavelengths. To this end, it is possible to use one or more laser beams, such as an expanded beam laser beam having a cross-sectional area of approximately 1-20 mm, and / or a plurality of laser beams. Detection preferably comprises multi-point fluorescence excitation by a laser, such as a dot matrix of laser points generated by a diffractive optical device (see WO 2002 / 097406) or a quantum well laser.

[0172] Fluorescence emission from multiple nucleic acid strands can be detected in parallel using a detection component (e.g., a detector matrix comprising, for example, an electronic detector matrix (e.g., a CCD camera), a CMOS detector matrix (e.g., a CMOS camera), or an avalanche photodiode matrix). Detection can be performed by simultaneously performing fluorescence excitation and detection on some or all of the nucleic acid strands to be investigated. Detection is preferably performed on fluorescence emitted substantially orthogonally from the surface of the support, through the reaction space, or through the body of the support.

[0173] For example, detection can be performed by means of single molecule detection, such as by fluorescence correlation spectroscopy, which involves the integration of very small, preferably confocal, volume elements (e.g., 10 -21 to 10 -101) exposure to excitation light from a laser or another suitable light source, which excites the receptors in this measurement volume, causing them to emit fluorescent light, the fluorescent light emitted from the measurement volume being measured by means of a photodetector and the change in the measured emission over time being correlated with the concentration of the analyte, making it possible to identify the individual molecules in the measurement volume at suitably high dilutions. Details of the procedures and apparatus for detection can be found in the disclosure of EP 0 679 251, the contents of which are incorporated herein by reference. Confocal determination of single molecules is further described in Rigler and Mets (Soc. Photo-Opt. Instrum. Eng. 1921 (1993), 239 ff.) and Mets and Rigler (J. Fluoresc. 4 (1994) 259-264), the contents of which are incorporated herein by reference.

[0174] Alternatively, or in addition, detection can also be performed by time-resolved decay measurement (called "time gating"), as described, for example, by Rigler et al. in "Picosecond Single Photon Fluorescence Spectroscopy of Nucleic Acids", in "Ultrafast Phenomena", D.H. Auston, Ed. Springer 1984, the contents of which are incorporated herein by reference. Here, fluorescent molecules are excited in the measurement volume and then, for example, the detection interval is opened on the photodetector at time intervals of ≥ 100 ps. In this way, it is possible to keep the background signal generated by the Raman effect at a sufficiently low level so that individual molecules can be detected in a substantially interference-free manner.

[0175] The methods and apparatus of the present disclosure are also suitable for analyzing other single molecule events where high throughput single molecule analysis is a high requirement, ie, individual biomolecules bound to selected sites for analysis.

[0176] In certain embodiments, the present disclosure relates to single molecule analysis of receptor-ligand interactions, e.g., encompassing binding of a receptor protein to a sample site and then studying the interaction with its ligand, e.g., in drug development.

[0177] In further embodiments, the present disclosure relates to single molecule analysis of hybridization events, for example, encompassing the attachment of short single-stranded nucleic acid molecules (e.g., DNA or RNA molecules), for example, ranging in length from 3 to 300 nucleotides, followed by the addition of a sample comprising a complementary nucleic acid molecule and observation of any hybridization events. Application areas may be, for example, the detection of viral RNA / DNA, the detection of bacterial DNA / RNA, and the detection of short fragments of DNA from cancer cells in the bloodstream.

[0178] Additionally, the present disclosure will be explained in detail with reference to the following specific examples.

[0179] Figure 1 An embodiment of the prior art is shown. A radiation source (1) directs a constant primary electromagnetic radiation (2) toward a molecule (4) bonded to a support (3). The molecule (4) immobilized on the support (3) undergoes a biomolecular process involving several states. In response to irradiation with the constant primary electromagnetic radiation (2), some of these states cause changes associated with the emission of secondary electromagnetic radiation (5). Certain biomolecular states result in unique emission profiles of the secondary electromagnetic radiation (5) that can be detected by a detector (6).

[0180] Figure 2 Embodiments of the prior art are shown. Single-molecule DNA or RNA sequencing can include generating a secondary DNA or RNA chain complementary to the primary DNA or RNA chain. An enzyme (e.g., a polymerase) catalyzes the extension of the secondary chain, wherein nucleotides complementary to the nucleotides in the primary chain are incorporated one by one. Each nucleotide emits fluorescence with a unique emission wavelength distribution. These emission distributions overlap to a certain extent, which causes limitations on the ability to accurately determine which nucleotide has been incorporated. Therefore, the accuracy of single-molecule DNA or RNA sequencing has encountered a barrier.

[0181] Figure 3 An embodiment of the present disclosure is shown. The source of primary electromagnetic radiation comprises a collection of three lasers with different wavelengths. During a molecular event, for example, the incorporation of a single nucleotide into a growing secondary nucleic acid chain, the primary electromagnetic radiation changes in real time during the molecular event. This in turn results in the detection of the secondary electromagnetic radiation, which can be plotted in a three-dimensional graph. Figure 2 Compared to the prior art embodiments, since three dimensions are now available, there is no overlap between the secondary electromagnetic radiation profiles of the individual nucleotides, which leads to increased accuracy compared to the prior art.

[0182] During the molecular event of incorporation of A, T, G or C, the radiation source switches between three laser wavelengths. Each of A, T, G and C is attached to a fluorescent marker, each with a characteristic excitation and emission wavelength distribution. Each of the three different laser wavelengths produces a unique response in terms of emission from the fluorescent marker. Based on this, a three-dimensional response as shown in the figure can be constructed. Since three dimensions are used, instead of the conventional method ( Figure 2 ), thus achieving greater precision in determining which of A, T, G, or C is incorporated. The limited space in which each tag provides its response is called the "fingerprint" of each of A, T, C, and G, respectively.

[0183] In this particular embodiment, the individual lasers are turned on and off so that only one laser is active at any given time. The lasers are activated in a sequential order over time. In more general embodiments, multiple lasers can be activated in combinations of two, three, or more lasers, for example, 2, 3, or 4 lasers, to achieve the best accuracy when the distance between "fingerprints" is greatest.

[0184] Figure 4 An embodiment of the present disclosure is shown. The detector and the source of radiation form a control system, wherein the detector can control the source of radiation.

[0185] The detector (in some embodiments, after consultation with hardware and / or software responsible for analyzing the detected secondary electromagnetic radiation) provides instructions and / or synchronization signals to the source of primary electromagnetic radiation (laser). These instructions can be, for example, adjustments, such as increasing the exposure time of a certain wavelength.

[0186] In some embodiments, the detector and the source of primary electromagnetic radiation synchronize changes made by the source of electromagnetic radiation to the amplitude, wavelength, and / or other spectral characteristics of the radiation source. In one embodiment, the detector precisely controls when and which wavelengths the radiation source emits at any given time. In another embodiment, the instructions the detector issues to the source of electromagnetic radiation depend on signals previously detected by the detector.

[0187] In another embodiment, the detector controls the source of radiation in real time based on data recorded during a molecular event. Thus, the detector includes software or hardware, or is connected to a computer running software, that analyzes the output signal and modifies the instructions to the detector during the analysis to optimize the accuracy of the determination of the relevant parameters during a particular molecular event. Such modifications could, for example, alter the time at which a laser is turned on and off.

Claims

1. A method for analyzing single molecule events, comprising: - emitting primary electromagnetic radiation from a radiation source towards a sample undergoing a single molecule event, wherein the primary radiation causes emission of secondary electromagnetic radiation from components of the single molecule event, and wherein the secondary electromagnetic radiation is distinguishable from the primary electromagnetic radiation, and - detecting said secondary electromagnetic radiation, wherein the method further comprises modifying at least one characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event, thereby emitting primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one further characteristic towards the sample during the time period, wherein the first characteristic is different from the at least one further characteristic, and The method further comprises modifying a characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event.

2. A method as claimed in claim 1, wherein the secondary electromagnetic radiation is detected separately for the first characteristic and the at least one further characteristic of the primary electromagnetic radiation, and wherein a combined signal of the secondary electromagnetic radiation is provided for the first characteristic and the at least one further characteristic of the primary electromagnetic radiation.

3. The method as claimed in claim 2, wherein the combined signal is a fingerprint of n different characteristics from the primary electromagnetic radiation, wherein n is in particular 2, 3 or 4.

4. The method according to any of the preceding claims, wherein the radiation source comprises at least one laser, in particular a plurality of lasers having different emission wavelengths.

5. A method according to any one of the preceding claims, wherein the at least one characteristic of the primary electromagnetic radiation is controlled and optionally adjusted in dependence on the detected secondary electromagnetic radiation emitted from the sample of the single molecule event, in, In particular, the controlling and optional adjusting is performed by a detector adapted to detect secondary electromagnetic radiation emitted from the sample of single molecule events, and Therein, more particularly, the control and optional adjustment is based on dynamic processing of previous measurements of secondary radiation emitted from the sample of single molecule events.

6. The method of any of the preceding claims, wherein the at least one sample comprises at least one sample site on a support, wherein the support comprises a substrate and a plurality of sample sites on a surface of the support, and wherein the sample sites are spatially separated from one another; wherein components of the single molecule event are immobilized to the at least one sample site, wherein the immobilized components particularly comprise biomolecules, such as nucleic acid polymerases, particularly DNA or RNA polymerases, and The at least one sample comprises at least one luminescent component of a single molecule event, such as a reactant, a reaction intermediate, and / or a reaction product, wherein the at least one luminescent component emits the secondary electromagnetic radiation. The method of claim 6 , wherein the at least one luminescent component is a fluorescent component.

8. A method as claimed in claim 6 or 7, wherein the sample comprises a plurality of different luminescent components, in particular fluorescent components, wherein at least some of the luminescent components, in particular fluorescent components, have distinguishable and partially overlapping luminescence emission spectra, in particular distinguishable and partially overlapping fluorescence emission spectra.

9. The method according to any of the preceding claims, comprising analyzing a plurality of subsequent single molecule events in one sample, in particular comprising analyzing a plurality of subsequent single molecule events in different samples separately and in parallel.

10. The method of claim 9, wherein the plurality of single molecule events comprises a subsequent step of single molecule nucleic acid sequence analysis, the analysis comprising a plurality of nucleic acid elongation steps wherein luminescent nucleotide building blocks are incorporated into nucleic acid molecules in the presence of a nucleic acid polymerase, and Secondary electromagnetic radiation generated by the incorporation of the luminescent nucleotide building block into the nucleic acid molecule is detected.

11. A method according to any one of the preceding claims, comprising modifying a characteristic of the primary electromagnetic radiation, said characteristic being selected from the group consisting of: -wavelength, -amplitude, - Pulsating operation, - polarization, and - A combination of two or more of the above characteristics.

12. A method as claimed in any preceding claim, comprising modifying a characteristic of the primary electromagnetic radiation within a time interval in the range of approximately 50 ns to approximately 10 s, approximately 1 μs to approximately 500 ms or approximately 10 μs to approximately 100 ms.

13. An apparatus for analyzing single molecule events, comprising: - means for providing at least one sample suitable for a unimolecular reaction; a radiation source adapted to emit primary electromagnetic radiation towards the sample, wherein the primary radiation causes emission of secondary electromagnetic radiation from components of the single molecule event, and wherein the secondary electromagnetic radiation is distinguishable from the primary electromagnetic radiation, and - means for detecting said secondary electromagnetic radiation, The radiation source is adapted to modify at least one characteristic of the primary electromagnetic radiation several times during a time period of the single molecule event, thereby emitting primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one further characteristic to the sample during the time period, wherein the first characteristic is different from the at least one further characteristic.

14. The apparatus of claim 13, further comprising: - means for controlling and optionally adjusting the primary electromagnetic radiation in dependence on detected secondary electromagnetic radiation emitted from the sample of single molecule events, wherein, in particular, the components comprise a detector adapted to detect secondary electromagnetic radiation emitted from the sample of the single molecule event, and Therein, more particularly, the components are adapted to control and optionally adjust the primary electromagnetic radiation by dynamic processing based on previous measurements of secondary radiation emitted from a sample of single molecule events.

15. Use of the method of any one of claims 1 to 12 or the device of any one of claims 13 to 14 for providing increased precision in the analysis of single molecule events.

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