Fingerprint analysis of single-molecule events

Abstract

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

Description

[Technical Field]

[0001] explanation The present disclosure relates to the analysis of molecular events observed by irradiating a sample with primary electromagnetic radiation and detecting secondary electromagnetic radiation emitted by the sample induced by the primary electromagnetic radiation, wherein the sample is irradiated with primary electromagnetic radiation and wherein at least one property of the primary electromagnetic radiation is altered several times during the duration of the event. [Background technology]

[0002] Sequencing the human genome or the genomes of other organisms and determining and comparing individual sequence variants requires the provision of sequencing methods that are firstly rapid and secondly can be used routinely and cost-effectively.

[0003] The high demand for cost-effective sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process and generate multiple sequences simultaneously. Examples of these sequencing technologies include massively parallel signature sequencing (Lynx Therapeutics), polony sequencing (Life Technologies), 454 pyrosequencing (Roche Diagnostics), Illumina sequencing (Solexa Inc), sequencing by ligation (Life Technologies), Ion Torrent semiconductor sequencing (Life Technologies), or DNA nanoball sequencing (Complete Genomics). These technologies enable rapid analysis of consensus sequences in nucleic acid populations. However, mutations present in minority sequences in the analyzed nucleic acid population, such as mutations present in the sequences of a minority cell genome, are hidden by the large number of other sequences present in the population and therefore go undetected.

[0004] To address these issues, single-molecule sequencing processes in several different formats have been developed. Some of these processes are in the field of fluorescence spectroscopy (FCS) and involve the detection and analysis of single molecules by fluorescence. Typically, nucleic acid polymerizing enzymes and / or nucleases, and fluorescently labeled nucleic acid and / or nucleotide building blocks, are used to individually determine the sequence of a single nucleic acid molecule based on the time-dependent changes in fluorescence as the nucleotide building blocks are incorporated into or cleaved from the nucleic acid molecule. Single-molecule sequencing processes and devices suitable for carrying out such processes are described, for example, in co-pending applications WO2002 / 097406, WO2003 / 052137, WO2006 / 013110, WO2013 / 131888, WO2015 / 104245, WO2017 / 001407, and WO2018 / 104301.

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

[0006] In practice, there is some overlap in the detected emission wavelength distributions of fluorescently labeled nucleotides, making it impossible, even theoretically, to identify all incorporated nucleotides with 100% accuracy. Similar overlaps are also observed in other parameters such as fluorescence lifetime, circular dichroism, and their combinations.

[0007] In addition to DNA and / or RNA sequencing, there are many additional application areas where high yield single molecule analysis is in high demand.

[0008] It was therefore one of several objectives of the present disclosure to provide improved means for distinguishing between the overlapping fluorescence emission spectra of labeled components involved in a single molecule event, e.g., spectroscopic methods such as fluorescence emission wavelength measurements, fluorescence lifetime measurements, circular dichroism measurements, absorption measurements, and / or combinations thereof. Summary of the Invention

[0009] In a first aspect, the present disclosure relates to a method for analyzing single molecule events, said method comprising the steps of: - emitting primary electromagnetic radiation from a radiation source to a sample in which a single molecular event occurs, wherein said primary radiation causes emission of secondary electromagnetic radiation from components of said single molecular event, and wherein said secondary electromagnetic radiation is distinguishable from said primary electromagnetic radiation; and - detecting said secondary electromagnetic radiation; wherein the method further comprises varying at least one property of the primary electromagnetic radiation several times during the duration of the single molecule event, whereby primary electromagnetic radiation having a first property and primary electromagnetic radiation having at least one further property are emitted into the sample during the period, wherein the first property is different from the at least one further property, and wherein the method further comprises varying the property of the primary electromagnetic radiation several times during the period of the single molecule event.

[0010] In certain embodiments, the single molecule event comprises the sequence analysis of a single nucleic acid molecule, hi further embodiments, the single molecule event comprises the analysis of a single molecule receptor-ligand interaction or the analysis of a single molecule hybridization event.

[0011] In a preferred embodiment, said at least one property of said primary electromagnetic radiation is controlled, and if necessary adjusted, in accordance with detected secondary electromagnetic radiation emitted from said sample of said single molecule event; wherein, in particular, said controlling and, if necessary, adjusting is performed by a detector configured to detect said secondary electromagnetic radiation emitted from said sample of said single molecule event; and Here, more particularly, said control and, if necessary, adjustment is a dynamic process based on prior measurement of secondary radiation emitted from said sample of said single molecule events.

[0012] A further aspect relates to an apparatus for analyzing single molecule events, said apparatus comprising: - means for providing at least one sample configured for a single molecule event; - a radiation source configured for emitting primary electromagnetic radiation to the sample, wherein the primary radiation causes emission of secondary electromagnetic radiation from components of the single-molecule event, and the secondary electromagnetic radiation is distinguishable from the primary electromagnetic radiation; and - means for detecting said secondary electromagnetic radiation, wherein the radiation source is configured to vary at least one property of the primary electromagnetic radiation several times during the duration of the single molecule event, such that primary electromagnetic radiation having a first property and primary electromagnetic radiation having at least one further property are emitted into the sample during the period, wherein the first property is different from the at least one further property.

[0013] In certain embodiments, the device is configured for said sequence analysis of a single nucleic acid molecule.

[0014] In a preferred embodiment, the device further comprises: - means for controlling, and if necessary adjusting, the primary electromagnetic radiation in accordance with the detected secondary electromagnetic radiation emitted from the sample of the single molecule event; wherein, in particular, the means includes a detector configured to detect the secondary electromagnetic radiation emitted from the sample of the single molecule event; and Here, more particularly, the means is configured to control and, if necessary, adjust the primary electromagnetic radiation according to a dynamic process based on prior measurements of the secondary radiation emitted from the sample of the single molecule event.

[0015] Further aspects relate to the use of the above method or the above device to provide increased precision in the analysis of single molecule events.

[0016] In certain embodiments, said use is to provide improved discrimination of different nucleobases incorporated during the polymerase reaction. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 shows an embodiment of the prior art. A radiation source (1) directs a primary electromagnetic radiation (2) toward a molecule (4) bound to a support (3). The molecule (4) immobilized on the support (3) undergoes a biomolecular process that includes several states. Some of these states result in an associated change in the emission of secondary electromagnetic radiation (5) in response to irradiation with the primary electromagnetic radiation (2). A given biomolecular state gives rise to a unique emission profile of secondary electromagnetic radiation (5) that is detected by a detector (6). [Figure 2]Figure 2 shows an embodiment of the prior art. Sequencing of single DNA or RNA molecules can involve generating a secondary DNA or RNA strand complementary to a primary DNA or RNA strand. An enzyme, e.g., a polymerase, catalyzes the extension of the secondary strand, such that nucleotides are sequentially incorporated one by one in a manner complementary to the nucleotides in the primary strand. Each nucleotide is fluorescent with a unique emission wavelength distribution. These emission wavelength distributions overlap to some extent, creating a limiting factor in the ability to accurately determine which nucleotides have been incorporated. As a result, the accuracy of single DNA or RNA molecule sequencing is limited. [Figure 3]FIG. 3 illustrates an embodiment of the present disclosure. The radiation source of the primary electromagnetic radiation includes a set of three lasers with different wavelengths. During a molecular event, such as the incorporation of a single nucleotide into an elongated secondary nucleic acid strand, the primary electromagnetic radiation changes in real time during the molecular event. This then results in the detection of secondary electromagnetic radiation, which can be plotted on a three-dimensional graph. Compared to the prior art embodiment according to FIG. 2, the availability of a third dimension eliminates overlap between the secondary electromagnetic radiation profiles of individual nucleotides, leading to improved accuracy compared to the prior art. During the molecular event of incorporation of an A, T, G, or C, the radiation source switches between three laser wavelengths. Each of the A, T, G, and C has a fluorescent label attached, each with a characteristic excitation and emission wavelength distribution. Each of the three different laser wavelengths generates a unique response in terms of emission from the fluorescent label. Based on this, a three-dimensional response can be constructed as shown. Because three dimensions are used instead of one as in the prior art (FIG. 2), much greater precision is achieved in determining whether an A, T, G, or C has been incorporated. The confined space in which each label provides a response is called the "fingerprint" of A, T, C, and G, respectively. In this particular embodiment, the individual lasers are turned on and off, with only one laser active at each given time. The lasers are activated in sequential order in time. In more general embodiments, multiple lasers, e.g., two, three, or four lasers, can be activated in combinations of two, three, or more, to maximize the distance between the "fingerprints" to achieve the highest precision. [Figure 4]FIG. 4 illustrates an embodiment of the present disclosure. The detector and the radiation source form a control system, in which the detector can control the radiation source. The detector (in some embodiments, in conjunction with hardware and / or software responsible for the analysis of the detected secondary electromagnetic radiation) provides instruction and / or synchronization signals to the source of primary electromagnetic radiation (laser). These instructions can be, for example, to adjust the exposure time of a certain wavelength, e.g., to increase the exposure time of a certain wavelength. In some embodiments, the detector and the source of primary electromagnetic radiation synchronize the changes made by the source of electromagnetic radiation amplitude, wavelength, and / or other spectral characteristics of the radiation source. In one embodiment, the detector precisely controls when and which wavelength should be emitted by the radiation source at any particular time. In another embodiment, the detector's instructions to the source of electromagnetic radiation depend on the signal previously detected by the detector. In further embodiments, the detector controls the source of radiation in real time based on data recorded during a molecular event. Thus, the detector has 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 in order to optimize the accuracy of the determination of relevant parameters during a particular molecular event. Such modifications could, for example, be to change the time for which a laser is on and the time for which a laser is off. DETAILED DESCRIPTION OF THE INVENTION

[0018] Items in this specification (Item 1) A method for analyzing single molecule events, the method comprising the steps of: - emitting primary electromagnetic radiation from a radiation source to a sample in which a single molecular event occurs, wherein said primary radiation causes emission of secondary electromagnetic radiation from components of said single molecular event, and wherein said secondary electromagnetic radiation is distinguishable from said primary electromagnetic radiation; and - detecting said secondary electromagnetic radiation; wherein the method further comprises modifying at least one property of the primary electromagnetic radiation during the time period of the single molecule event, whereby primary electromagnetic radiation having a first property and primary electromagnetic radiation having at least one additional property are emitted into the sample during the time period, wherein the first property is different from the at least one additional property. (Item 2) The method of item 1, wherein the secondary electromagnetic radiation is detected separately for the first characteristic and the at least one additional characteristic of the primary electromagnetic radiation. (Item 3) The method of item 2, wherein a signal combining secondary electromagnetic radiation for the first characteristic of the primary electromagnetic radiation and secondary electromagnetic radiation for the at least one further characteristic of the primary electromagnetic radiation is provided and analyzed. (Item 4) The method of item 3, wherein the combined signal is a fingerprint from n different characteristics of the primary electromagnetic radiation, where n is particularly 2, 3, or 4. (Item 5) The method of any one of the preceding items, wherein at least one property of the primary electromagnetic radiation is altered between at least two different states during the duration of the single molecule event. (Item 6) The method of Item 5, wherein at least one property of the primary electromagnetic radiation is altered between about 2 to 10 different states during the duration of the single molecule event. (Item 7) The method of any one of Items 5 to 6, wherein the spacing of one or more individual states of the primary electromagnetic radiation is selectively varied, for example, increased relative to other individual states. (Item 8) The method of any one of the preceding items, wherein the radiation source comprises at least one laser, particularly multiple lasers having different emission wavelengths. (Item 9) The method of any one of the preceding items, wherein a plurality of individual samples, e.g., at least 10, at least 100, at least 1,000, or at least 10,000 individual samples, are provided. (Item 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. (Item 11) The method of item 10, wherein separate beams of radiation are directed at each sample or group of individual samples. (Item 12) The method of any one of the preceding items, wherein the at least one sample comprises at least one sample spot on a support. (Item 13) The method of Item 12, wherein the support includes a substrate and a plurality of sample spots on a surface of the support, the sample spots being spatially separated from one another. (Item 14) The method of items 12 or 13, wherein a component of the single molecule event is immobilized on the at least one sample spot. (Item 15) The method of Item 14, wherein the immobilized component comprises a biological element, e.g., a biomolecule. (Item 16) The method of Item 15, wherein the immobilized reaction component comprises a nucleic acid polymerizing enzyme, particularly a DNA polymerase or an RNA polymerase. (Item 17) The method of any one of the preceding items, wherein the at least one sample contains at least one luminescent component, particularly at least one fluorescent component, such as a reactant, reaction intermediate, and / or reaction product, of the single molecule event, wherein the at least one luminescent component emits the secondary electromagnetic radiation. (Item 18) The method of Item 17, wherein the at least one luminescent component is a compound containing a luminescent group, e.g., a luminescent labeling group, particularly a fluorescent group, e.g., a compound containing a fluorescent labeling group. (Item 19) The method of items 17 or 18, wherein the sample contains a plurality of different luminescent components, wherein at least some of the luminescent components are distinguishable from secondary electromagnetic radiation and have partially overlapping emission spectra. (Item 20) The method of any one of Items 17 to 19, wherein the sample contains a plurality of different luminescent components, particularly fluorescent components, wherein at least some of the luminescent components, particularly fluorescent components, have distinguishable and overlapping luminescence emission spectra, particularly distinguishable and overlapping fluorescence emission spectra. (Item 21) The method of any one of Items 17 to 20, wherein the at least one luminescent component is present in the sample in a free form. (Item 22) The method of any one of the preceding items, comprising separately detecting multiple single molecule events in different samples, particularly separately detecting multiple single molecule events in different samples in parallel. (Item 23) The method of any one of the preceding items, comprising detecting multiple consecutive single molecule events in one sample, and in particular, separately detecting multiple consecutive single molecule events in parallel in different samples. (Item 24) The method of Item 23, wherein the multiple single molecule events comprise successive nucleic acid extension and / or nucleic acid degradation steps of single molecule nucleic acid sequence analysis. (Item 25) The method of Item 24, wherein the single molecule nucleic acid sequence analysis includes multiple nucleic acid extension steps, in which a luminescent nucleotide building block, for example, a luminescent nucleoside polyphosphate containing 3 to 15 phosphate groups, is incorporated into the nucleic acid molecule in the presence of a nucleic acid polymerization enzyme, for example, a DNA polymerase or an RNA polymerase. (Item 26) The method of Item 25, comprising detecting secondary electromagnetic radiation resulting from incorporation of the light-emitting nucleotide building block, e.g., the light-emitting nucleoside polyphosphate, into the nucleic acid molecule. (Item 27) The method of any one of the preceding items, comprising varying the properties of the primary electromagnetic radiation several times during the duration of the single molecule event. (Item 28) The method of any one of the preceding items, comprising altering the properties of the primary electromagnetic radiation during each of several successive single-molecule events. (Item 29) The method of any one of the preceding items, wherein the alteration includes temporarily ceasing the primary electromagnetic radiation for the duration of the single molecule event. (Item 30) The method of any one of the preceding items, comprising modifying a property of the primary electromagnetic radiation selected from the group consisting of: - wavelength, - amplitude, - Pulse operation, - Polarized light, and - A combination of two or more of the above characteristics. (Item 31) The method of any one of the preceding items, comprising changing the wavelength of the primary electromagnetic radiation during the single-molecule reaction. (Item 32) The method of any one of the preceding items, comprising varying the characteristics of the primary electromagnetic radiation over a time interval ranging from about 50 ns to about 10 s, from about 1 μs to about 500 ms, or from about 10 μs to about 100 ms. (Item 33) A method according to any one of the preceding items, wherein at least one characteristic of the primary electromagnetic radiation is controlled and, if necessary, adjusted according to the secondary electromagnetic radiation emitted and detected from the sample of the single molecule event. (Item 34) The method of Item 33, wherein the control and optional adjustment is performed by a detector configured to detect the secondary electromagnetic radiation emitted from the sample of the single molecule event. (Item 35) The method of items 33 or 34, wherein the control and, if necessary, adjustment is a dynamic process based on prior measurement of secondary radiation. (Item 36) An apparatus for analyzing single molecule events, comprising: - means for providing at least one sample configured for a single molecule event; - a radiation source configured to emit primary electromagnetic radiation to 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, wherein the radiation source is configured to modify at least one property of the primary electromagnetic radiation at least once during the time period of the single molecule event, such that primary electromagnetic radiation having a first property and primary electromagnetic radiation having at least one further property are emitted at the sample during the time period, wherein the first property is different from the at least one further property. (Item 37) The apparatus of Item 36 configured to perform the method of any one of Items 1 to 35. (Item 38) The device of item 36 or 37 configured to perform single molecule nucleic acid sequence analysis. (Item 39) Use of the method of any one of items 1 to 35 or the device of any one of items 36 to 38 to provide improved accuracy in the analysis of single molecule events. (Item 40) Use of item 39 to provide improved discrimination of different incorporated nucleobases during a polymerase reaction.

[0019] Detailed Description The present disclosure provides methods and devices for analyzing single molecule events, wherein the single molecule events involve the emission of electromagnetic radiation from a sample, e.g., from a sample spot on a support, that contains components of the single molecule event. In certain embodiments, the single molecule events involve the response of a biological component to the emission of electromagnetic radiation having a characteristic.

[0020] The present disclosure addresses the need for improved accuracy in the analysis of single molecule events observed by irradiating a sample with primary electromagnetic radiation and simultaneously detecting the resulting secondary electromagnetic radiation emitted by the sample as a result of the irradiation.

[0021] In particular, the present disclosure provides apparatus and processes for improving accuracy in determining each kinetic step of a single molecule event by allowing the source of electromagnetic radiation irradiating the sample to be varied in time between observations of any single kinetic step of a single molecule reaction.

[0022] For this purpose, at least one characteristic of the primary radiation is changed during the duration of the single molecule event being analyzed. Thus, primary electromagnetic radiation having a first characteristic and primary electromagnetic radiation having at least one additional characteristic are radiated into the sample of the single molecule event during the duration of the single molecule event. The first characteristic of the primary electromagnetic radiation is different from the at least one additional characteristic. The characteristic may be selected from, for example, wavelength, amplitude, pulsed operation, polarization, and a combination of two or more of the characteristics. Thus, the first wavelength (or any other characteristic or combination of characteristics) of the primary electromagnetic radiation is different from the at least one additional wavelength (or any other characteristic or combination of characteristics).

[0023] According to certain embodiments, the secondary electromagnetic radiation emitted by components associated with the single molecule event is detected separately from the first characteristic and the 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 a signal of the secondary electromagnetic radiation induced by the first characteristic of the primary electromagnetic radiation and a signal of the secondary electromagnetic radiation induced by the at least one additional characteristic of the primary electromagnetic radiation. By analyzing the combined signal, detection accuracy is significantly improved.

[0024] In certain embodiments, the combined signal is a fingerprint from n different properties of the primary electromagnetic radiation, where n is particularly 2, 3, or 4. The combined signal may be represented in an n-dimensional diagram or plot, where n is an integer of at least 2.

[0025] In an exemplary embodiment, a single-molecule DNA sequencing procedure is performed. In particular, a polymerization reaction catalyzed by a DNA or RNA polymerase occurs, in which a DNA or RNA molecule binds to a single polymerase molecule, and nucleotide building blocks are incorporated one by one, thus extending the DNA or RNA molecule. The source of electromagnetic radiation irradiating the sample is changed in at least one characteristic once or several times during the period in which each nucleotide is incorporated into the DNA or RNA strand.

[0026] In some embodiments, the properties of the primary electromagnetic radiation are altered several times during the duration of the single molecule event, for example, 2, 3, 4, 5 or more times, for example, up to 100 times.

[0027] When analyzing multiple single molecule events, the properties of the primary electromagnetic radiation may be altered between several consecutive single molecule events. In these embodiments, the alteration scheme may be the same for each of the consecutive single molecule events or may differ between the consecutive single molecule events. In some embodiments, the alteration may be a temporary cessation of the primary electromagnetic radiation between the duration of the single molecule events, i.e., the sample is irradiated with primary electromagnetic radiation only during certain intervals between the duration of the single molecule events.

[0028] In some embodiments, the property of the primary electromagnetic radiation that is altered is selected from the group consisting of: - wavelength, - amplitude, - Pulse operation, - Polarized light, and - A combination of two or more of the above characteristics.

[0029] In certain embodiments, the wavelength of the primary electromagnetic radiation is changed at least once during the duration of the single molecule event.

[0030] According to the present disclosure, at least one property of the primary electromagnetic radiation is altered between at least two different states, e.g., between at least two different wavelengths or wavelength combinations, during the duration of the single molecule event. In some embodiments, the property of the primary electromagnetic radiation is altered between about 2 to 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, different states during the duration of the single molecule event.

[0031] According to the present disclosure, at least one property of the primary electromagnetic radiation is altered at least once during the duration of a single molecule event, hi some embodiments, the at least one property of the primary electromagnetic radiation is altered over a time interval ranging from about 50 ns to about 10 s, from about 1 μs to about 500 ms, or from about 10 μs to about 100 ms.

[0032] In some embodiments, at least one characteristic of the primary electromagnetic radiation is controlled and optionally adjusted according to the detected secondary electromagnetic radiation emitted from the sample of the single molecule event. In some embodiments, the control and optional adjustment is performed by a detector configured to detect the detected secondary electromagnetic radiation emitted from the sample of the single molecule event. In certain embodiments, the control and optional adjustment is a dynamic process based on prior measurements of secondary radiation. In certain embodiments, the control and optional adjustment is based on real-time analysis of the detected secondary electromagnetic radiation. This control and optional adjustment may provide a feedback mechanism for optimization of the scheme for altering at least one characteristic of the primary electromagnetic radiation.

[0033] In one embodiment, the detector (optionally in combination with appropriate hardware and / or software) provides indications of said modification of the primary radiation source according to a scheme depending on the type of molecular event currently being analyzed, e.g., as indicated by secondary emissions measured previously in the analysis procedure of said event.

[0034] In one embodiment, the detector (in combination with appropriate hardware and / or software as necessary) provides indication of said modification of the primary radiation source according to a scheme dependent on prevailing conditions, such as temperature, type and concentration of reagents, etc., which may be, for example, as indicated by previously measured secondary radiation in one or several prior event analysis procedures.

[0035] In specific embodiments, the detector (optionally in combination with appropriate hardware and / or software) can instruct the primary radiation source to become completely inactive at predetermined intervals during the duration of the single molecule event, and this instruction can be provided based on an analysis of the initially measured secondary radiation.

[0036] In a further embodiment, the change in the primary electromagnetic radiation is predetermined without a detector analyzing the secondary electromagnetic radiation in real time.

[0037] In certain embodiments, the wavelength of the primary electromagnetic radiation is changed several times during each incorporation event in a single-molecule nucleic acid synthesis procedure. Each A, C, T / U, and G nucleotide building block is differentially labeled, e.g., chemically linked to a different fluorescent moiety, each having an emission spectrum with a unique maximum, where each emission spectrum has a distribution around the maximum that overlaps with each other. In such cases, a radiation source can be provided that can emit multiple different wavelengths, e.g., a set of lasers with specific, unique output wavelengths. The wavelength of the primary electromagnetic radiation emitted by the radiation source can thereby be changed between several different states during the single-molecule event, e.g., the incorporation of a nucleotide building block into the extending nucleic acid molecule. For example, in a first state, a first laser is turned on while the other lasers are turned off; in a second state, a second laser is turned on while the other lasers are turned off; etc. The change between different states is a sufficiently rapid and continuous process that the excitation of each laser can be performed several times during each single event, for example, several times during each integration event. This allows the signal generated by the secondary radiation to be recorded as each laser is turned on several times during the integration event by a detector. The detector is synchronized with the lasers, and thus knows which laser is activated and when. This then allows a specific response curve for each laser wavelength to be provided.

[0038] These specific response curves for each event can be combined to obtain a multidimensional response plot or diagram for each event. Thus, the lack of precision associated with the overlapping emission distributions of individual labeled components can be overcome by providing a combined signal of the emissions recorded and collected for each laser during one integration event. This combined signal can generally be represented as an n-dimensional plot or diagram, or "fingerprint." In an exemplary, but non-limiting, embodiment, where three different lasers are used (n=3), a three-dimensional fingerprint can be provided, as shown in Figure 3 below. According to this fingerprint, there is no longer any overlap between the emission spectra of the labeled nucleotide building blocks, and each labeled nucleotide is clearly detected within a certain space in the three-dimensional plot.

[0039] In further exemplary embodiments, the number of lasers may be increased from n=3 to a larger number, for example 4, 5, or even more, allowing for further separation of the different signals within the n-dimensional fingerprint.

[0040] A corresponding fingerprint may also be provided by modifying other properties or a combination of properties of the primary electromagnetic radiation.

[0041] Furthermore, the spacing of one or more individual states of the primary electromagnetic radiation may be selectively varied, e.g., increased relative to other individual states, e.g., the length of the active period of one or more selected wavelengths used for the primary electromagnetic radiation may be varied, e.g., increased relative to the active periods of other wavelengths, depending on the emission characteristics of the luminescent component.

[0042] In some embodiments, the introduction of periods of zero primary electromagnetic radiation between active periods, for example different active periods, may be used.

[0043] In some embodiments, the individual states of the primary electromagnetic radiation may include combinations of different wavelengths and their relative amplitudes. In some embodiments, the individual states of the primary electromagnetic radiation may include combinations of different wavelengths and polarizations.

[0044] The sample of the single molecule event typically comprises a sample spot on a support. The support may comprise a substrate and a plurality of sample spots on the support surface that are spatially separated from one another. In some embodiments, the support surface is formed by the substrate and the sample spots. In some embodiments, the substrate forms a continuous area in which the sample spots are distributed. Each sample spot on the support surface is surrounded by the substrate, which differs from the sample spots, for example, in terms of material and / or surface. Typically, the substrate is configured to inhibit and / or prevent attachment of biomolecules, such as polypeptides, while the sample spots are configured to allow attachment of desired biomolecules.

[0045] On the sample spot, the component, e.g., biological component, involved in the single molecule event is immobilized. In one embodiment, the component comprises a biomolecule, in particular 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 component.

[0046] In some embodiments, the support is a substantially planar support, i.e., it does not contain any peaks or valleys of about 1000 nm or more, or about 100 nm or more. In further embodiments, the support is a structured support, e.g., a surface area of ​​about 5×10 -24 liters to approximately 1 x 10 -15The support includes a well-like recess having a volume of 1 liter or a pillar having a height of about 1 nm to about 500 nm. In principle, the support may have any design as long as a reaction space can be formed that allows the occurrence of a single molecule event on the at least two sample spots where the single biomolecule is immobilized.

[0047] 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., an absolute refractive index of about 1.5 to about 3 in the visible range, or about 1.5 to about 4 in the near-infrared range. In further embodiments, the substrate is an optically opaque material, e.g., a metal or a metalloid such as silicon.

[0048] In certain embodiments, the substrate comprises a non-conductive material. Specific examples include glass, quartz, plastic, a metal oxide-based material, e.g., a silicon dioxide-based material (such as glass, silica, or quartz), or a composite material comprising such materials. In further embodiments, the substrate comprises a conductive material, e.g., an optically transparent material such as indium tin oxide.

[0049] Typically, the substrate has a thickness of about 10 μm to about 5 mm, particularly about 20 μm to about 2 mm.

[0050] The surface of the support includes a plurality of sample spots spatially separated from one another by the substrate surface. The sample spots are configured for attachment of biomolecules. In some embodiments, the support includes a plurality of sample spots, e.g., at least 10, at least 100, at least 1,000, at least 10,000 sample spots, or at least 100,000 spots. In some embodiments, the support includes up to 10 6 or 10 9 , or even more sample spots.

[0051] In some embodiments, the sample spot comprises or consists of at least one conductive material, such as a single metal, or a combination of metals, such as an alloy or mixture of multiple different metals. For example, metals that can be attached to sulfur-containing elements in the form of thiols or disulfides, or metals that can be attached to chelating elements such as polyhistidine tags, 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 foregoing metals.

[0052] In some embodiments, the sample spot comprises or consists of at least one metal oxide, including a single metal oxide or a combination of metal oxides. For example, metal oxides that can be attached to phosphorus-containing elements, such as phosphonic acid or phosphonate ester forms, or metal oxides that can be attached to chelating elements, such as polyhistidine tags, are suitable. Specific examples of suitable metal oxides include TiO2 and NiO.

[0053] In a further embodiment, the sample spot comprises or consists of at least one non-conductive material.

[0054] Sample spots can be prepared by deposition of a metal vaporized on the support covered with a grid mask, which can be fabricated by electron beam lithography or an equivalent technique. The size of the openings in the grid mask can correspond to the size of the spots on the support surface. Alternatively, the spots on the support can be prepared by site-specific deposition of nanoparticles, e.g., having a size of 2-10 nm, onto the support, particularly onto supports with planar surfaces, by precision pipetting of the particles.

[0055] In certain embodiments, the sample spots have a size suitable for said attachment of a single biomolecule, hi such embodiments, the sample spots have a diameter of about 1 nm to about 30 nm, particularly about 2 nm to about 20 nm.

[0056] In some embodiments, the sample spot is a discrete structure on the surface of the substrate. In some embodiments, the sample spot has a lower surface proximal to the substrate and an upper surface distal to the substrate, where the distance between the lower surface and the upper surface defines the height of the sample spot. In certain embodiments, the height is about 50 pm to about 500 nm, particularly about 100 pm to about 20 nm, and more particularly about 500 pm to about 10 nm, e.g., about 2 nm.

[0057] The sample spots on the support are configured to accommodate the attachment of biomolecules, for example, by covalent or non-covalent bonds. The biomolecules can be selected from polypeptides, nucleic acids, carbohydrates, and any combination thereof, for example, glycosylated polypeptides or ribonucleoproteins. In some embodiments, the biomolecule is a complex consisting of several individual units, for example, several polypeptide units, or several polypeptide and nucleic acid units.

[0058] In a specific embodiment, the biomolecule is a nucleic acid polymerizing enzyme, particularly a DNA polymerase or an RNA polymerase, or a nucleic acid-polymerizing molecule complex, particularly a DNA polymerizing complex or an RNA polymerizing complex comprising a nucleic acid polymerizing enzyme and a nucleic acid molecule. In a specific embodiment, the biomolecule is a DNA polymerase with a DNA binding cleft, particularly a family A DNA polymerase, including but not limited to Klenow, Taq, or T7 DNA polymerase, or any genetically modified version thereof, or a family B polymerase, including but not limited to Therminator, Phi29, RB-69, or T4 DNA polymerase, or any genetically modified version thereof. In this context, reference is made to US Pat. No. 7,745,116 B2, the contents of which are incorporated herein by reference.

[0059] In a further embodiment, the biomolecule is a nuclease, in particular an exonuclease or a nucleic acid-degrading molecule complex, in particular a DNA-degrading molecule complex or an RNA-degrading molecule complex comprising a nuclease and a nucleic acid molecule.

[0060] In a further embodiment, the biological molecule is a gene editing enzyme, in particular a Cas nuclease such as a Cas3, Cas9, Cas10, or Cas12 nuclease, or any genetically modified form 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.

[0061] The single molecule event to be detected occurs in a sample containing the required components of the event, e.g., biomolecules and, optionally, small molecules. At least one of the components is a luminescent component comprising a luminescent group, i.e., a group capable of emitting secondary electromagnetic luminescent radiation in response to irradiation by the primary electromagnetic radiation. In some embodiments, the luminescent component is a compound bearing a luminescent labeling group. In some embodiments, the luminescent component is a compound that is itself capable of emitting light.

[0062] In some embodiments, the luminescent component of the 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 fluorescent radiation in response to being irradiated by the primary electromagnetic radiation. In some embodiments, the fluorescent component is a compound carrying a fluorescent labeling group. In some embodiments, the fluorescent component is a compound that can itself fluoresce.

[0063] In certain embodiments, the sample of the single molecule event comprises a plurality of different luminescent components, where at least some of the luminescent components have overlapping emission spectra of secondary electromagnetic radiation. For example, the sample may comprise a plurality of different fluorescent components, where at least some of the fluorescent components have distinguishable and overlapping fluorescence emission spectra.

[0064] In certain embodiments, the fluorescent moieties have overlap in their fluorescence wavelengths, their fluorescence lifetimes, their polarization properties, eg, circular dichroism, and any combination thereof.

[0065] In some embodiments, the fluorescence lifetime is measured several times during each molecular event. Other spectroscopic methods, such as circular dichroism analysis, absorbance methods in which the absence of electromagnetic radiation is observed in at least one state during the several spectroscopic measurements during a single molecular event, are also encompassed by the present invention. Combinations of several spectroscopic methods are also embodiments of the present invention.

[0066] The at least one luminescent component may be present in the sample in immobilized and / or free form. In certain embodiments, the at least one luminescent component is present in free form.

[0067] The disclosed methods can include separately analyzing multiple single molecule events in different samples, particularly in different sample spots on a support, hi certain embodiments, the methods include separately analyzing multiple single molecule events in parallel in different samples, particularly in different sample spots on a support.

[0068] In some embodiments, the method of the present disclosure includes analyzing multiple consecutive single molecule events in one sample, particularly analyzing multiple consecutive single molecule events in different samples in parallel separately. The term "multiple single molecule events" encompasses a series of consecutive single molecule events occurring in the same sample, for example, at the sample spot on the support. In some embodiments, the series 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 consecutive nucleic acid extension and / or nucleic acid degradation steps of single molecule nucleic acid sequence analysis.

[0069] In certain embodiments, single-molecule nucleic acid sequencing involves multiple steps of nucleic acid extension, in which a light-emitting nucleotide building block, e.g., a light-emitting labeled nucleoside polyphosphate containing 3 to 15 phosphate groups, is incorporated into a nucleic acid molecule in the presence of a nucleic acid polymerizing enzyme, e.g., a DNA polymerase or an RNA polymerase. These embodiments can include detecting secondary electromagnetic radiation from the incorporation of the light-emitting nucleotide building block, e.g., a light-emitting labeled nucleoside polyphosphate, into the nucleic acid molecule.

[0070] According to the present disclosure, the sample in which the single molecule event occurs is irradiated with primary electromagnetic radiation, wherein at least one property of the primary electromagnetic radiation is altered during the duration of the single molecule event.

[0071] The present disclosure further provides an apparatus for analyzing single molecule events, said apparatus comprising: - means for providing at least one sample configured for a single molecule event; - a radiation source configured to emit primary electromagnetic radiation to 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, wherein the radiation source is configured to vary at least one property of the primary electromagnetic radiation at least once during the duration of the single molecule event, whereby primary electromagnetic radiation having a first property and primary electromagnetic radiation having at least one further property are emitted into the sample during the duration of the single molecule event, wherein the first property is different from the at least one further property.

[0072] In some embodiments, the device is configured to perform the methods described above, for example, to perform single molecule nucleic acid sequencing.

[0073] The disclosed methods and devices are useful for providing increased precision in the analysis of single molecule events, and in certain embodiments, they are useful for providing improved discrimination between different nucleobases incorporated during the polymerase reaction.

[0074] Methods and apparatus for analyzing single molecule events are disclosed, for example, in WO2002 / 097406, WO2003 / 052137, WO2006 / 013110, WO2013 / 131888, WO2015 / 104245, WO2017 / 001407, and WO2018 / 104301, the contents of which are incorporated herein by reference.

[0075] For the analysis of single molecule events, the biomolecules can be placed in the sample spots on the support, where they are contacted with a sample solution containing free reaction partners, thereby defining one or more reaction spaces, particularly at least 100, at least 1000, or at least 10,000, and up to 10 6 Many more molecules can be analyzed on a single support, for example, on a single planar support.

[0076] The nucleic acid molecules to be sequenced can be selected from DNA molecules, such as genomic DNA fragments, cDNA molecules, plasmids, or RNA molecules, such as mRNA molecules. The nucleic acid molecules can be derived from a genomic or expression library generated from a cell or organism, e.g., a eukaryotic or prokaryotic cell or organism. This can be achieved by using a plurality of different nucleic acid template molecules, e.g., at least 10, 100, 1,000, or 10,000, and up to 100,000, 10 6 , or 10 7 This allows for the parallel sequencing of , or even more, different nucleic acid molecules.

[0077] The nucleic acid molecule to be sequenced can be linear or circular, for example, a covalently linked circular single-stranded nucleic acid molecule.To obtain a circular nucleic acid template, the linear nucleic acid molecule can be subjected to a circularization procedure and, if necessary, a strand separation procedure during sample preparation.Circularization can be achieved by ligation according to known protocols, for example, using DNA ligase or RNA ligase.In some embodiments, an adapter molecule and / or an identifier molecule, i.e., a nucleic acid molecule of known sequence, can be attached to the nucleic acid molecule.

[0078] The sequencing may include nucleic acid extension and / or nucleic acid degradation. The sequencing process may include one or more sequencing cycles.

[0079] The nucleic acid synthesizing enzyme molecule is capable of extending a primer annealed to a nucleic acid template molecule. Primer extension can be carried out by stepwise incorporation of individual nucleotide building blocks into 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 synthesizing enzyme is selected from polymerases capable of template-specific nucleic acid polymerization, preferably natural or modified polymerases including DNA polymerases and RNA polymerases, such as thermostable DNA polymerases.

[0080] Specific examples of suitable DNA polymerases include Taq polymerase, exonuclease-deficient Taq polymerase, E. coli DNA polymerase I, Klenow fragment, reverse transcriptase, Φ29-related polymerases, including wild-type Φ29 polymerase, and derivatives of such polymerases, such as exonuclease-deficient versions, T7 DNA polymerase, T5 DNA polymerase, RB69 polymerase, and the like.

[0081] Nuclease molecules can stepwise cleave individual nucleotide building blocks from nucleic acid molecules. Preferably, exonucleases are used, more preferably single-stranded exonucleases that degrade in the 3'→5' or 5'→3' direction. Particularly preferred exonucleases are 3'→5' exonucleases such as E. coli exonuclease I and E. coli exonuclease III, and 5'→3' exonucleases such as T7 exonuclease, E. coli exonuclease II, and E. coli exonuclease VIII. In addition, the exonuclease activity of various polymerases, such as Klenow fragment, Taq polymerase, or T4 polymerase, can be used.

[0082] The nucleic acid synthesizing enzyme molecule is contacted with a linear or circular nucleic acid template molecule, such as a single-stranded DNA or RNA molecule, and a primer molecule that is annealed to or can anneal to the nucleic acid template molecule. The primer molecule is preferably a single-stranded nucleic acid or nucleic acid analog molecule with a free 3' end that can be extended by an enzymatic reaction catalyzed by the immobilized nucleic acid synthesizing enzyme molecule. The length of the primer molecule is selected to enable effective annealing to the template under reaction conditions. Typically, the length of the primer molecule is at least 8, at least 10, at least 12, or at least 15 nucleotides, and can be, for example, up to 20, 25, 50, or 100 nucleotides, or even longer. In some embodiments, the primer is resistant to digestion by the nuclease molecule, for example, by incorporating a nucleotide analog building block and / or an analog bond between the nucleotide building blocks that is stable against degradation. In other embodiments, the primer is susceptible to digestion by the nuclease molecule.

[0083] The sequence of the primer is selected so that it anneals effectively to the template molecule under the reaction conditions. For example, the primer can be a universal degenerate primer that can statistically anneal to unknown nucleic acid sequences. In other embodiments, the primer can be capable of annealing to a known sequence portion of the nucleic acid template molecule. In this embodiment, a known adapter and / or identifier sequence can be incorporated into the nucleic acid template molecule. The primer can be unlabeled or can include a fluorescent labeling group.

[0084] Furthermore, the presence of luminescent nucleotide building blocks, e.g., nucleotide building blocks carrying at least one fluorescent labeling group, is essential, and preferably each different nucleotide building block (A, G, C, T / U) contains a different fluorescent labeling group.

[0085] The fluorescent labeling group may be selected from known fluorescent labeling groups used for labeling biopolymers, particularly nucleic acids, such as fluorescein dyes, rhodamine, oxazines such as Evoblue or Gnothis Blue, phycoerythrin, Cy3, Cy5, IR dyes, or derivatives thereof.

[0086] The nucleotide building blocks may carry (i) a fluorescent labeling group that remains on the building block when the building block is incorporated into a nucleic acid molecule during primer extension catalyzed by a nucleic acid synthesizing enzyme molecule, and / or (ii) a fluorescent labeling group that is cleaved from the building block when the building block is incorporated into a nucleic acid molecule during primer extension catalyzed by a nucleic acid synthesizing enzyme molecule. The fluorescent labeling group that remains on the building block is preferably attached to the α-phosphate group, sugar, and / or nucleobase.

[0087] In certain embodiments, the fluorescent labeling group remaining in the building block is attached to the nucleobase via a linker, which may have a chain length of up to 15, preferably 10-12, carbon atoms, optionally including a heteroatom, such as an N, O, or S atom. The fluorescent labeling group that is cleaved upon incorporation of the building block into a nucleic acid molecule may be attached to a terminal phosphate group, such as the γ-phosphate group of a triphosphate building block, of a polyphosphate building block, including, but not limited to, a hexa-, penta-, tetra-, or triphosphate building block. In certain embodiments, a building block is selected that contains both (i) a fluorescent labeling group that remains after incorporation and (ii) a fluorescent labeling group that is cleaved during incorporation. In this case, fluorescent groups that can interact with each other by, for example, quenching and / or energy transfer, may be selected.

[0088] The nucleic acid molecule to be sequenced comprises a fluorescent labeling group if the nucleic acid molecule is subjected to direct sequencing using a nuclease molecule, whereas the nucleic acid molecule to be sequenced may not comprise a fluorescent labeling group if the nucleic acid molecule is used as a template in primer extension.

[0089] The sequencing procedure may include a step of generating a nucleic acid molecule having incorporated nucleotide building blocks during primer extension catalyzed by the nucleic acid synthesizing enzyme molecule, and / or a second step of cleaving individual nucleotide building blocks from the generated nucleic acid molecule catalyzed by a nuclease molecule. Depending on the type of fluorescent label, nucleic acid sequencing may be performed during primer extension and / or degradation.

[0090] Sequencing during primer extension involves the use of nucleotide building blocks that carry a fluorescent labeling group that is cleaved from the building block when the nucleotide building block is incorporated into a nucleic acid molecule. In this case, a time-dependent change in fluorescence caused by the cleavage of the fluorescent labeling group from the nucleotide building block can be determined. Sequencing during nucleic acid degradation involves the use of nucleotide building blocks that carry a fluorescent labeling group that remains on the building block when the nucleotide building block is incorporated into a nucleic acid molecule. The stepwise cleavage of individual nucleotide building blocks from the nucleic acid molecule causes a time-dependent change in fluorescence as the labeled nucleotide building block is released from the nucleic acid molecule. In some embodiments, performing sequencing during extension and degradation is also possible, i.e., when using nucleotide building blocks that carry both a fluorescent labeling group that remains on the building block and a fluorescent labeling group that is cleaved from the building block when the building block is incorporated into a nucleic acid molecule. In this embodiment, both fluorescent groups may be the same or different.

[0091] In some embodiments, the method comprises one or more cycles of nucleic acid synthesis and nucleic acid degradation to determine the base sequence of a nucleic acid molecule template. The nucleic acid synthesis comprises extension of the primer annealed to the nucleic acid template molecule, catalyzed by the nucleic acid synthesizing enzyme molecule, whereby 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 nuclease molecule.

[0092] When a nucleotide building block is incorporated into an extended nucleic acid molecule, a time-dependent change in the fluorescence can occur, which can be detected as described above. Preferably, incorporation of the nucleotide building block into an extended nucleic acid molecule is associated with a detectable increase in the fluorescence, preferably a transient increase in the fluorescence. For example, a nucleotide building block can be used that carries a fluorescent labeling group on the portion of the molecule that is cleaved when the building block is incorporated into the primer, e.g., the γ-phosphate.

[0093] When nucleotide building blocks are cleaved from the synthesized nucleic acid molecule, the fluorescent labeling group incorporated into the nucleic acid chain interacts, for example, with chemical groups in the nucleic acid, in particular with nucleic acid bases such as G, or / and with adjacent fluorescent labeling groups, resulting in a change in fluorescence, particularly in fluorescence intensity, due to quenching or / and energy transfer processes, compared to the so-called "isolated" form of the fluorescent labeling group, and this time-dependent change in fluorescence can be measured. The removal of individual nucleotide building blocks by cleavage changes the overall fluorescence, for example the fluorescence intensity of the immobilized nucleic acid chain, and this change is a function of the removal of individual nucleotide building blocks by cleavage, i.e., a function of time.

[0094] In certain embodiments, the association of a labeled nucleotide with a biomolecular complex is detected by measuring the polarization of the emitted photons. The polarization of excited-state photons is altered by the rotational motion of the light-emitting nucleotide label and can be used to identify free-floating, unbound labeled nucleotides in the polymerization process.

[0095] This time-dependent change in fluorescence during extension and / or degradation can be recorded in parallel for multiple nucleic acid molecules and correlated with the base sequence of the individual nucleic acid strands. Preferably, fluorescent labeling groups are used that are at least partially quenched when incorporated into the nucleic acid strand, such that the fluorescence intensity increases after the nucleotide building block containing the labeling group or an adjacent building block that causes quenching is removed by cleavage.

[0096] During incorporation and / or removal of individual nucleotide building blocks, it is possible to measure the change in fluorescence intensity of the nucleic acid strand and / or the incorporated or cleaved nucleotide building blocks due to quenching or energy transfer processes. This change in fluorescence intensity over time depends on the base sequence of the nucleic acid strand being studied and can therefore be correlated with said sequence.

[0097] The complete sequence of the nucleic acid molecule can be determined by using a mixture of nucleotide building blocks labeled with all four different bases, e.g., A, G, C, and T, or a mixture of nucleotide building blocks labeled with combinations of two or three different bases. If appropriate, a "sequence identifier," i.e., a labeled nucleic acid of known sequence, can be attached to the nucleic acid strand to be studied, e.g., by enzymatic reaction using ligase and / or terminal transferase, so that a known fluorescence pattern is obtained initially at the start of sequencing, followed by a fluorescence pattern corresponding to the unknown sequence to be studied.

[0098] The detection comprises irradiating the support with primary electromagnetic radiation from a radiation source, preferably by means of a laser or other suitable light source, to cause excitation of the fluorescent labeling group. In one embodiment, the radiation source comprises a plurality of different lasers emitting radiation at different wavelengths. In this regard, it is possible to use one or more laser beams, e.g., an expanded laser beam having a cross section of about 1 to 20 mm, and / or multiple laser beams. The detection preferably comprises multipoint fluorescence excitation by laser, e.g., a dot matrix of laser dots generated via diffractive optics (see WO 2002 / 097406) or a quantum well laser.

[0099] The fluorescence emission of multiple nucleic acid strands can be detected in parallel using a detection means such as, for example, an electronic detector matrix, for example, a CCD camera, a CMOS detector matrix, for example, a detector matrix including an avalanche photodiode matrix. The detection can be performed in such a way that fluorescence excitation and detection are performed in parallel for some or all of the nucleic acid strands being studied. Preferably, detection is performed on fluorescent light emitted from the support surface in an essentially perpendicular direction through the reaction space or the support.

[0100] The detection can be carried out, for example, by means of single molecule detection, for example, by fluorescence correlation spectroscopy. -21 From 10 -10 The method involves exposing a very small, preferably confocal, volume element of about 1 μm to excitation light from a laser or other suitable light source. This excitation light excites receptors present in the measurement volume, causing them to fluoresce. The fluorescence emitted from this measurement volume is measured by means of a photodetector, and the measured change in emission over time is correlated with the concentration of the analyte, thereby making it possible to identify individual molecules in the measurement volume at suitable high dilutions. Details of the procedure and the equipment used for detection can be found in the disclosure of EP 0 679 251, the contents of which are incorporated herein by reference. The 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.

[0101] Alternatively or additionally, detection can also be performed by a method of time-resolved decay measurement called "time gating," as described, for example, by Rigler et al., "Picosecond Single Photon Fluorescence Spectroscopy of Nucleic Acids," in "Ultrafast Phenomena," D.H. Australianton, Ed., Springer 1984, the contents of which are incorporated herein by reference. Here, the fluorescent molecules are excited in the measurement volume, followed by a detection interval of the photodetector, e.g., a time interval of ≥ 100 ps. In this way, the background signal generated by the Raman effect can be kept sufficiently low, allowing for the detection of single molecules in an essentially interference-free manner.

[0102] The disclosed methods and apparatus are also suitable for the analysis of additional single molecule events, where there is a high demand for high yield single molecule analysis, i.e., the analysis of single biomolecules bound to spots selected for analysis.

[0103] In certain embodiments, the present disclosure relates to said single molecule analysis of receptor-ligand interactions, including, for example, binding of a receptor protein to a sample spot and subsequent study of its interaction with its ligand, for example in drug development.

[0104] In further embodiments, the present disclosure relates to single molecule analysis of hybridization events, including the attachment of short single-stranded nucleic acid molecules, such as DNA or RNA molecules, having lengths in the range of 3-300 nucleotides, followed by the addition of a sample containing a complementary nucleic acid molecule and the observation of any hybridization events. Applications can include, for example, viral RNA / DNA detection, bacterial DNA / RNA detection, and the detection of short DNA fragments from cancer cells in the bloodstream.

[0105] The present disclosure will be further described in detail by reference to the following specific embodiments. [Example]

[0106] Figure 1 shows an embodiment of the prior art. A radiation source (1) directs a primary electromagnetic radiation (2) toward a molecule (4) bound to a support (3). The molecule (4) immobilized on the support (3) undergoes a biomolecular process that includes several states. Some of these states result in an associated change in the emission of secondary electromagnetic radiation (5) in response to irradiation with the primary electromagnetic radiation (2). A given biomolecular state gives rise to a unique emission profile of secondary electromagnetic radiation (5) that is detected by a detector (6).

[0107] Figure 2 shows an embodiment of the prior art. Sequencing of single DNA or RNA molecules can involve generating a secondary DNA or RNA strand complementary to a primary DNA or RNA strand. An enzyme, e.g., a polymerase, catalyzes the extension of the secondary strand, such that nucleotides are sequentially incorporated one by one in a manner complementary to the nucleotides in the primary strand. Each nucleotide is fluorescent with a unique emission wavelength distribution. These emission wavelength distributions overlap to some extent, creating a limiting factor in the ability to accurately determine which nucleotides have been incorporated. As a result, the accuracy of single DNA or RNA molecule sequencing is limited.

[0108] Figure 3 shows an embodiment of the present disclosure. The radiation source of the primary electromagnetic radiation includes a set of three lasers with different wavelengths. During a molecular event, for example, during the incorporation of a single nucleotide into an elongated secondary nucleic acid strand, the primary electromagnetic radiation varies in real time during the molecular event. This then results in the detection of secondary electromagnetic radiation that can be plotted on a three-dimensional graph. Compared to the prior art embodiment according to Figure 2, the availability of a third dimension eliminates overlap between the secondary electromagnetic radiation profiles of individual nucleotides, leading to improved accuracy compared to the prior art.

[0109] 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 has a fluorescent label attached, each with a characteristic excitation and emission wavelength distribution. Each of the three different laser wavelengths produces a unique response in terms of the emission from the fluorescent label. Based on this, a three-dimensional response can be constructed as shown. Because three dimensions are used instead of one dimension as in the prior art (Figure 2), much greater accuracy is achieved in determining whether A, T, G, or C has been incorporated. The confined space in which each label provides a response is called the "fingerprint" of A, T, C, and G, respectively.

[0110] In this particular embodiment, the individual lasers are turned on and off, with only one laser active at each given time. The lasers are activated in sequential order in time. In more general embodiments, multiple lasers, for example, two, three, or four lasers, can be activated in combinations of two, three, or more to maximize the distance between the "fingerprints" to achieve the highest accuracy.

[0111] Figure 4 shows an embodiment of the present disclosure, wherein the detector and the radiation source form a control system, where the detector can control the radiation source.

[0112] The detector (in some embodiments in conjunction with hardware and / or software responsible for the analysis of the detected secondary electromagnetic radiation) provides instruction and / or synchronization signals to the source of primary electromagnetic radiation (laser). These instructions may be, for example, to adjust the exposure time of a certain wavelength, for example, to increase the exposure time of a certain wavelength.

[0113] In some embodiments, the detector and the source of primary electromagnetic radiation synchronize the changes made by the source of electromagnetic radiation amplitude, wavelength, and / or other spectral characteristics of the radiation source. In one embodiment, the detector precisely controls when and which wavelength must be emitted by the radiation source at any particular time. In another embodiment, the detector's instruction of the source of electromagnetic radiation depends on the signal previously detected by the detector.

[0114] In a further embodiment, the detector controls the source of radiation in real time based on data recorded during a molecular event. Thus, the detector has 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 relevant parameters during a particular molecular event. Such modifications can be, for example, to change the time for which a laser is on and the time for which a laser is off.

Claims

1. 1. A method for analyzing single molecule events, said method comprising the steps of: - emitting primary electromagnetic radiation from a radiation source to a sample in which a single molecular event occurs, wherein said primary radiation causes emission of secondary electromagnetic radiation from components of said single molecular event, and wherein said secondary electromagnetic radiation is distinguishable from said primary electromagnetic radiation; and - detecting said secondary electromagnetic radiation, wherein the method further comprises varying at least one property of the primary electromagnetic radiation several times during the period of the single molecule event, whereby primary electromagnetic radiation having a first property and primary electromagnetic radiation having at least one additional property are emitted into the sample during the period, wherein the first property is different from the at least one additional property; and wherein the method further comprises altering a property of the primary electromagnetic radiation several times during the duration of the single molecule event.

2. 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, and wherein a signal combining the secondary electromagnetic radiation for the first characteristic and the at least one further characteristic of the primary electromagnetic radiation is provided.

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

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

5. at least one said characteristic of said primary electromagnetic radiation is controlled, and if necessary adjusted, according to said detected secondary electromagnetic radiation emitted from said sample of said single molecule event; wherein in particular said controlling and optionally adjusting is performed by a detector configured to detect said secondary electromagnetic radiation emitted from said sample of said single molecule events; and More particularly, the method of any one of the preceding claims, wherein said controlling and optionally adjusting is a dynamic process based on prior measurements of secondary radiation emitted from said sample of said single molecule events.

6. the at least one sample comprises at least one sample spot on a support, wherein the support comprises a substrate and a plurality of sample spots on the surface of the support, and wherein the sample spots are spatially separated from one another; wherein a component of the single molecule event is immobilized on at least one of the sample spots, wherein the immobilized component comprises, in particular, a biomolecule, such as a nucleic acid polymerizing enzyme, in particular a DNA polymerase or an RNA polymerase; and 10. The method of any one of the preceding claims, wherein said at least one sample comprises at least one luminescent component of said single molecule event, such as a reactant, reaction intermediate, and / or reaction product, and wherein said at least one luminescent component emits said secondary electromagnetic radiation.

7. 7. The method of claim 6, wherein said at least one luminescent moiety is a fluorescent moiety.

8. 8. The method of claim 6 or 7, wherein the sample comprises a plurality of different luminescent, particularly fluorescent, components, wherein at least some of the luminescent, particularly fluorescent, components have distinguishable and overlapping luminescence emission spectra, particularly distinguishable and overlapping fluorescence emission spectra.

9. 10. The method of any one of the preceding claims, comprising analysing multiple consecutive single molecule events in one sample, in particular comprising separately analysing multiple consecutive single molecule events in parallel in different samples.

10. The plurality of single molecule events comprises sequential steps of single molecule nucleic acid sequencing including multiple steps of nucleic acid extension, wherein a light-emitting nucleotide building block is incorporated into a nucleic acid molecule in the presence of a nucleic acid polymerase; and 10. The method of claim 9, wherein secondary electromagnetic radiation resulting from the incorporation of the light-emitting nucleotide building block into the nucleic acid molecule is detected.

11. 10. The method of any one of the preceding claims, comprising modifying a property of the primary electromagnetic radiation selected from the group consisting of: - wavelength, - amplitude, - pulsed operation, - Polarization, and - A combination of two or more of the above properties.

12. 10. The method of any one of the preceding claims, comprising modifying the characteristics of the primary electromagnetic radiation over a time interval ranging from about 50 ns to about 10 s, from about 1 μs to about 500 ms, or from about 10 μs to about 100 ms.

13. 1. An apparatus for analyzing single molecule events, comprising: means for providing at least one sample configured for a single molecule reaction; a radiation source configured to emit primary electromagnetic radiation at 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, wherein the radiation source is configured to vary at least one property of the primary electromagnetic radiation several times during the time period of the single molecule event, such that primary electromagnetic radiation having a first property and primary electromagnetic radiation having at least one further property are emitted at the sample during the time period, wherein the first property is different from the at least one further property.

14. 14. The apparatus of claim 13, further comprising means for controlling, and optionally adjusting, said primary electromagnetic radiation in accordance with said detected secondary electromagnetic radiation emitted from said sample of said single molecule event, wherein in particular, said means comprises a detector configured to detect said secondary electromagnetic radiation emitted from said sample of said single molecule event; and More particularly, the means is configured to control and optionally adjust the primary electromagnetic radiation according to a dynamic process based on prior measurements of the secondary radiation emitted from the sample of the single molecule event.

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

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