Dye dimer and kit for use in an imaging procedure

Abstract

dye dimer, containing - a single strand of DNA (nucleotide strand) formed by a sequence of nucleotides, and - one molecule each of a dye capable of emitting fluorescence radiation, bound to the opposite terminal regions of the nucleotide strand, - wherein the dye dimer in a first conformation is slightly excitable to emit fluorescence radiation; and - is readily excitable to emit fluorescence radiation in a second conformation, wherein the fluorescence intensity in the second conformation is at least 5 times higher, in particular at least 8 times higher, advantageously at least 10 times higher, than in the first conformation, characterized in that - the dye is ATTO 520.

Description

[0001] The invention relates to a dye dimer and a kit.

[0002] For high-resolution imaging, especially in living cells and tissues (samples), as well as for the investigation of processes in living cells, a number of imaging techniques are known that are based on the marking of structures of interest with markers and their excitation to emit detection radiation.

[0003] In recent years, the method known as DNA-PAINT (DNA-point accumulation for imaging in nanoscale topography) has been established and further developed (Chung, KKH et al., 2022, Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging, Nature Methods 19: 554-559). For example, a structure to be examined (antigen) in the sample is coated with an antibody that specifically binds to it. The antibody is coated with a single-stranded sequence of deoxyribonucleic acid (DNA), which is referred to as a "docking strand" (hereinafter also: binding molecule).

[0004] To image antibodies bound to an antigen, a number of dye molecules are added to the medium containing the sample to be imaged. These dye molecules contain a dye, specifically a fluorophore, bound to a short piece of single-stranded DNA (nucleotide strand, "imager strand"). The nucleotide sequence of the nucleotide strand is compatible with the binding molecule, meaning the dye molecule can bind to the binding molecule. It is advantageous if the dye molecules are designed in such a way that they only exhibit strong fluorescence emission when the dye molecule in question is attached to the binding molecule, i.e., when the imager strand and docking strand are attached to one another.

[0005] Recent work shows that unwanted background radiation can be significantly reduced by attaching a dye to each end of the nucleotide strand of the dye molecule (TDI, "two-dye-imager"; Kessler, LF et al. 2023, Self-quenched flurophore dimers for DNA-Paint and STED microscopy, Angew. Chem. Int. Ed. 62, e202307538). Such dye molecules, i.e. the combination of a nucleotide strand and two dyes, are henceforth referred to as dye dimers. The dyes are chosen such that they largely prevent each other from emitting fluorescence radiation when the dyes are spatially close to each other (self-quenching). When not coupled to a binding molecule, the dye dimer exhibits a compact shape. This state (hereinafter also referred to as the first conformation) is also referred to as the "H-dimer."

[0006] If the dye dimer binds to the binding molecule, i.e., if the "image strand" and "docking strand" align, the nucleotide strand is stretched and the dyes are so far apart that the effect of self-quenching is no longer relevant. Therefore, the dyes can be excited in this second conformation, for example, using suitable excitation radiation to emit fluorescent radiation with a higher intensity than in the first conformation.

[0007] The desired high spatial resolution when investigating interactions between cells and cell components can be achieved if only a narrow area of ​​the sample in the direction of the detection axis of a detection optics used (z-direction) is illuminated with excitation radiation and, if possible, fluorescence radiation is excited only in the limited illuminated area (Kessler, LF et al. 2023, Self-quenched flurophore dimers for DNA-Paint and STED microscopy, Angew. Chem. Int. Ed. 62, e202307538).

[0008] However, the state-of-the-art solutions require long measurement times to image a volume of the sample. In addition, the unwanted background radiation must be kept within an acceptable range by using only small amounts of dye molecules.

[0009] The invention is based on the object of proposing a dye dimer by means of which disadvantages of the prior art are reduced and in particular excitation of undesired background signals is reduced and thus the signal-to-background ratio is increased.

[0010] This object is achieved with a dye dimer according to the main claim. Provisions of the invention are proposed in the form of kits. Advantageous further developments are specified in the dependent claims.

[0011] The dye dimer comprises a single DNA strand formed by a sequence of nucleotides. One molecule of the dye ATTO 520 ( Fig. 2) is bound to one of the opposite terminal regions of the nucleotide strand.

[0012] ATTO 520 is based on the well-known Rhodamine G6. Its absorption wavelength is 517 nm, and its emission wavelength is 538 nm.

[0013] The dye dimer can adopt at least two conformations. In a first conformation, the dye dimer is barely excitable to emit fluorescent radiation. Upon excitation, the dye dimer exhibits only a relatively low fluorescence emission. The dye dimer can, for example, undergo self-quenching of the two dye molecules and be formed, for example, as an H-dimer. In a second conformation, the dye dimer can be excited to emit fluorescent radiation and emits with significantly higher fluorescence intensity than in the first conformation. The fluorescence intensities in the second conformation are, under otherwise identical conditions, at least 5 times higher, in particular at least 8 times higher, and advantageously at least 10 times higher than in the first conformation.In the second conformation, the nucleotide strand can be stretched, so that the two dyes are as far apart as possible and no or no significant self-quenching occurs. The second conformation is particularly present when the dye dimer is bound to a binding molecule.

[0014] Such a binding molecule is, in particular, a single DNA strand (“docking strand”) whose nucleotide sequence is compatible with the sequence of the nucleotide strand (“imager strand”) of the dye dimer according to the invention and thus enables binding between the two nucleotide strands. It is sufficient if the nucleotide strand and binding molecule are sufficiently compatible to enable reversible binding. The dye dimer and binding molecule can therefore bind to one another. This binding is preferably reversible, so that only a portion of the dye dimers present are in the second conformation and can be detected. Dye dimers in the first conformation do contribute to the background signal, but diffuse too quickly through the medium surrounding the sample to be precisely localized.

[0015] In one embodiment of the dye dimer according to the invention, the nucleotide strand (“imager strand”) has a length of ten nucleotides with the sequence 5′-GTA ATGAAG A-3′ (also designated P3). A compatible binding molecule (“docking strand”) has the sequence methyltetrazine-5′-TTT CTT CAT TA-3′ (Schnitzbauer, J. et al., 2017, Super-resolution microscopy with DNA-PAINT, Nature Protocols 12: 1198-1228).

[0016] In a further embodiment, the dye dimer according to the invention can have a nucleotide strand with seven nucleotides and a sequence 5'-TGT GTG T-3' (also designated R4). A compatible binding molecule can have the sequence methyltetrazine-5'-ACACACACACACACACACA (Reinhardt, SCM et al., 2023, Ångström-resolution fluorescence microscopy, Nature 617: 711-716; Narayanasamy, KK et al., 2021, Visualizing synaptic multi-protein patterns of neuronal tissue with DNA-assisted single-molecule localization microscopy, Frontiers in Synaptic Neuroscience 13: 671288, doi: 10.3389 / fnsyn.2021.671288).

[0017] The dye dimers and / or the antibodies, in particular their modified form with the corresponding binding molecules methyltetrazine-5'-TTT CTT CAT TA-3' or methyltetrazine-5'-ACACACACACACACACACA, can be provided to a user in the form of a kit.

[0018] A kit for use in a TDI DNA-PAINT method, particularly for characterizing CD20, comprises a quantity of a first dye dimer containing two molecules of the dye ATTO Oxa 14 or ATTO 520. Rituximab (RTX), ocrelizumab (ORC), ofatumumab (OFA), or obinutuzumab (OBZ) is present as an antibody specific for the CD20 antigen, wherein the antibody is provided with a binding molecule that is compatible with at least a portion of the nucleotide strand of the dye dimer and allows reversible binding of the nucleotide strand of the dye dimer to the binding molecule. The kit may additionally contain other components such as buffers, enzymes, and excipients.

[0019] Another kit for use in a two-color TDI DNA PAINT procedure provides a quantity of a first dye dimer and a second dye dimer. The first dye dimer comprises two molecules of the ATTO Oxa 14 dye, while the second dye dimer comprises two molecules of the ATTO 520 dye. The kit may also contain other components such as buffers, enzymes, and excipients.

[0020] Both kits can be intended for use in a process in which the excitation radiation is shaped and irradiated as a lattice light sheet.

[0021] Such methods and their possible uses are described below.

[0022] The imaging process is carried out using a dye dimer. During the process, structures (antigens) of a sample to be imaged are bound to antibodies. These antibodies are provided with first binding molecules, also known as "docking strands." These first binding molecules are compatible with at least a portion of the dye dimer's nucleotide strand, meaning that the dye dimer and binding molecule can bind to each other. This binding is preferably reversible.

[0023] The sample to be imaged is illuminated with excitation radiation suitable for exciting the emission of fluorescent radiation from the dye dimer. The excitation radiation is preferably shaped in a planar manner using optical elements of an illumination optics, so that it propagates essentially in a plane obliquely or transversely to the optical axis of the illumination optics, and the sample is illuminated with a corresponding light sheet. The dye dimers present in the area illuminated by the light sheet and in the second conformation are excited to emit fluorescent radiation. The emitted fluorescent radiation is captured with two-dimensional resolution as detection radiation and evaluated for imaging. In particular, the excitation radiation is designed as a lattice light sheet and directed into the sample.

[0024] State-of-the-art light sheets are generated by compressing illuminating radiation in a direction perpendicular to its propagation direction using optical lenses, for example cylindrical lenses (static light sheet). It is also possible to quickly move the illuminating radiation, shaped into a thin light beam, back and forth in a plane, for example using a deflection device (scanner) (dynamic light sheet). Both approaches can also be combined. In contrast to the state of the art, in a lattice light sheet, such as the Lattice Light Sheet 7 from Carl Zeiss Microscopy GmbH, the illuminating radiation is shaped into a defined grid or network. A spatial light modulator (SLM) can be used for this purpose. The illuminating radiation thus shaped can be moved in a plane using a scanner.In addition, the illumination radiation can be provided in the form of non-diffractive beams, such as Bessel beams or Sinc3 beams. The special shape of the illumination radiation creates a very thin light sheet, which also exhibits few or no unwanted side lobes.

[0025] The thin light sheet thus generated can be moved through the sample by inducing a relative movement between the light sheet and the sample. To achieve this, the light sheet can be moved using a deflection device and / or the sample can be moved using a movable sample stage. A three-dimensional image of the sample can be generated from a multitude of acquired two-dimensional images arranged consecutively in the z-direction (z-stack).

[0026] Using a combination of dye dimers and a lattice light sheet, this method can achieve high spatial resolution with an improved signal-to-noise ratio (SNR) compared to the state of the art. The method can be described as LLS-TDI-DNA-PAINT. The high fluorescence intensity of the dye dimers excited in the second conformation advantageously supports the accuracy of localizing the individual detected signals.

[0027] In one embodiment of the process according to the invention, the dye of the dye dimer ATTO Oxa14 ( Fig. 1). This oxazine dye, which is a derivative of the dye MR121 (CAS 185213-55-2) with a side group of the form R = CH2COOH bonded to a nitrogen atom ( Fig. 1), is proven and readily available. The dye absorbs and emits in aqueous solution at 660 nm and 680 nm, respectively (similar to: Marmé, N. et al., 2005, Aggregation behavior of the red-absorbing oxazine derivative MR 121: A new method for determination of pure dimer spectra, Chemical Physics Letters 408: 221 - 225).

[0028] The use of ATTO 520 (e.g. ATTO-TEC GmbH, Siegen, Germany; Fig. 2) as a dye of the dye dimer is possible in a further embodiment of the process according to the invention.

[0029] It is also possible to use the dye AF 647, whose absorption wavelength is 647 nm and its emission wavelength is 667 nm.

[0030] As already described above, the dye dimers can be bound via their nucleotide strand to antibodies modified with corresponding binding molecules. The antibodies can bind to specific antigens. In one embodiment of the method, the B lymphocyte antigen CD20, hereinafter also referred to as CD20 for short, is chosen as the sample structure acting as the antigen.

[0031] CD20 is a transmembrane protein of B lymphocyte cells (B cells). As an essential part of the adaptive immune system, B cells are capable of producing and releasing antibodies. CD20 can induce changes in B cells, even leading to cell death (see below). Different pathways for B cell destruction have been postulated. These include the direct induction of cell death through interaction with CD20, phagocytosis, Fc receptor-mediated antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Ghosh, A. et al. (in press), Decoding the molecular interplay of endogenous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science ; Rouge, L. et al., 2020, Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab, Science 367: 1224-1230).

[0032] Several monoclonal antibodies (mAbs) are known that bind to the CD20 antigen and can be used in the method according to the invention. The antibodies rituximab (RTX, CAS 174722-31-7) and 2H7 (OCR, ocrelizumab; CAS 637334-45-3) bind to almost the same epitope of CD20. The monoclonal antibodies ofatumumab (OFA; CAS 679818-59-8) and obinutuzumab (OBZ; CAS 949142-50-1) can also be used.

[0033] The mAbs rituximab RTX, ocrelizumab OCR, and ofatumumab OFA are classified as Class I (Rouge, L. et al., 2020, Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab, Science 367: 1224-1230). These mAbs are designed to cross-link CD20 and trigger the CDC pathway to destroy B cells.

[0034] The monoclonal antibody obinutuzumab OBZ, on the other hand, is classified as Class II. It is currently assumed in the literature that it does not cause cross-linking and that the mode of action of the ADCC rather than the CDC is more relevant.

[0035] The high resolution and the SNR, which is also increased compared to the state of the art, allow the method to simultaneously use at least two different dye dimers and to perform a two-color DNA PAINT using a lattice light sheet (two-color LLS-TDI-DNA-PAINT).

[0036] Instead of just one antibody, another antibody is used, which is equipped with second binding molecules. In this way, a first dye dimer can bind to the first binding molecules, while a second dye dimer can bind to the second binding molecules. The second dye dimer differs from the first dye dimer in terms of its excitation wavelength, its emission wavelength, and / or its compatibility with the existing binding molecules. It is possible to use different antibodies and equip them with different binding molecules. It is also conceivable to use the same antibodies but couple them with different binding molecules, so that different dye dimers can be bound.

[0037] It has been found that it is advantageous to use ATTO Oxa14 as the dye for the first dye dimer and ATTO 520 as the dye for the second dye dimer in such a two-color DNA-PAINT variant. The two dyes, ATTO Oxa 14 and ATTO 520, are sufficiently far apart in terms of their excitation and emission wavelengths to reliably separate signals from the detection radiation. However, with regard to their spectrum and their behavior upon addition of an excess of binding molecules (see below), both dyes behave very similarly ( Fig. 3 and Fig. 4), so that simultaneous use is easily possible.

[0038] The advantages of a two-color LLS-TDI-DNA-PAINT application are highlighted by another experiment conducted by the inventors. Raji cells were labeled with 5 µg / mL of either anti-CD20 RTX-AF647 or 2H7-AF647 and anti-CD45 HI30-CF568 (562 / 584 nm). After the incubation phase with RTX / 2H7, the cells were fixed. The cells were scanned (sampled) using a Lattice Light Sheet 7 from Carl Zeiss Microscopy GmbH. The results showed that RTX accumulated in some areas of the cells and was particularly present in cell protrusions. Furthermore, the RTX signal is located primarily on one side of the cell, meaning that the cells became polarized, which confirms the results of other studies conducted by the inventors.

[0039] Furthermore, Raji cells were labeled with 2H7-AF647 and with SPY-555 (555 / 580 nm) against actin, and the living cells were observed and imaged using LLS-TDI-DNA-PAINT.

[0040] The method has also led to further findings, which are presented in detail in the currently in-press publication Ghosh, A. et al, Decoding the molecular interplay of endogenous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science, and are only briefly outlined below.

[0041] It has been shown that, contrary to previous assumptions, therapeutic antibodies of both type I and type II crosslink CD20 molecules on the cell membrane of B cells. The efficiency of crosslinking varies greatly from antibody to antibody. However, it always occurs, for example, when the cells are incubated with the antibodies at room temperature or at 37°C. The efficiency of crosslinking also increases significantly with the concentration of the added antibodies. This made it possible to precisely determine the number of therapeutic antibodies binding to the cell membrane of B cells.

[0042] The method also showed that CD20 is highly concentrated on the microvilli of the cells (cell extensions) and that the antibodies bind mainly there.

[0043] This method allows the molecular distribution of CD20 / antibody signals to be imaged three-dimensionally throughout the entire cell. Compared to the current state of the art, it is now also possible to capture their distribution on the upper side of the cell with molecular precision.

[0044] It has also been recognized that type I antibodies strongly polarize B cells and stabilize the microvilli, meaning the microvilli are stable, very long, and all extend in one direction. Type II antibodies, on the other hand, do not polarize B cells, although they also cross-link the CD20 molecules. The length of the microvilli stabilized by the antibodies depends strongly on the concentration and type of antibody. This result can be used to classify antibodies.

[0045] The invention, particularly in conjunction with the described imaging method, also allows for the examination of large volumes of a sample. Due to the high fluorescence intensity, low noise, and high localization precision, unlike the prior art, the illumination does not have to be limited to a small area of ​​the sample, for example, using TIRF. Since in previous prior art methods, for example, when using TIRF technology, the concentration of dye molecules must also be kept very low, such images take several hours.

[0046] The higher increase in fluorescence intensity during transient binding of the dye dimers to the binding molecules, however, allows the method to be used for applications without TIRF excitation. High-resolution images of entire cells, not just the lower (basal) membrane, can be generated. A 3D image of a whole cell takes only 4 hours. A measurement time of 10 to 20 minutes per image plane is sufficient. Furthermore, the measurements can be performed with an imager concentration 10 to 50 times higher than is currently available.

[0047] The method can therefore be used to obtain high-resolution images of a cell and to study interactions between an antibody, an antigen and a cell.

[0048] In addition, the efficacy of an antibody can be assessed after its binding to an antigen by determining the degree of cross-linking of addressed tumor-associated receptors and / or induced cell polarization.

[0049] The invention thus opens up new ways to test new therapeutic antibodies and to better understand their mode of action.

[0050] During the work on the invention, it has been shown that an excess of binding molecules by a factor of at least 10 3 , but advantageously of at least 10 4 , leads to an approximately 6 to 9-fold increase in the fluorescence intensity of the bound dye dimers ( Fig. 3 and Fig. 4). Character description In the Fig. 1 shows the structural formulas of the dyes MR121 and ATTO Oxa14. The Fig. Figure 2 shows the structural formula of the dye ATTO 520.

[0051] The behavior of the dye dimers with a nucleotide strand of form R4 and the dye ATTO 520 is expressed as relative intensity (normalized to 1) over a wavelength range from approximately 530 nm to 650 nm in Fig. 3 (simplified redrawn from Ghosh, A. et al. (in press), Decoding the molecular interplay of endogenous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science). The curve with a dashed solid line shows the intensity curve without the addition of an excess of binding molecules. The solid line shows the curve in the presence of a 10 4 -fold excess of binding molecules.

[0052] The behavior of the dye dimers with a nucleotide strand of form R4 and the dye ATTO Oxa14 is expressed as relative intensity (normalized to 1) over a wavelength range of approximately 650 nm to 750 nm in Fig.4 (reproduced from Ghosh, A. et al. (in press), Decoding the molecular interplay of endogenous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science). The curve with a dashed solid line shows the intensity curve without the addition of an excess of binding molecules. The solid line shows the curve in the presence of a 10 4 -fold excess of binding molecules. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited non-patent literature

[0000] Chung, K. K. H. et al, 2022, Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging, Nature Methods 19: 554 - 559

[0003] Kessler, L. F. et al. 2023, Self-quenched flurophore dimers for DNA-Paint and STED microscopy, Angew. Chem. Int. Ed. 62, e202307538 [0005, 0007] Schnitzbauer, J. et al., 2017, Super-resolution microscopy with DNA-PAINT, Nature Protocols 12: 1198-1228

[0015] Reinhardt, S. C. M. et al., 2023, Ångström-resolution fluorescence microscopy, Nature 617: 711 -716

[0016] Narayanasamy, K. K. et al., 2021, Visualizing synaptic multi-protein patterns of neuronal tissue with DNA-assisted singlemolecule localization microscopy, Frontiers in Synaptic Neurosciene 13: 671288, doi: 10.3389 / fnsyn.2021.671288

[0016] Marmé, N. et al., 2005, Aggregation behavior of the red-absorbing oxazine derivative MR 121: A new method for determination of pure dimer spectra, Chemical Physics Letters 408: 221 - 225

[0027] Ghosh, A. et al (im Druck), Decoding the molecular interplay of endogeneous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science [0031, 0051, 0052] Rouge, L. et al., 2020, Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab, Science 367: 1224-1230 [0031, 0033] Ghosh, A. et al, Decoding the molecular interplay of endogeneous CD20 and therapeutic antibodies with fast volumetric nanoscopy, Science

[0040]

Claims

[1] Dye dimer, comprising - a single strand of DNA formed by a sequence of nucleotides (nucleotide strand), and - one molecule of a dye capable of emitting fluorescent radiation, bound to the opposite terminal regions of the nucleotide strand, - wherein the dye dimer in a first conformation is slightly excitable to emit fluorescent radiation; and - in a second conformation, it can be readily excited to emit fluorescent radiation, the fluorescence intensity in the second conformation being at least 5 times higher, in particular at least 8 times higher, advantageously at least 10 times higher, than in the first conformation, characterized by , that - the dye is ATTO 520. [2] Dye dimer according to claim 1, characterized bya nucleotide strand with a length of ten nucleotides, in particular with the sequence GTAATGAAGA-3'. [3] Dye dimer according to claim 1, characterized by a nucleotide strand with a length of seven nucleotides, in particular with the sequence: 5'-TGTGTGT-3'. [4] A kit for use in a TDI DNA PAINT method comprising an amount of the dye dimer according to any one of claims 1 to 3. [5] Kit for use in a TDI DNA PAINT method, in particular for characterising CD20, comprising an amount of a first dye dimer, in which - a single strand of DNA (nucleotide strand) formed by a sequence of nucleotides is present, and - one molecule of a dye which can be excited to emit fluorescent radiation is bound to the opposite terminal regions of the nucleotide strand, - wherein the dye dimer in a first conformation is slightly excitable to emit fluorescent radiation; and - in a second conformation, it can be readily excited to emit fluorescent radiation, the fluorescence intensity in the second conformation being at least 5 times higher, in particular at least 8 times higher, advantageously at least 10 times higher, than in the first conformation, characterized by , that - the dye dimer comprises two molecules of the dye ATTO Oxa 14 or ATTO 520, and - Rituximab, Ocrelizumab, Ofatumumab (OFA) or Obinutuzumab (OBZ) is present as an antibody specific for the CD20 antigen, wherein the antibody is provided with a binding molecule which is compatible with at least a portion of the nucleotide strand and allows reversible binding of the nucleotide strand of the dye dimer to the binding molecule. [6] Kit for use in a two-color TDI DNA PAINT method comprising an amount of a first dye dimer and a second dye dimer, each of which - have a single strand of DNA formed by a sequence of nucleotides (nucleotide strand), and - one molecule of a dye which can be excited to emit fluorescent radiation is bound to the opposite terminal regions of the nucleotide strand, - wherein each dye dimer in a first conformation is slightly excitable to emit fluorescence radiation; and - can be excited in a second conformation to emit fluorescent radiation, wherein the fluorescence intensity in the second conformation is at least 5 times higher, in particular at least 8 times higher, advantageously at least 10 times higher than in the first conformation, characterized by , that - a first dye dimer comprises two molecules of the dye ATTO Oxa 14, and - a second dye dimer contains two molecules of the dye ATTO 520.

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