Calcium independent phosphatidylserine binding compounds for detecting phosphatidylserine positive cells

Abstract

The present invention relates to phosphatidylserine (PS) binding compound derivatives and fusions of PS binding compounds and arm compounds. Described herein are methods of producing and using PS-binding compound derivatives and fusions of PS-binding compounds with arm compounds by providing functional groups to couple to functional group-containing fluorophores for multicolor flow cytometry and multiplex imaging; providing functional groups to be conjugated to the magnetic beads for depleting PS-positive cells; functional groups are provided to couple to oligonucleotides comprising PCR processing sequences and unique DNA barcodes of PS-positive cells, as well as capture sequences, for distinguishing between positive and living cells in single cell RNA sequencing.

Description

Background Technology

[0001] Phosphatidylserine (PS), a phospholipid with a negatively charged head group, is an important component of the cell bilayer and is typically found in the inner layer. Under physiological conditions, PS exposure on the outer layer not only functions as a signal for the phagocytosis of apoptotic and dead cells but also participates in the clearance of unwanted cells during early development and in promoting tumor growth and metastasis. In routine practice, PS-positive dead cells can lead to false positives due to their strong autofluorescence and increased nonspecific antibody binding, which can affect data quality, especially in flow cytometry experiments. Therefore, PS binders have been widely used as important biomarkers for detecting, monitoring, and consuming PS-positive cells in heterogeneous cell populations.

[0002] Annexin V (or annexin A5), a recombinant protein, is the most widely used PS binder, specifically binding PS in the presence of calcium (Vermes et al., A novel assay for apoptosis: Flowcytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of Immunological Methods. 1995; Volume 184, Issue 1, July 17). Recent studies have compared the effects of fluorescein on PS in the presence of calcium using sum-frequency vibrational spectroscopy. 2+ Dependence and Ca 2+ Different annexin V binding behaviors under non-dependent conditions (Ma et al., Calcium-dependent and-independent annexin V binding: distinct molecular behaviors at cell membrane interfaces. Chem Commun. 2020; Feb 6; 56(11):1653-1656). The data in this paper indicate that the addition of Ca... 2+ After that, the original Ca 2+The calcium-independent binding process has undergone a shift, with annexin V repositioning to a more stable state. This indicates that a high concentration of calcium is always necessary for annexin V staining. If cell staining with annexin V is required, the cell buffer must be replaced with an annexin V binding buffer, which consists of 0.1 M HEPES (pH 7.4), 1.4 M NaCl, and 25 mM CaCl2. During buffer replacement, cell state may change, and dead cells may be lost during centrifugation. If sample fixation is required for post-staining, PS-positive cells stained with annexin V may not be retained. Therefore, a new generation of calcium-independent PS binding agents is needed.

[0003] A recent report describes how Apo-15 selectively recognizes apoptotic and dead cells by binding to negatively charged phospholipids exposed on the surface of apoptotic and dead cells in a calcium-independent manner, thereby enabling the detection of PS-positive cells by flow cytometry and microscopy (Barth et al., A fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis. Nature Communications. 2020; 11, Article number: 4027). Apo-15 is a cyclic peptide containing the sequence SEQ ID NO:1 [RKKWFW(BODIPY)G], wherein the amino acids Arg, Lys, Trp, Phe, and Gly each contain a carboxyl group (COOH) and an amino group (NH2). The Trp(BODIPY) is a Fmoc-labeled amino acid of Trp linked to the BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indene) dye via a spacerless C / C bond (Subiros-Funosas et al., A Trp-BODIPY cyclic peptide for fluorescence labelling of apoptotic bodies. Chem. Commun. 2017; 53945–948; and Mendive-Tapia et al., Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue. Nature Communications. 2016; 7, 10940; and Mendive-Tapia et al., Preparation of a Trp-BODIPY fluorogenic aminoacid to label peptides for enhanced live-cell fluorescence imaging. 2017. NatProtoc. Aug; 12(8): 1588-1619). Trp(BODIPY) functions as a single element for imaging detection and retains the natural properties of Trp. The Arg and Lys groups contain not only amino and carboxyl groups but also a second amino group on the side chain. Since Arg and Lys are positively charged and are key elements for binding negatively charged phosphatidylserine, the second amino group was not considered for chemical labeling.Therefore, Apo-15 lacks available functional groups for conjugation with commonly used fluorophores (such as Alexa dyes). If different excitation and emission BODIPY dyes are used, new Trp(BODIPY) must be prepared, followed by the synthesis of a new form of Apo-15. Currently, only one commercially available form of Trp(BODIPY), commercially named ApotrackerGreen, is available, excites at 500 nm and emits at 520 nm, and is used for flow cytometry applications. Apo-15 has some limitations in terms of selecting different fluorophore forms for multicolor flow cytometry and multiplex imaging. Therefore, an advanced novel PS-binding compound capable of conjugating with different fluorophore forms is needed.

[0004] Dead cell removal is a rapid and direct method for eliminating dead cells from cell cultures to tissue preparations. The most common method for consuming PS-positive cells is using annexin V-conjugated particles, or labeling PS-positive cells with biotinylated annexin V followed by calcium-dependent binding of streptavidin-conjugated particles. Using buffers containing high concentrations of calcium can cause cells to aggregate, which affects the yield of viable cells. MojoSort TM Human Dead Cell Removal Kit and MojoSort TM Mouse dead cell removal kits (BioLegend, catalog numbers: 480159 and 480157) have recently been launched. These two kits do not require Ca... 2+ In cases where PS-positive cells need to be consumed, an additional step of pre-incubating Apo-monomer recombinant protein and streptavidin nanobeads for 5 minutes is required before contact with the cells. Therefore, a one-step calcium-free PS-positive cell removal kit is urgently needed to shorten the experimental process with high purity and yield. Apo-15 can bind to PS-positive cells, but it lacks available functional groups for conjugation with solid-phase carriers. Therefore, advanced novel PS-binding compounds with functional groups for conjugation with solid-phase carriers are needed to consume PS-positive cells.

[0005] Single-cell RNA sequencing (scRNA-seq) has become the state-of-the-art method for simultaneously measuring surface protein and gene expression within single cells using oligonucleotide-conjugated antibodies. It provides high-resolution snapshots of complex cell populations and a better understanding of the function of individual cells in the context of their microenvironment (Eberwine et al., The promise of single-cell sequencing. Nature Methods. 2014; 11, pages 25–27; and Pennisi et al., Chronicling embryos, cell by cell, gene by gene. Science. 2018; Vol 360, Issue 6387; and Saliba et al., Single-cell RNA-seq: advances and future challenges. Nucleic Acids Res. 2014; 18; 42(14): 8845–8860). Single-cell sequencing technology requires four main steps: (1) isolating individual cells from a cell population into each droplet; (2) extracting, processing, and amplifying the genetic material of each isolated cell; (3) preparing a “sequencing library” containing the genetic material of the isolated cells; and (4) sequencing the library using a next-generation sequencer (Pennisi et al., Chronicling embryos, cell by cell, gene by gene. Science. 2018; Vol 360, Issue 6387). Each droplet carries a DNA “barcode” to uniquely label the cDNA derived from the single cell. After reverse transcription, the cDNA from many cells is mixed together for sequencing, and transcripts from specific cells can also be identified by the unique barcode.

[0006] Single-cell RNA sequencing is gaining widespread application in biological disciplines such as immunology, oncology, and developmental biology. However, the entire process, from cell preparation to data analysis, is time-consuming and costly. Typically, scRNA-seq experiments generate a subset of low-quality data from damaged, dead, or mixed cells, which can hinder downstream analysis and lead to misinterpretation of the data (Chen et al., Single-Cell RNA-Seq Technologies and Related Computational Data Analysis. Front Grnet. 2019; 10:317; and Deleersnijder et al., Current Methodological Challenges of Single-Cell and Single-Nucleus RNA-Sequencing in Glomerular Diseases. J Am Soc Nephrol. 2021; Aug; 32(8):1838–1852). Due to technological limitations and biological factors, the analysis, integration, and interpretation of single-cell omics data, such as how to identify non-living cell contamination in single-cell sequencing methods, remain significant challenges.

[0007] An innovative method is needed to prepare oligonucleotide-tagged PS binders that distinguish PS-positive cells from live cells by using unique barcodes as identifiers. This would allow bioinformaticians to filter out PS-positive cells and analyze only live cells, improving the accuracy and efficiency of cell indexing of transcriptomes and epitopes via sequencing (CITE-Seq). Typically, oligonucleotide-containing reagents require at least 0.1 mM EDTA (ethylenediaminetetraacetic acid) to prevent DNA digestion by nucleases. When the oligonucleotides come into contact with cells, EDTA chelates calcium in the cell buffer, affecting the PS-binding function of annexin V. Therefore, a novel calcium-independent PS binder conjugated to an oligonucleotide containing a unique barcode for PS-positive cells is urgently needed.

[0008] Therefore, there is still room in the field to expand the application of existing compounds in distinguishing and eliminating PS cells by using substituted amino acids containing specific functional groups to modify or change the structure of existing compounds. Summary of the Invention

[0009] This invention generally relates to methods for preparing phosphatidylserine (PS) binding compound derivatives and fusions of PS binding compounds with arm compounds. It also relates to methods for using PS binding compound derivatives and fusions of PS binding compounds with arm compounds to detect PS-positive cells for multicolor flow cytometry and multiplex imaging, for consuming PS-positive cells in a separation system, and for distinguishing positive cells from live cells in single-cell RNA sequencing.

[0010] A phosphatidylserine (PS) conjugated compound wherein the residue Trp (BODIPY) at position 6 of the amino acid sequence of SEQ ID NO:1 has been replaced by the natural amino acid Trp to remove fluorescence, and wherein the residue Gly at position 7 has been replaced by the amino acid Cys or the synthetic amino acid propargylglycine (Pra).

[0011] The present invention provides a PS-binding compound derivative (Item 2) comprising the sequence SEQ ID NO:2[RKKWFWC], wherein the amino acid Cys is used to provide a thiol group for coupling with a maleimide-containing tag by means of a thiol-maleimide reaction.

[0012] A PS-binding compound derivative, Project 2, containing the sequence SEQ ID NO:2, was synthesized and validated by mass spectrometry. Project 2 is conjugated to a maleimide-containing fluorophore, wherein the fluorophore is Atto 647 containing maleimide. Atto 647-conjugated Project 2 (Atto647 Project 2) was validated by mass spectrometry, flow cytometry, and fluorescence microscopy.

[0013] Experimental results show that the PS-binding compound derivative item 2, containing the sequence SEQ ID NO:2, not only retains the PS-binding properties but also provides a thiol group for coupling with maleimide-containing fluorophores for use in multicolor flow cytometry and multiplex imaging.

[0014] Item 2, a PS-binding compound derivative containing the sequence SEQ ID NO:2, contains a thiol group, allowing the PS-binding compound derivative to be coupled to a maleimide-containing tag, wherein the tag is a detectable unit, including but not limited to fluorophores, solid-phase supports, oligonucleotides, biotin, avidin, proteins, enzymes, and radionuclides. Furthermore, the tag can also be a maleimide-containing bifunctional linker, wherein the maleimide-containing bifunctional linker includes, but is not limited to, maleimide-Pol-maleimide, maleimide-Pol-thiol, maleimide-Pol-azide, maleimide-Pol-alkyne, maleimide-Pol-NHSter, maleimide-Pol-amine, and maleimide-Pol-COOH; wherein the maleimide is used for linkage with the thiol group in Cys of Item 2, and the other functional group in the maleimide-containing bifunctional linker is used for chemical labeling; wherein the Pol is a polymer, including but not limited to (PEG). n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is an integer from 1 to 60, preferably from 1 to 24, and more preferably from 1 to 12.

[0015] In this invention, item 2 of the PS-binding compound derivative containing the sequence SEQ ID NO:2 provides a thiol group for coupling with a maleimide-containing tag. Alternatively, the tag can also be a maleimide-containing bifunctional connector, which can provide additional functional groups, such as amines, NHSter, thiols, maleimides, alkynes, azides, and carboxyl groups, for chemical labeling.

[0016] The present invention provides a PS-binding compound derivative (Item 3) comprising the sequence SEQ ID NO:3 [RKKWFWPra], wherein the synthesized amino acid Pra is used to provide an alkyne group for coupling with an azide-containing tag by means of an azide-alkyne reaction.

[0017] A PS-binding compound derivative, item 3, containing the sequence SEQ ID NO:3, was synthesized and conjugated to an azide-containing fluorophore, wherein the fluorophore is fluorescein (FITC) containing an azide. The PS-binding compound derivative item 3 was verified by mass spectrometry, and the FITC-conjugated item 3 (FITC item 3) was verified by flow cytometry.

[0018] Experimental results show that the PS-binding compound derivative item 3, containing the sequence SEQ ID NO:3, not only retains the PS-binding properties, but also provides an alkyne group for coupling with azide-containing fluorophores for use in multicolor flow cytometry.

[0019] Item 3, a PS-binding compound derivative containing the sequence SEQ ID NO:3, contains an alkyne group, thereby allowing PS-binding compound derivative Item 3 to be coupled to an azide-containing tag, wherein the tag is a detectable unit, including but not limited to fluorophores, solid-phase supports, oligonucleotides, biotin, avidin, proteins, enzymes, and radionuclides. Furthermore, the tag may be an azide-containing amino acid, including but not limited to azido-lysine, azido-propargylglycine, azido-L-propargylglycine, azido-histidine, azido-tryptophan, azido-phenylalanine, azido-arginine, azido-glutamine, azido-glycine, azido-valine, and azido-alanine, wherein the azide group is used for linkage with the alkyne in Pra of Item 3, and wherein the functionalized amino acid is used for chemical labeling. Furthermore, the label can also be a bifunctional connector containing an azide, including but not limited to azido-Pol-maleimide, azido-Pol-thiol, azido-Pol-alkyne, azido-Pol-azido compound, azido-Pol-amine, azido-Pol-NHSter, and azido-Pol-COOH, wherein the azide group is used for linkage with Pra of Item 3, and the other functional group in the bifunctional connector containing the azide is used for chemical labeling, wherein the Pol is a polymer used as the connector, including but not limited to (PEG). n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is an integer from 1 to 60, preferably from 1 to 24, and more preferably from 1 to 12.

[0020] In this invention, item 3 of the PS-binding compound derivative containing the sequence SEQ ID NO:3 provides an alkyne group for coupling with an azide-containing tag. Furthermore, the tag can also be an azide-containing amino acid or an azide-containing bifunctional linker, which can provide additional functional groups, such as amines, NHSter, thiols, maleimides, alkynes, azides, and carboxyl groups, for chemical labeling.

[0021] This invention provides a fusion of a PS-binding compound and an arm compound, comprising SEQ ID NO:5 [RKKWFWPra-Xa1(azido)-Xa2-(Pol-Xa3)] m The sequence [[Xa1(azido)-Xa2-(Pol-Xa3]] (item 5) contains SEQ ID NO:4[Xa1(azido)-Xa2-(Pol-Xa3]]. m The arm of the compound (item 4) of the sequence of SEQ ID NO:3 is linked to Pra in the PS-binding compound derivative item 3 to provide at least one functional group for coupling with at least one tag containing the functional group.

[0022] A fusion compound containing the sequence SEQ ID NO:5, coupled with a fluorophore containing a functional group, was synthesized and verified by mass spectrometry. The variable amino acid [Xa1 (azido)] was Lys (azido), the variable amino acid (Xa2) was Lys, and the variable amino acid (Pol-Xa3) was... m The formulation is (PEG4-Lys)3, wherein the PEG is Fmoc-NH-PEG-COOH, and the three Lys groups in (PEG4-Lys)3 provide three amino groups that are coupled to three NHSter-containing fluorophores, wherein the functionalized fluorophores are NHSter-containing fluorescein (FITC). Project 5 (FTIC Project 5) was verified by flow cytometry for NHSter-containing FITC coupling.

[0023] Experimental results show that the fusion product 5 containing the sequence SEQ ID NO:5 not only retains the PS binding properties, but also provides three amine groups to be coupled with three NHSter-containing fluorophores for multicolor flow cytometry.

[0024] The fusion product item 5, comprising the sequence of SEQ ID NO:5 described herein, contains an arm compound item 4 comprising the sequence of SEQ ID NO:4, wherein Xa1 is an azide-containing amino acid, including but not limited to azido-lysine, azido-propylated glycine, azido-L-propylated glycine, azido-histidine, azido-tryptophan, azido-phenylalanine, azido-arginine, azido-glutamine, azido-glycine, azido-valine, and azido-alanine, wherein the azide group is used for linkage with the alkyne in Pra of item 3, wherein the amino acid having the functional group is used for linkage with Xa2; Xa2 is an amino acid used as a linker for linkage with Xa1 and (Pol-Xa3), including but not limited to Lys, Arg, His, Ser, Thr, Cys, Asn, Gln, Pra, and Tyr; and (Pol-Xa3) mLet Pol be a separate variable used for the connection with Xa2, where Pol is the polymer used as the connector, including but not limited to (PEG). n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n The Xa3 is an amino acid having an amine, thiol, or alkyne functional group, used for tag coupling with a tag containing an NHSter, maleimide, or azide group, wherein the amino acid includes, but is not limited to, Lys, Arg, His, Met, Cys, and Pra. The number of polymers can be selected and is represented by n, where n is an integer from 1 to 60, preferably from 1 to 24, more preferably from 1 to 12. The variable m is the quantity of (Pol-Xa3), where m is an integer from 1 to 10.

[0025] In this invention, the fusion of a PS-binding compound containing the sequence of SEQ ID NO:5 and its arm can provide amine, thiol, or alkyne functional groups for coupling with a tag containing NHSter, maleimide, or azide, wherein the tag serves as a detectable unit, including but not limited to fluorophores, oligonucleotides, solid-phase carriers, biotin, avidin, proteins, enzymes, and radionuclides. Furthermore, the functional groups provided by fusion item 5 can be selected for chemical labeling, and the number of functional groups provided by fusion item 5 is adjustable.

[0026] This invention provides a fusion of a PS-binding compound and an arm compound comprising the sequence SEQ ID NO:7 [RKKWFWPra-Xa1(azido)-Xa2-Pol-Xa3] (item 7), wherein an arm compound comprising the sequence SEQ ID NO:6 [Xa1(azido)-Xa2-Pol-Xa3] (item 6) is linked to Pra in a PS-binding compound derivative comprising the sequence SEQ ID NO:3 to provide a functional group for coupling with a tag containing the functional group.

[0027] Fusion item 7 containing the sequence SEQ ID NO:7 was synthesized and verified by mass spectrometry, wherein variable Xa1 (azido group) is Lys (azido group), variable Xa2 is Lys, Pol is PEG12, PEG is Fmoc-NH-PEG-COOH, and variable Xa3 is Cys to provide an alkyne group for coupling with a maleimide-containing tag.

[0028] The fusion compound 7 was conjugated with maleimide-containing protein fluorophore R-phycoerythrin (PE) (PE compound 7) and validated by flow cytometry.

[0029] Project 7 was conjugated with maleimide-coated magnetic particles and validated using a magnetic separation system.

[0030] Fusion item 7 was coupled with a maleimide-containing oligonucleotide containing the sequence of SEQ ID NO:8 (poly-T oligonucleotide item 7) and verified by flow cytometry.

[0031] Experimental results show that the fusion of the PS binding compound and arm of SEQ ID NO:7, item 7, can be coupled with maleimide-containing protein fluorophores (PE) for multicolor flow cytometry, coupled with maleimide-coated magnetic particles for consuming PS-positive cells, and coupled with maleimide-containing oligonucleotides for binding PS-positive cells. The oligonucleotides contain a PCR operation sequence, a unique DNA barcode sequence for PS-positive cells, and a capture sequence, which can distinguish positive cells from live cells in single-cell RNA sequencing.

[0032] The fusion product item 7 described herein, containing the sequence of SEQ ID NO:7, contains an arm containing the sequence of SEQ ID NO:6, wherein Xa1 is an azide-containing amino acid, including but not limited to azido-lysine, azido-propargylglycine, azido-L-propargylglycine, azido-histidine, azido-tryptophan, azido-phenylalanine, azido-arginine, azido-glutamine, azido-glycine, azido-valine, and azido-alanine, wherein the azide group is used for linkage with the alkyne in Pra of item 3, and wherein the functionalized amino acid is used for linkage with Xa2; Xa2 is an amino acid used as a linker to link with Xa1 and Pol, including but not limited to Lys, Arg, His, Ser, Thr, Cys, Asn, Gln, Pra, and Tyr; and Pol is a polymer containing amine and carboxyl groups used as a linker to link with Xa2 and Xa3, including but not limited to (PEG). n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is the number of polymers, and n is an integer from 1 to 60, preferably from 1 to 24, more preferably from 1 to 12; the Xa3 is an amino acid containing an amine, thiol, or alkyne functional group for coupling with a tag containing NHSter, maleimide, or azide, wherein the amino acid includes, but is not limited to, Cys, Pra, Met, Lys, Arg, and His.

[0033] In this invention, fusion item 7, consisting of a PS-binding compound and an arm compound, provides amine, thiol, or alkyne functional groups for coupling with tags containing NHSter, maleimide, or azides, wherein the tags serve as detectable units, including but not limited to fluorophores, oligonucleotides, solid-phase supports, biotin, avidin, proteins, enzymes, and radionuclides. Furthermore, the functional groups provided by fusion item 7 can be optionally used for chemical labeling. The length of the connector is adjustable, which facilitates coupling with large-sized tags.

[0034] In this invention, PS binding compound derivatives items 2 and 3, and fusion items 5 and 7 provide functional groups for coupling with functional group-containing fluorophores for multicolor flow cytometry and multiplex imaging, for coupling with functional group-containing solid-phase carriers for consuming PS-positive cells, and for coupling with functional group-containing oligonucleotides for binding PS-positive cells. These oligonucleotides can distinguish PS-positive cells from live cells by recognizing unique DNA barcode sequences in single-cell sequencing, wherein the oligonucleotides comprise PCR manipulation sequences, unique DNA barcode sequences for PS-positive cells, and capture sequences.

[0035] Other advantages and novel features of the invention will become apparent when considered in conjunction with the accompanying drawings, based on the following detailed description of various non-limiting embodiments of the invention. If this specification contains conflicting and / or inconsistent disclosures with any other document incorporated by reference, this specification shall prevail. If two or more documents incorporated by reference contain conflicting and / or inconsistent disclosures, the document with the later effective date shall prevail. Attached Figure Description

[0036] To gain a more complete understanding of the invention, reference is now made to the accompanying drawings (Figures) which illustrate in detail, and to the embodiments described below by way of examples of the invention, wherein:

[0037] Figure 1A-1 The molecular weight of the linear PS-binding compound derivative (Item 2) containing the sequence SEQ ID NO:2, as measured by mass spectrometry, is shown.

[0038] Figure 1A-2 The molecular weights of the cyclic PS-binding compound derivative containing the sequence SEQ ID NO:2, as measured by mass spectrometry, are shown.

[0039] Figure 1A-3 The molecular weight of Atto 647 Project 2, as measured by mass spectrometry, is shown.

[0040] Figure 1BThe staining patterns and frequencies of PS-positive cells stained with Atto 647 Project 2, ApotrackerGreen, or Alexa 647 Annexin V on thymocytes of one-day-old C57BL / 6 mice are shown.

[0041] Figure 1C The staining patterns and frequencies of PS-positive cells simultaneously stained with Atto 647 Project 2 and Apotracker Green on U937 cells stimulated with IL-4 for 2 days are shown.

[0042] Figure 1D The staining patterns and frequencies of PS-positive cells stained with Atto 647 Project 2 were shown before and after fixation, after U937 cells were stimulated with fresh and IL-4 for 2 days.

[0043] Figure 1E Multiple images are shown of simultaneous staining with calcein AM, Atto 647 Project 2, and fixable viability dye-405.

[0044] Figure 2A-1 The molecular weight of the linear PS-binding compound derivative (Item 3) containing the sequence SEQ ID NO:3, as measured by mass spectrometry, is shown.

[0045] Figure 2A-2 The molecular weights of the cyclic PS-binding compound derivative containing the sequence SEQ ID NO:3, as measured by mass spectrometry, are shown.

[0046] Figure 2B The staining patterns and frequencies of PS-positive cells simultaneously stained with FITC Project 3 and Atto647 Project 2 on U937 cells stimulated with IL-4 for 2 days are shown.

[0047] Figure 3A The molecular weight of the PS-binding compound containing the sequence SEQ ID NO:5 and the fusion of the arm (Item 5) coupled with FITC is shown by mass spectrometry (Item 5).

[0048] Figure 3B The staining patterns and frequencies of PS-positive cells stained with FITC Project 5 or FITC Project 3 on thymocytes of one-day-old C57BL / 6 mice are shown.

[0049] Figure 3C The staining intensity of different concentrations of FITC item 5 and FITC item 3 is shown on overgrown U937 cells.

[0050] Figure 4AThe molecular weight of the PS-binding compound containing the sequence SEQ ID NO:7 and its fusion with the arm (Item 7) is shown by mass spectrometry.

[0051] Figure 4B The staining patterns and frequencies of PS-positive cells stained with PE Project 7 or Atto 647 Project 2 on a mixture of fresh and heat-shock-treated Jurkat cells are shown.

[0052] Figure 4C The frequency of PS-positive and PS-negative cells was shown before and after the consumption of a mixture of freshly prepared and heat-shock-treated C57BL / 6 mouse thymocytes.

[0053] Figure 4D The staining patterns and frequencies of PS cells stained with pre-hybridized Alexa 647 poly-T oligonucleotide project 7 (Alexa 647 poly-T oligonucleotide project 7), Atto 647 project 2, or FITC project 5 on one-day-old C57BL / 6 thymocytes are shown.

[0054] Figure 4E The staining patterns and frequencies of PS-positive cells stained with pre-hybridized Alexa 647 poly-T oligonucleotide Project 7 or FITC Project 5 on a mixture of live and heat-shock-treated Jurkat cells are shown.

[0055] Figure 4F The staining patterns and frequencies of PS-positive cells simultaneously stained with pre-hybridized Alexa 647 poly-T oligonucleotide project 7 and propidium iodide (PI) or FITC project 5 are shown.

[0056] Figure 4G A schematic diagram illustrating the single-cell sequencing process is provided. Detailed Implementation

[0057] The following detailed description illustrates certain preferred embodiments of the invention and is therefore representative only, not depicting the actual scope of the invention. It should be understood that the invention is not limited to the specific embodiments described, nor to the particular methods, schemes, and reagents described herein, as these can vary.

[0058] Unless otherwise stated, all scientific and technical terms used herein have their common meaning in the art. The definitions provided herein are intended to aid in understanding certain terms frequently used herein and are not intended to limit the scope of this disclosure.

[0059] As used herein, the terms “conjugate,” “conjugation,” “labeling,” “couple,” “coupling,” “coupled,” “link,” “linking,” “linked,” and “linkage” are used interchangeably. These terms refer to the chemical conjugation of two or more elements, components, or molecules together. Methods of chemical conjugation (e.g., using click chemistry) are known in the art. The terms “conjugate,” “labeling,” or “coupled” as used herein refer to the collinear covalent bonding or attachment of two or more proteins, fluorescent dyes, oligonucleotides / oligonucleotides, avidin, streptavidin, enzymes, solid-phase carriers (particles, microbubbles), or fragments thereof through a peptide backbone.

[0060] The term "PS-binding compound" as used herein refers to a compound that specifically binds phosphatidylserine (PS). In the context of this invention, a PS-binding compound should be understood as one capable of binding PS on the outer layer of the cell membrane of apoptotic or dying cells, dead cells or cell debris, activated platelets, and extracellular vesicles (EVs).

[0061] Phosphatidylserine (PS) conjugates

[0062] The reference cyclic amphiphilic peptide Apo-15 comprises the sequence of SEQ ID NO:1 (Item 1), wherein Arg and Lys are hydrophilic and positively charged amino acids, and Trp, Phe, and Gly are hydrophobic and neutrally charged amino acids. Each amino acid in the Apo-15 sequence contains an amino group (NH2) and a carboxyl group (COOH). Of the amino acids in the Apo-15 sequence, Arg also has a complex side chain (CH2-CH2-CH2-NH-CNH-NH2), and Lys also has a long side chain of four CH2 groups and ends with an amino group. Because Arg and Lys are positively charged, and positive charge is a key element for binding to negatively charged PS, the amino groups on the side chains are not considered for chemical labeling. The Trp(BODIPY) is based on the fluorescent amino acid Fmoc-Trp(C2-BODIPY)-OH, which contains a fluorescent BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indenobenzene) core, wherein BODIPY is linked to Trp via a C-C bond without spacers. Gly is used to promote head and tail cyclization.

[0063] Apo-15 is a novel PS binder, similar to the commonly used PS binder Annexin V, for detecting PS-positive cells using flow cytometry or fluorescence microscopy. An advantage of this reagent is that Apo-15 binds to PS in a calcium-independent manner. As an imaging reagent, it typically requires different forms of fluorophores to provide options for selecting different fluorophores in multicolor flow cytometry or multiplex imaging. To date, Apo-15 has only one form that excites at 500 nm and emits at 520 nm for use in multicolor flow cytometry. Since Apo-15 lacks functional groups that can be used for conjugation with commonly used fluorophores, novel derivatives of Trp(BODIPY) with different excitation and emission are needed before synthesizing new forms of Apo-15. To meet market demands, PS binders should possess functional groups capable of conjugating different forms of fluorophores using standard and well-defined conjugation methods, while maintaining a simple and cost-effective manufacturing process.

[0064] To overcome the limitations of Apo-15 and enable its conjugation to different forms of fluorophores while maintaining its PS-binding ability, the structure of the cyclic peptide needs to be modified and altered by replacing certain amino acids in the sequence with alternative amino acids without significant loss of activity. The amino acid substitutions can be considered based on the relative similarity of the amino acids, such as their hydrophobicity, hydrophilicity, and charge. To achieve this goal, the novel method of the present invention comprises: (1) maintaining the PS-binding ability of Apo-15; (2) removing the fluorescent amino acid Trp(BODIPY); and (3) creating at least one additional functional group for chemical labeling.

[0065] The “PS-binding compound derivative” used in this article refers to a phosphatidylserine (PS)-binding compound in which the residue Trp (BODIPY) at position 6 of the amino acid sequence of SEQ ID NO:1 (Apo-15) has been replaced by the natural amino acid Trp to remove fluorescence, and the residue Gly at position 7 has been replaced by the amino acid Cys or the synthetic amino acid propargylglycine (Pra).

[0066] The present invention provides a PS-binding compound derivative comprising the sequence of SEQ ID NO:2 (Item 2), wherein the amino acid Cys is used to provide a thiol group for coupling with a maleimide-containing tag by means of a thiol-maleimide reaction.

[0067] The method for preparing PS-binding compound derivatives in Project 2 includes: (1) replacing the fluorescent amino acid Trp(BODIPY) at position 6 of the amino acid sequence of SEQ ID NO:1 with the natural amino acid Trp to remove fluorescence and retain the natural properties of Trp; and (2) replacing the amino acid Gly at position 7 of the amino acid sequence of SEQ ID NO:1 with the amino acid Cys, wherein the Cys contains three functional groups, including an amino group and a carboxyl group for promoting head-tail cyclization, and a thiol group on the side chain for chemical labeling by using a thiol-maleimide reaction. The thiol-maleimide reaction is a simple and rapid reaction and is one of the most popular methods in current bioconjugation techniques.

[0068] Project 2 is used as an example of preparation and use of Project 2 by coupling a PS-binding compound derivative with a maleimide-containing fluorophore, wherein the maleimide-containing fluorophore Atto 647 is coupled with Project 2 by using a thiol-maleimide reaction, and the fluorophore is used as a detectable unit by flow cytometry or fluorescence microscopy for multicolor flow cytometry and multiplex imaging.

[0069] Project 2, a PS-binding compound derivative, was synthesized and validated by mass spectrometry. The observed molecular weight of the cyclic peptide in Project 2 was 1034.4 g / mol (see [link to Project 2]). Figure 1A-2 The molecular weight is 1052.4 g / mol, which is the molecular weight of linear item 2 (see [reference]). Figure 1A-1 Subtract one H2O molecular weight. Experimental results show that Project 2 was successfully synthesized. Also... Figure 1A-3 As shown, Project 2 was successfully conjugated with Atto 647 maleimide (Atto 647 Project 2) by using the thiol-maleimide reaction.

[0070] Whether a reagent is considered a PS binder or has PS binding activity can be determined by using flow cytometry to detect its PS binding activity, as has been described in the art. Therefore, according to the present invention, PS-positive cells can be used for such assays, wherein the number of stained cells can be compared with reference reagents Apotracker Green and / or annexin V.

[0071] To verify the staining patterns and frequencies of PS-positive dying and dead cells, Atto647 Project 2 or Apotracker Green (trade name for Apo-15) (in Ca2+-free) was used. 2+ One-day-old C57BL / 6 mouse thymocytes were stained in parallel with either PBS (in PBS) or Alexa 647 Annexin V (in annexin binding buffer). Sytox Blue is an opaque, blue-light-emitting nucleic acid indicator used to indicate dead cells. Figure 1BAs shown, Project 2 - Sytox - For the live cells shown in Q4, Project 2 + Sytox - The membrane-intact dying cells shown in Q1, and item 2 + Sytox + The dead cells are shown in Q2. The staining patterns and frequencies of PS-positive dying and dead cells stained with Atto647 Project 2 or Apotracker Green or Alexa 647 are comparable to each other.

[0072] When U937 cells were stimulated for 2 days with IL-4 simultaneously stained with Atto 647 Project 2 and Apotracker Green, the frequencies of PS-positive dying cells and dead cells stained with Atto 647 Project 2 and Apotracker Green were very similar. Furthermore, cells stained with Atto 647 Project 2 and Apotracker Green showed a diagonal distribution in the two-parameter density plot (see...). Figure 1C ).

[0073] To further investigate the relationship between PS-positive dying cells and dead cells stained with Atto 647 Project 2 and Apotracker Green, correlation coefficients were used. Table 1 shows the frequencies of live cells, PS-positive dying cells, and dead cells from four independent experiments, where Ca2+ staining was performed in the absence of Ca2+. 2+ Different cell types were simultaneously stained in PBS with different concentrations of Atto 647 Project 2 and Apotracker Green. As shown in Table 2, the correlation coefficients were very close to 1.0, indicating that live cells, PS-positive dying cells, and dead cells stained with Atto 647 Project 2 and Apotracker Green showed a very strong positive correlation and an almost perfect linear relationship. The experimental results validated the high correlation between PS-positive dying cells and dead cells stained with Atto 647 Project 2 and Apotracker Green.

[0074] To verify whether PS-positive cells stained in Project 2 could be fixed for subsequent staining, in Ca-free... 2+ Fresh U937 cells were stained with Atto 647 Project 2 in PBS and stimulated with IL-4 for 2 days, followed by fixation with 2% paraformaldehyde (PFA). Figure 1DAs shown, the staining patterns and frequencies of cells in Q1, Q2, Q3, and Q4 were similar before and after fixation. The fluorescence intensity of fixed cells was slightly lower than that of unfixed cells, which is due to the washing that occurred after fixation. The experimental results demonstrate that PS-positive dying and dead cells stained with Atto647 Project 2 are fixable and support the claim that Project 2 binding of PS is Ca2+. 2+ Independent.

[0075] To further verify whether PS cells stained with Atto 647 Project 2 were visible under a microscope, adherent human lung adenocarcinoma cell line (Calu-6) cells were simultaneously stained with Atto 647 Project 2, calcein AM, and fixable viability dye-405. Figure 1E As shown, calcein AM + Atto647 Project 2 - Motile dye-405 - The cell is a living cell, calcein AM 弱 / - Atto 647 Project 2 + Motile dye-405 - These are dying cells, and calcein AM - Atto 647 Project 2 + Motile dye-405 + These are dead cells. Calcein AM is a membrane-permeable live-cell labeling dye that labels live cells. Viable dye-405 can be immobilized to react with the amine groups of proteins in damaged cell membranes, thus labeling dead cells. Experimental results indicate that Atto 647 Project 2 can be used for multiplex imaging.

[0076] As described elsewhere in this document, experimental results demonstrate that the present invention has successfully synthesized PS-binding compound derivative item 2. Item 2 not only retains the PS-binding ability but also provides a thiol group for coupling with maleimide-containing fluorophores, offering more options for the selection of different fluorophores in multicolor flow cytometry and multiplex imaging. Since different maleimide-containing fluorophores are commercially available and the conjugation methods are well-described, the risks associated with the manufacturing process and costs should be reduced.

[0077] PS-binding compound derivative item 2 is coupled to a maleimide-containing tag, wherein the maleimide-containing tag may also be a maleimide-containing bifunctional linker, including but not limited to maleimide-Pol-maleimide, maleimide-Pol-thiol, maleimide-Pol-azide, maleimide-Pol-alkyne, maleimide-Pol-NHSter, maleimide-Pol-amine, and maleimide-Pol-COOH; wherein the maleimide is used to link to the thiol group in Cys of item 2, wherein the other functional group in the maleimide-containing bifunctional linker, thiol, or maleimide, or azide, or alkyne, or NHSter, or amine, or carboxyl group, is used for chemical labeling; wherein the linker may be a polymer or an amino acid, wherein the polymer includes (PEG). n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is the number of polymers, and n is an integer from 1 to 60, preferably from 1 to 24, and more preferably from 1 to 12.

[0078] In this invention, PS-binding compound derivative item 2, containing the sequence SEQ ID NO:2, provides a thiol group for coupling with a maleimide-containing tag, wherein the tag serves as a detectable unit, including but not limited to fluorophores, solid-phase supports, oligonucleotides, biotin, avidin, proteins, enzymes, and radionuclides. Furthermore, when the tag is a maleimide-containing bifunctional linker, the maleimide-containing bifunctional linker can provide amine, or NHSter, or carboxyl, or thiol, or maleimide, or alkyne, and azide groups for chemical labeling.

[0079] In this invention, when a fluorophore contains a functional group, the fluorophore can be any fluorescent dye. It includes, but is not limited to, PE, APC, PerCp and their tandem dyes such as Cy3, Cy5, Cy7, A596, A640, A750 and A810, 350 / 405 / 488 / 647 / 594 / 700 / 750, DL545 / 570 / 585 / 590 / 685, FITC, NIR dyes and Pacific Blue, Pacific Orange, BV421, BV510, BV605, BV650, BV710, Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, Atto633, Atto635, Atto647, Atto655, Atto 680, Atto700, Atto725, functionalized BODIPY, and bioluminescent dyes. Such fluorophores can be used as detectable units by flow cytometry and fluorescence microscopy.

[0080] The present invention provides another PS-binding compound derivative (item 3) containing the sequence of SEQ ID NO:3, wherein the synthesized amino acid Pra is used to provide an alkyne group for coupling with an azide-containing tag by means of an azide-alkyne reaction.

[0081] The method for preparing PS-binding compound derivative item 3 includes: (1) replacing the fluorescent amino acid Trp (BODIPY) at position 6 of the amino acid sequence of SEQ ID NO:1 with the natural amino acid Trp to remove fluorescence and retain the natural properties of Trp; (2) replacing the amino acid Gly at position 7 of the amino acid sequence of SEQ ID NO:1 with the synthetic amino acid Pra, wherein the Pra is an Fmoc-protected Gly derivative containing amine and carboxyl groups for promoting head-tail cyclization, and alkyne groups for azide-alkyne reactions, which provides high flexibility in incorporation into peptides.

[0082] The PS-binding compound derivative of Project 3 is coupled with an azide-containing fluorophore as an example of the preparation and use of Project 3, wherein the azide-containing fluorophore fluorescein azide is coupled with Project 3 by using an azide-alkyne reaction. This fluorophore is used as a detectable unit by flow cytometry for multicolor flow cytometry.

[0083] PS-binding compound derivatives (Project 3) were synthesized, and their molecular weights were measured by mass spectrometry. Figure 2A-1As shown, the observed molecular weight of linear item 3 is 1044.5 g / mol. The observed molecular weight of cyclic item 3 is 1026.5 g / mol, which is due to the elimination of one H2O molecule during head-to-tail cyclization (see...). Figure 2A-2 Experimental results confirmed the successful synthesis of Project 3. The fluorescein azide was coupled to Project 3, and the coupling of fluorescein azide to Project 3 (FITC Project 3) was verified by flow cytometry.

[0084] To verify FITC Project 3, in the absence of Ca 2+ Cells stimulated with IL-4 for 2 days were simultaneously stained with FITC Project 3 and Atto647 Project 2 in PBS. Figure 2B As shown, the staining patterns and frequencies of PS-positive dying and dead cells stained with FITC Project 3 and Atto 647 Project 2 were similar. Furthermore, PS-positive cells stained with FITC Project 3 and Atto 647 Project 2 showed a high correlation.

[0085] As described elsewhere in this document, experimental results demonstrate that the present invention has successfully synthesized PS-binding compound derivative item 3. Item 3 not only retains PS-binding ability but also allows for coupling with azide-containing fluorophores for multicolor flow cytometry, potentially providing more options for selecting different forms of azide-containing fluorophores in multicolor flow cytometry and multiplex imaging. Since different azide-containing fluorophores are commercially available and the conjugation methods are well-described, the risks associated with the manufacturing process and costs should be reduced.

[0086] PS is coupled with a azido-containing tag to a compound derivative project 3, wherein the azido-containing tag may be an amino acid containing an azido compound, including but not limited to azido-lysine, azido-propargylglycine, azido-L-propargylglycine, azido-histidine, azido-tryptophan, azido-phenylalanine, azido-arginine, azido-glutamine, azido-glycine, azido-valine, and azido-alanine, wherein the azido group is used to link with the alkyne group in Pra, and the amino acid with the functional group is used for chemical labeling.

[0087] PS is coupled with a tag containing an azide to a compound derivative of Project 3, wherein the tag containing the azide can also be a bifunctional connector containing an azide, including but not limited to azide-Pol-maleimide, azide-Pol-thiol, azide-Pol-alkyne, azide-Pol-azide, azide-Pol-amine, azide-Pol-NHSter, and azide-Pol-COOH, wherein the azide is used to connect with the alkyne in Pra of Project 3, wherein the other functional group in the bifunctional connector containing the azide, such as amine, NHSter, carboxyl, maleimide, thiol, alkyne, or azide, is used for chemical labeling; wherein Pol is a polymer used as a connector, including but not limited to (PEG). n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is the number of polymers, and n is an integer from 1 to 60, preferably from 1 to 24, and more preferably from 1 to 12.

[0088] In this invention, a PS-binding compound derivative item 3 containing the sequence SEQ ID NO:3 is coupled to an azide-containing tag, wherein the tag is a detectable unit, including but not limited to fluorophores, solid-phase supports, oligonucleotides, biotin, avidin, proteins, enzymes, and radionuclides. Furthermore, when the tag is an azide-containing amino acid or an azide-containing bifunctional linker, the azide-containing amino acid or bifunctional linker can provide amines, or NHSter, or thiols, or maleimides, or azides, or alkynes and carboxyl groups for chemical labeling.

[0089] Items 2 and 3 of this invention can be used for imaging by labeling fluorophores containing maleimide or azide. Arg and Lys in Items 2 and 3 cannot be used for chemical labeling of NHSter-containing fluorophores because Arg and Lys are key elements for PS binding. Furthermore, both Items 2 and 3 have only one functional group to bind one fluorophore. The ratio of fluorophore to Items 2 or 3 is 1:1. This poses a challenge for weak dyes used for imaging, such as FITC. To generate amine groups for the amine-NHSter reaction and improve fluorescence intensity, the inventors designed a fusion of a PS-binding compound and an arm compound, wherein the arm compound provides at least one amine functional group for chemical labeling.

[0090] This invention provides a fusion of a PS-binding compound and an arm compound, comprising SEQ ID NO:5 [RKKWFWPra-Xa1(azido)-Xa2-(Pol-Xa3)]m The sequence [[Xa1(azido)-Xa2-(Pol-Xa3]] (item 5) contains SEQ ID NO:4[Xa1(azido)-Xa2-(Pol-Xa3]]. m The arm compound (item 4) of the sequence is linked to item 3, wherein arm compound item 4 provides at least one functional group to be coupled to at least one tag containing a functional group.

[0091] The method for preparing fusion compound 5 and its coupling with a tag includes: (1) synthesizing 3 according to the sequence of SEQ ID NO:3; (2) synthesizing arm compound 4 according to the sequence of SEQ ID NO:4; (3) linking 4 with 3 and then coupling it with a tag containing maleimide or azide; (4) coupling 4 with a tag containing NHSter and then linking it with 3.

[0092] Fusion item 5 is coupled with an NHSter-containing fluorophore as an example of the preparation and use of item 5, wherein the NHSter-containing fluorophore is coupled with item 4 by means of an amine-NHSter reaction, and then linked with item 3. This fluorophore is used as a detectable unit for detection by flow cytometry in multicolor flow cytometry.

[0093] An NHSter-containing fluorophore coupled to fusion compound 5 was synthesized and validated by mass spectrometry and flow cytometry, wherein the variable amino acid [Xa1 (azido)] is Lys (azido); the variable amino acid (Xa2) is Lys; and the variable amino acid (Pol-Xa3) is... m The PEG is (PEG4-Lys)3; the PEG is Fmoc-NH-PEG-COOH, and the Lys in (PEG4-Lys)3 provides three amine groups that are coupled to three NHSter-containing fluorophores, wherein the fluorophores are NHSter-containing fluorescein (FITC). The NHSter-containing fluorescein (FITC) coupled to Project 5 was verified by flow cytometry.

[0094] like Figure 3A As shown, the observed molecular weight of item 5 is 3535.4 g / mol, which indicates that item 3 has been linked to item 4, and each item 5 is coupled with three FITCs.

[0095] To verify FITC Project 5, in the absence of Ca 2+ In PBS solution, thymocytes from one-day-old C57BL / 6 mice were stained in parallel using FITC assay 5 or FITC assay 3. Figure 3B As shown, the staining patterns and frequencies of PS-positive cells stained with FITC item 5 and FITC item 3 are comparable.

[0096] Therefore, the ratio of FITC to item 5 is 3:1, and thus the fluorescence intensity of FITC item 5 should be stronger than that of FITC item 3. Figure 3C As shown, at different concentrations, the staining intensity of FITC item 5 was higher than that of FITC item 3.

[0097] As described elsewhere in this paper, experimental results demonstrate that the present invention has successfully synthesized FITC item 5, which makes it possible to provide amine functional groups for coupling with NHSter-containing fluorophores via the amine-NHSter reaction. Item 5 not only retains PS binding ability but also can be coupled with multiple fluorophores to enhance fluorescence intensity for use in multicolor flow cytometry and multiplex imaging. Furthermore, the number of amine functional groups can be modified by adding or removing short repeats (PEG4-Lys).

[0098] The arm compound item 4 contains the sequence of SEQ ID NO:4, wherein Xa1 (azido) is an azide-containing amino acid, including but not limited to azide-lysine, azide-propargylglycine, azide-L-propargylglycine, azide-histidine, azide-tryptophan, azide-phenylalanine, azide-arginine, azide-glutamine, azide-glycine, azide-valine, and azide-alanine, wherein the azide group is used to connect with the alkyne in Pra of item 3, and the amino acid having the functional group is used to connect with X2a.

[0099] Compound item 4 contains the arm of the sequence SEQ ID NO:4, wherein the variable Xa2 is an amino acid containing an amino and a carboxyl group, used in conjunction with Xa1 and (Pol-Xa3). m The connector is connected, and the amino acids mentioned therein include, but are not limited to, Lys, Arg, His, Ser, Thr, Cys, Asn, Gln, Pra, and Tyr.

[0100] Compound item 4 contains the arm of the sequence SEQ ID NO:4, wherein (Pol-Xa3) m are independent variables, where Pol is used as a connector, including but not limited to (PEG). n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n], wherein the variable n is the number of polymers, and n is an integer from 1 to 60, preferably from 1 to 24, more preferably from 1 to 12; the variable Xa3 is an amino acid having a functional group for chemical labeling, wherein the functional group includes, but is not limited to, amines, thiols and alkynes, and wherein the amino acid includes, but is not limited to, Lys, Arg, His, Cys and Pra; the variable m is the number of (Pol-Xa3), wherein m is an integer from 1 to 10.

[0101] In this invention, the fusion of a PS-binding compound containing the sequence of SEQ ID NO:5 and its arm can provide an amine, thiol, or alkyne functional group for coupling with a tag containing NHSter, maleimide, or azide, wherein the tag serves as a detectable unit, including but not limited to fluorophores, oligonucleotides, solid-phase carriers, biotin, avidin, proteins, enzymes, and radionuclides. Furthermore, the functional group provided by item 5 can be selected for chemical coupling, and the number of functional groups provided by item 5 in the fusion is adjustable.

[0102] The molecular weights of Projects 2 and 3 are 1034.4 g / mol and 1026.4 g / mol, respectively, which are smaller than those of protein dyes, such as phycoerythrin (PE) dye (molecular weight: 240,000 Daltons). To label large-sized tags, such as PE or particles, the inventors designed a second fusion of a PS-binding compound with an arm, wherein the arm provides functional groups for coupling functional tags.

[0103] This invention provides a fusion of a PS-binding compound and an arm compound comprising the sequence SEQ ID NO:7 [RKKWFWPra-Xa1(azido)-Xa2-Pol-Xa3], wherein an arm compound comprising the sequence SEQ ID NO:6 [Xa1(azido)-Xa2-Pol-Xa3] is linked to item 3, wherein the arm compound item 4 provides a functional group for coupling with a tag containing a functional group.

[0104] The method for preparing item 7 includes: (1) synthesizing item 3 containing the sequence of SEQ ID NO:3; (2) synthesizing item 6 containing the sequence of SEQ ID NO:6; (3) linking item 6 to item 3 and then coupling it to a tag containing maleimide or azide group; (4) coupling item 6 to a tag containing NHSter and then linking it to item 3.

[0105] The coupling of fusion item 7 with a thiol-containing tag serves as an example of the preparation and use of item 7, wherein, after item 6 is linked to item 3, the thiol-containing tag is coupled to item 7 by using a thiol-maleimide reaction.

[0106] Fusion Item 7 was synthesized and verified by mass spectrometry, wherein the variable Xa1 is Lys (azide group), the variable Xa2 is Lys, the Pol is PEG12, the PEG is Fmoc-NH-PEG-COOH, and the variable Xa3 is Cys to provide a thiol group for coupling with a maleimide-containing tag.

[0107] like Figure 4A As shown, the observed molecular weight of item 7 is 2029 g / mol. The experimental results confirm that items 3 and 6 were successfully linked.

[0108] Examples of methods for preparing fluorophore-labeled Item 7 using maleimide-containing R-phycoerythrin (PE) and methods for using Item 7 in multicolor flow cytometry. PE maleimide is directly conjugated to Item 7 using a thiol-maleimide reaction and purified using an S-200 column. (See example...) Figure 4B As shown, the staining patterns and frequencies of PS-positive cells stained with PE Project 7 or Atto 647 Project 2 were comparable in a calcium-independent manner. The experimental results indicate that Project 7 can be used for coupling with protein dyes.

[0109] The magnetic particles conjugated in Project 7 are another example of the method for preparing magnetic particles conjugated in Project 7 for consuming PS-positive cells. For example... Figure 4C As shown, in a mixture of freshly prepared and heat-shocked mouse thymocytes in cell separation buffer (buffer composition: PBS and 0.5% BSA and 2mM EDTA), the magnetic particles conjugated to Project 7 consumed PS-positive cells, and through two separations, the percentage of live cells increased from 53.3% to 96.27%, and the recovery rate of live cells was 84%.

[0110] Item 7, an oligonucleotide-tagged designation, is another example of a method for preparing oligonucleotide-conjugated Item 7 for binding PS-positive cells in single-cell sequencing. The oligonucleotide is a designed short DNA sequence comprising: (1) a PCR operation sequence serving as the starting point for DNA synthesis; (2) a unique DNA barcode serving as a PS-positive cell identifier that can be easily recovered from a single transcriptome; and (3) a capture sequence serving as a sequence that binds to a complementary sequence on a cell capture bead. Item 7 is used to bind PS-positive cells, enabling the differentiation of PS-positive cells from live cells in single-cell RNA sequencing.

[0111] The experimental procedure for single-cell RNA sequencing includes: (1) contacting oligonucleotide / DNA barcode-tagged item 7 with a heterogeneous cell population containing PS-positive cells; (2) capturing each cell in a fluid flow device; (3) lysing each single cell; (4) sequencing each cell; and (5) analyzing the sequence.

[0112] Entry 7 is an oligonucleotide-tagged sequence, wherein the oligonucleotide comprises the sequence of SEQ ID NO:8, wherein the sequence of SEQ ID NO:9 is a PCR operation sequence, which is a constant sequence identical on all primers to allow PCR amplification after the formation of a single-cell transcriptome (STAMP) attached to the microparticle, and wherein the sequence of SEQ ID NO:10 is a unique cell barcode, which is a unique identical sequence for PS-positive cells, allowing for retrieval of the cell's origin. In this invention, the unique cell barcode is used to identify PS-positive cells. The sequence of SEQ ID NO:11 is a 30 bp oligo-A capture sequence, which serves as a sequence that binds to a complementary sequence on the cell capture bead, encapsulating each cell with a different barcoded microparticle in a microdroplet. In the sequence, "B" represents nucleotide C, G, or T, and "*" represents a phosphate thioester bond to prevent nuclease degradation. Once individual cells are isolated into individual droplets, they are lysed, resulting in the release of mRNA from the cells, which hybridizes with primers to generate STAMPs (single-cell transcriptomes attached to microparticles). STAMPs are then amplified, and subsequently sequenced and analyzed using STAMP barcoding to infer the cell of origin for each transcript. Oligonucleotides can be designed with different sequences for different single-cell sequencing systems, including but not limited to 10x single-cell sequencing systems, Luminex single-cell analysis systems, and BD Rhapsody single-cell analysis systems.

[0113] To verify whether oligonucleotide project 7 could specifically bind to PS-positive cells, Alexa 647-conjugated poly-T was prepared to hybridize with the complementary sequence of poly-A. First, 50 pM of oligonucleotide project 7 was complementary to 37.5 pM of Alexa 647-conjugated poly-T (Alexa 647 poly-T oligonucleotide project 7) at room temperature for 20 minutes, followed by incubation with different heterogeneous cell populations for an additional 15 minutes, and then a wash. Cells stained with Atto 647 project 2 or FITC project 5 served as controls.

[0114] Figure 4D The staining patterns and frequencies of PS-positive cells stained with Alexa 647 poly-T oligonucleotide project 7, Atto647 project 2, or FITC project 5 on thymocytes of one-day-old mice were shown to be comparable.

[0115] Figure 4E The staining patterns and frequencies of PS-positive cells simultaneously stained with Alexa647 poly-T oligonucleotide project 7 and Sytox Blue or FITC project 5 and Sytox Red on a mixture of live and heat-shock-treated Jurkat cells were shown to be similar.

[0116] To further validate the specificity of oligonucleotide project 7, a mixture of live and heat-shocked Jurkat cells was simultaneously stained with Alexa 647 poly-T oligonucleotide project 7 and propidium iodide (PI) or FITC project 5. Figure 4F As shown, PS-positive cells stained with 647 poly-T oligonucleotide project 7 are highly correlated with PS-positive cells stained with propidium iodide or FITC project 5.

[0117] As described elsewhere in this document, experimental results demonstrate that the present invention has successfully synthesized fusion item 7. This fusion item 7 not only retains PS binding ability but also provides tunable linkers for thiol groups, enabling coupling with maleimide-containing protein dyes for multicolor flow cytometry; coupling with maleimide-coated magnetic particles for consuming PS-positive cells; and coupling with maleimide-containing oligonucleotides for binding PS-positive cells. This allows for the differentiation of PS-positive cells from live cells in single-cell RNA sequencing, wherein the oligonucleotides contain PCR manipulation sequences, unique barcodes for PS-positive cells, and capture sequences.

[0118] Figure 4G This diagram illustrates the single-cell sequencing analysis process. Oligonucleotide Project 7 is mixed with different oligonucleotide conjugated antibodies containing unique barcodes to stain heterogeneous cell populations. Individual cells are captured in a fluidic flow apparatus, each cell co-encapsulated in a microdroplet with a different barcoded microparticle by hybridizing a polyA capture sequence with complementary polyT beads. Once the cells are isolated into the droplets, they are lysed, releasing mRNA, which is then hybridized with primers. STAMPS are generated, then amplified and sequenced. Bioinformaticians can use the STAMPS barcodes to infer the source cell of each transcript, enabling biological data analysis, such as sequence analysis. PS-positive cells are identified and filtered out via unique barcodes before complex biological data analysis. Therefore, bioinformaticians will analyze only live cells, effectively saving time and improving data quality.

[0119] Arm compound item 6, wherein the variable Xa1 (azido) is an azide-containing amino acid, including but not limited to azido-lysine, or azido-propargylglycine, azido-L-propargylglycine, azido-histidine, azido-tryptophan, azido-phenylalanine, azido-arginine, azido-glutamine, azido-glycine, azido-valine, and azido-alanine, wherein the azide group is used to connect with the alkyne group in Pra of item 3, and the amino acid having a functional group is used to connect with Xa2.

[0120] Arm compound project 6, wherein variable Xa2 is an amino acid containing an amino group and a carboxyl group, which serves as a linker to Xa1 and Pol, wherein the amino acid includes, but is not limited to, Lys, Arg, His, Ser, Thr, Cys, Asn, Gln, Pra and Tyr.

[0121] Compound 6, where Pol is used as a connector to link Xa2 and Xa3, wherein Pol includes, but is not limited to, PEG. n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where the variable n is the quantity of polymers, and n is an integer from 1 to 60, preferably from 1 to 24, more preferably from 1 to 12.

[0122] Arm compound project 6, wherein variable Xa3 is an amino acid for providing a functional group for chemical labeling, wherein variable Xa3 is a functional group-containing amino acid, wherein the functional group includes, but is not limited to, amines, thiols and alkynes, and wherein the amino acid includes, but is not limited to, Cys, Pra, Met, Lys, Arg and His.

[0123] In this invention, the fusion item 7, comprising a PS-binding compound containing the sequence of SEQ ID NO:7 and an arm, can provide amine, thiol, or alkyne functional groups for coupling with tags containing NHSter, maleimide, or azide, wherein the tag serves as a detectable unit, including but not limited to fluorophores, oligonucleotides, solid-phase carriers, biotin, avidin, proteins, enzymes, and radionuclides. Furthermore, the functional groups provided by fusion item 7 can be selected for chemical coupling. The length of the connector can be adjusted, which can facilitate coupling with large-sized tags.

[0124] In this invention, PS-binding compound derivatives items 2 and 3, and fusions of PS-binding compounds and arm compounds items 5 and 7, can be coupled to tags containing different functional groups, wherein the tags serve as detectable units. The tags are fluorophores for multicolor flow cytometry and multiplex imaging, solid-phase carriers for consuming PS-positive cells, and oligonucleotides for binding PS-positive cells, which can distinguish PS-positive cells from live cells in single-cell RNA sequencing, wherein the oligonucleotides contain a PCR manipulation sequence, a unique DNA barcode for PS-positive cells, and a capture sequence.

[0125] Example

[0126] To better illustrate the present invention, the following embodiments are provided. Specific materials mentioned are for illustrative purposes only and are not intended to limit the scope of the invention.

[0127] All commercially available chemicals were of analytical grade and ready for use without further purification. All amino acids were purchased from Sigma. Atto 647N maleimide was purchased from Sigma (catalog number: 05316), fluorescein azide from Sigma (catalog number: 910147), R-phycoerythrin (PE) from Agilent (catalog number: PB32B), oligonucleotides were synthesized by Integrated DNA Technologies, and maleimide-coated magnetic particles were purchased from Ocean NanoTech (catalog number: SM0200-10).

[0128] Example 1

[0129] Synthesis and Validation of Project 2

[0130] Based on the sequence of SEQ ID NO:2, the PS-binding compound derivative Item 2 was synthesized using a standard Fmoc chemistry protocol. The crude peptide was then purified by HPLC, and its molecular weight was measured by mass spectrometry. Atto 647N maleimide was conjugated to Item 2 using a standard thiol-maleimide reaction chemistry protocol. In this invention, Item 2 conjugated with Atto 647N maleimide is named Atto 647 Item 2. Item 2 and Atto 647 Item 2 were synthesized and conjugated by Innopep Inc.

[0131] Figures 1A-1 to 1A-3 The molecular weight of item 2, measured by mass spectrometry, is shown. (Example) Figure 1A-1 As shown, the molecular weight of linear item 2 is 1052.4 g / mol. During head-tail cyclization, the carboxylic acid reacts with the amine during amidation. Water molecules are removed in the reaction to form an amide. For example... Figure 1A-2 As shown, the observed molecular weight of the cyclic peptide in Project 2 is 1034.4 g / mol, which is obtained by subtracting the molecular weight of H2O from the molecular weight of the linear Project 2. Mass spectrometry results confirm that Project 2 has been successfully synthesized, and the purity of Project 2 is >95%. Furthermore, as... Figure 1A-3 As shown, Item 2 is coupled with Atto 647, and the observed molecular weight of Atto 647 Item 2 is 1763.9 g / mol. The purity of Atto 647 Item 2 is >95%.

[0132] To validate Atto 647 Project 2 by flow cytometry, thymocytes from one-day-old C57BL / 6 mice were subjected to a calcium-free environment. 2+Cells were incubated in parallel for 15 minutes at room temperature with 28 pM / test of Atto 647 Project 2 and Sytox Blue or 400 nM / test of Apotracker and Sytox Blue in PBS, or 5 μL / test of Alexa 647 Annexin V and Sytox Blue in Annexin V binding buffer, and then analyzed by NovoCyte Quanteon flow cytometry without washing. Debris was gated according to cell density maps at low levels of forward scattering. Two-parameter density maps show Atto 647 Project 2 or Apotracker Green or Annexin V relative to Sytox Blue. Sytox Blue is an impermeable, blue-emitting nucleic acid indicator used to indicate dead cells. In this study, Sytox Blue was used at 20 nM / test.

[0133] like Figure 1B As shown, Atto 647 Project 2 - Sytox - The cells are the living cells shown in Q4, Atto647 Project 2 + Sytox - It is the dying cell shown in Q1, and item 2 + Sytox + These are the dead cells shown in Q2. In thymocytes of one-day-old C57BL / 6 mice, atto 647 or Apotracker Green (in Ca2+-free) were used. 2+ The frequencies of PS-positive cells stained with either PBS (in PBS) or annexin V (in annexin V binding buffer) were roughly equal. Cells were analyzed using NovoCyte Quanteon flow cytometry without washing.

[0134] To verify the relationship between Atto 647 Project 2 and Apotracker Green staining in PS-positive cells, U937 cells stimulated with IL-4 for 2 days were simultaneously stained for 5 minutes in pristine cell culture medium with 28 pM / test Atto 647, 400 nM Apotracker Green, and 50 nM Sytox Blue. Cells were then analyzed by NovoCyteQuanton flow cytometry without washing. Figure 1C As shown, the staining patterns and frequencies of live cells, PS-positive dying cells, and dead cells stained with Atto 647 Project 2 and Apotracker Green are similar. In the two-parameter density map of Atto 647 Project 2 versus Apotracker Green, PS-positive dying cells and dead cells show a diagonal distribution.

[0135] To further investigate the relationship between PS-positive dying cells and dead cells stained with Atto 647 Project 2 and Apotracker Green, correlation coefficients were used. Table 1 shows the results in Ca-free... 2+ The frequencies of live cells, PS-positive dying cells, and dead cells from different cell types were determined in four independent experiments in PBS at different concentrations of Atto647 Project 2 and Apotracker Green. Cells were analyzed using NovoCyte Quanteon flow cytometry without washing.

[0136] Table 1

[0137]

[0138] The correlation was assessed to evaluate the strength of the relationship between PS-positive dying cells and dead cells stained with Atto 647 Project 2 and Apotracker Green.

[0139] Correlation can be calculated using the following formula:

[0140]

[0141] in:

[0142] · ρ(X,Y) - Correlation between variables X and Y

[0143] • Cov(X,Y) - Covariance between variables X and Y

[0144] ·σ x -Standard deviation of the variable X

[0145] ·σ y -Standard deviation of the Y variable

[0146] The covariance between two variables can be calculated as follows:

[0147]

[0148] in:

[0149] ·X i The value of the -X variable

[0150] ·Y j The value of the Y variable

[0151] ·X avg -The mean (average) of variable X.

[0152] ·Y avg- Mean (average) of the Y variable

[0153] n - the number of data points

[0154] Based on the experimental results shown in Table 1, the correlations between live cells, PS-positive dying cells, and dead cells stained with Atto 647 Project 2 and Apotracker Green were calculated, as shown in Table 2:

[0155] Table 2

[0156]

[0157] The correlation coefficient is very close to 1.0, which means that live cells, PS-positive dying cells, and dead cells stained with Atto 647 Project 2 and Apotracker Green show a very strong positive correlation and an almost perfect linear relationship. Both experimental and computational results confirm the high correlation between PS-positive dying cells and dead cells stained with Atto 647 Project 2 and Apotracker Green.

[0158] To verify the fixation of PS-positive cells stained with Atto 647 Project 2, fresh U937 cells stimulated with IL-4 for 2 days were simultaneously stained with 28 pM / test Atto 647 Project 2 and 20 nM Sytox Blue. The cells were then divided into two groups. One group of cells was analyzed immediately by flow cytometry without washing after staining. The other group of cells was fixed for 10 minutes at room temperature by adding 16% paraformaldehyde (PFA) to a final concentration of 2% PFA, followed by centrifugation at 350 g for 5 minutes. The cells were then resuspended in Ca2+-free medium. 2+ Fresh and stimulated cells, in fixed or unfixed conditions, were analyzed on day 0 and day 2, respectively, in PBS. Cells were analyzed using NovoCyte Quanteon flow cytometry without washing.

[0159] like Figure 1D As shown, the staining pattern and frequency of PS-positive cells were similar in both stimulated and unstimulated samples before and after fixation. Due to the washing step performed after fixation, the fluorescence intensity of fixed cells was slightly lower than that of unfixed cells. The experimental results confirm that PS-positive cells bound to Project 2 are Ca... 2+ The PS-positive cells stained with Project 2 are non-dependent and can be fixed.

[0160] To further verify whether PS cells stained with Atto 647 Project 2 were visible under a fluorescence microscope, adherent human lung adenocarcinoma cell line (CaLu-06) cells were exposed to UV light for 20 minutes, and then incubated at room temperature in a Ca-free environment. 2+ Cells were simultaneously stained in PBS with 1 μg / 100 μL of calcein AM, 84 pM / 100 μL of Atto 647 Project 2, and 1 μL / 100 μL of immobilizable viability dye 405 for 15 minutes, followed by a single wash. Cells were then imaged using a Leica DM18 microscope. Calcein AM is a membrane-permeable live-cell labeling dye that labels live cells. Immobilizable viability dye 405 reacts with protein amine groups in damaged cell membranes, labeling dead cells. Images were captured using individual filters, and all individual images were overlaid using LAS X software.

[0161] like Figure 1E As shown, calcein AM + Atto 647 Project 2 - Fixed Viscosity Dye 405 - The cells are living cells, containing calcein AM. 弱 / - Atto 647 Project 2 + Positive and fixable active dye 405 - The cells are dying cells, containing calcein AM. - Atto647 Project 2 + Fixed Viscosity Dye 405 + The cells were dead. Experimental results indicate that Atto 647 Project 2 can be used for multiplex imaging.

[0162] Example 2

[0163] Synthesis and Validation of Project 3

[0164] Based on the sequence of SEQ ID NO:3, the PS-binding compound derivative item 3 was synthesized using a standard Fmoc chemistry protocol. The crude peptide was then purified by HPLC, and its molecular weight was measured by mass spectrometry. FITC item 3 was conjugated using a standard azide-alkyne reaction chemistry protocol. Item 3 was synthesized by Innopep Inc.

[0165] Figure 2A-1 and Figure 2A-2 The molecular weight of item 3, measured by mass spectrometry, is shown. For example... Figure 2A-1 As shown, the molecular weight of linear item 3 is 1044.5 g / mol. The observed molecular weight of cyclic item 3 is 1026.5 g / mol, because one H2O molecule was eliminated during head-to-tail cyclization (see...). Figure 2A-2Experimental results confirmed that Project 3 was successfully synthesized. The purity of Project 3 is >95%.

[0166] To characterize FITC Project 3, U937 cells stimulated with IL-4 for 2 days were used in a Ca-free environment. 2+ Cells were simultaneously stained for 5 minutes in PBS buffer with 68 pM / test FITC Project 3, 28 pM / test Atto 647 Project 2, and 20 nM Sytox Blue, and then analyzed by NovoCyte Quanteon flow cytometry without washing.

[0167] like Figure 2B As shown, the staining patterns and frequencies of PS-positive dying cells and dead cells stained with FITC Project 3 and Atto 647 Project 2 were similar. Furthermore, PS-positive dying cells and dead cells stained with FITC Project 3 and Atto 647 Project 2 showed a high correlation.

[0168] Example 3

[0169] Synthesis and Verification of FITC Project 5

[0170] A fusion of a PS-binding compound and an arm compound coupled with FITC was synthesized, comprising: (1) synthesizing Item 3 according to the sequence of SEQ ID NO:3 using a standard Fmoc chemistry protocol; (2) synthesizing arm compound Item 4 according to the sequence of SEQ ID NO:4 using a standard Fmoc chemistry protocol; (3) coupling FITC containing NHSter to Item 4 using a standard amine-NHSter reaction chemistry protocol; and (4) then linking Item 3 to FITC-coupled Item 4 using a standard azide-alkyne reaction chemistry protocol. The crude peptide was then purified by HPLC, and its molecular weight was measured by mass spectrometry. FITC Item 5 was synthesized by Wuxi AppTec.

[0171] Figure 3A The molecular weight of FITC item 5, as measured by mass spectrometry, is shown. (Example) Figure 3A As shown, the observed molecular weight of item 5 is 3535.4 g / mol. Item 4 is linked to item 3, and each item 5 is coupled with three FITC molecules. Experimental results confirm the successful synthesis of FITC item 5. The purity is 94.7%.

[0172] To characterize FITC Project 5, one-day-old mouse thymocytes were subjected to calcium-free... 2+Cells were simultaneously stained with 5 nM SytoxRed and 14 pM FITC Project 5 or 68 pM FITC Project 3 in PBS for 5 minutes, without washing. Cells were then analyzed by NovoCyteQuanton flow cytometry. Figure 3A The data shown indicate that item 5 was coupled with three FITCs, and the staining intensity of FITC item 5 should be stronger than that of FITC item 3. To further compare the staining intensity of FITC item 5 and FITC item 3, overgrown U937 cells were stained parallel to each other with different concentrations of FITC item 5 and FITC item 3 for 5 minutes, without washing. easyCyte TM Flow cytometers (Luminex Corporation) are used to analyze cells.

[0173] like Figure 3B As shown, the staining patterns and frequencies of PS cells stained by FITC Project 5 and FITC Project 3 were similar in thymocytes of one-day-old C57BL / 6 mice.

[0174] like Figure 3C As shown, on overgrown U937 cells, the fluorescence staining intensity of FITC Project 5 was higher than that of FITC Project 3 at different concentrations. The optimal concentrations for FITC Project 5 and FITC Project 3 were 14 pM and 68 pM, respectively. easyCyte TM Flow cytometers (Luminex Corporation) are used to analyze cells.

[0175] Example 4

[0176] Synthesis and Validation of Project 7

[0177] Item 7, a fusion of a PS-binding compound and an arm compound, was synthesized, comprising: (1) synthesizing Item 3 according to the sequence of SEQ ID NO:3 using a standard Fmoc chemical protocol; (2) synthesizing Item 6 according to the sequence of SEQ ID NO:6 using a standard Fmoc chemical protocol; and (3) linking Item 6 to Item 3 using a standard azide-alkyne reaction chemical protocol. The crude peptide was purified by HPLC and its molecular weight was measured by mass spectrometry. Item 7 was synthesized by Innopep Inc.

[0178] like Figure 4A As shown, the observed molecular weight of item 7 is 2029 g / mol. Experimental results confirm that items 3 and 6 have been successfully linked. The purity of item 7 is >94%.

[0179] Phycoerythrin (PE) maleimide was conjugated to Project 7 using a standard thiol-maleimide reaction protocol, and PE Project 7 was purified by S-200 column fractionation. To characterize PE Project 7, a mixture of live and heat-shock-treated Jurkat cells was stained in pristine cell culture medium with 2.4 μg / 100 μL PE Project 7 and 5 nM Sytox Red or 28 pM / test Atto 647 Project 2 and 20 nM Sytox Blue in parallel for 15 min, followed by one wash. Cells were then analyzed by NovoCyte Quanteon flow cytometry.

[0180] like Figure 4B As shown, the staining patterns and frequencies of PS cells stained with PE Project 7 and Atto 647 Project 2 were similar on a mixture of live and heat-shock-treated Jurkat cells.

[0181] To verify whether Project 7 could be conjugated with a solid-phase carrier to consume PS-positive cells, maleimide-coated magnetic particles were conjugated to Project 7 using a standard thiol-maleimide chemistry protocol. The conjugated magnetic particles were washed and sonicated to remove aggregated particles. A mixture of freshly prepared and heat-shock-treated C57BL / 6 mouse thymocytes was then processed at 1 x 10⁻⁶ cells. 7 / mL was suspended in cell separation buffer (buffer composition: PBS, 0.5% FCS, and 2mM EDTA). Then 1x10 6 100 μL of cells were transferred to an FACS tube and incubated with 10 μL of Project 7 conjugated magnetic particles at room temperature for 15 minutes. The cells were then diluted to 3 mL with cell separation buffer and placed in a separator for 5 minutes. The suspended cells were then poured into a new collection tube. To further improve viable cell purity, the cells in the collection tube were placed in the separator for another 5 minutes, and the suspended cells were poured into another new collection tube. After both separations, the unseparated cells and the separated cells in the collection tubes were centrifuged and then resuspended in 100 μL of Ca-free buffer. 2+ The cells were then placed in PBS. The resuspended cells were then stained with 14 pM FITC and 5 nM Sytox Red for 5 minutes at room temperature, without washing. easyCyte TM Flow cytometers (Luminex Corporation) are used to analyze cells.

[0182] like Figure 4C As shown, the magnetic particles conjugated in Project 7 consumed PS-positive cells and increased the percentage of live cells from 53.3% to 96.27%, with a live cell yield of 84%.

[0183] The maleimide-containing oligonucleotide containing the sequence of SEQ ID NO:8 includes the PCR operation sequence of SEQ ID NO:9, the unique DNA barcode sequence of SEQ ID NO:10, and the capture sequence of SEQ ID NO:11 (polyA). Oligonucleotide Project 7 was conjugated using a standard thiol-maleimide chemistry protocol and purified by an S-200 column. To verify whether Oligonucleotide Project 7 could specifically bind to PS-positive cells, an Alexa 647-conjugated polyT was prepared, which hybridized to the complementary sequence of SEQ ID NO:11 (polyA). 50 pM of Oligonucleotide Project 7 was first complementary to 37.5 pM of Alexa 647-conjugated polyT at room temperature for 20 min, then incubated with different heterogeneous cell populations for an additional 15 min, followed by a wash. Cells stained with 28 pM / test Atto 647 Project 2 or 14 pM / test FITC Project 5 served as controls. 20 nM Sytox Blue was used to stain PS-positive dead cells. The cells were then analyzed using a NovoCyte Quanteon flow cytometer.

[0184] like Figure 4D As shown, the staining pattern and frequency of PS-positive cells stained with Alexa 647 poly-T oligonucleotide Project 7 on thymocytes of one-day-old C57BL / 6 mice were similar to those stained with Atto 647 Project 2 and FITC Project 5.

[0185] A mixture of live and heat-shock-treated Jurkat cells was stained in pristine cell culture medium with 50 pM pre-hybridized Alexa 647 poly-T oligonucleotide project 7 and 20 nM Sytox Blue or 14 pM FITC project 5 and 5 nM Sytox Red for 15 min, and then analyzed using a NovoCyte Quanteon flow cytometer without washing. The staining patterns and frequencies of PS-positive cells stained with Alexa 647 poly-T oligonucleotide project 7 and FITC project 5 were also comparable (see [link to original text]). Figure 4E ).

[0186] A mixture of live and heat-shocked Jurkat cells was simultaneously stained in pristine cell culture medium with 50 pM pre-hybridized Alexa 647 poly-T oligonucleotide Project 7 and 2 μL / 100 μL propidium iodide or 14 pM FITC Project 5 for 15 minutes. Then... easyCyte TM Flow cytometry (Luminex Corporation) is used to analyze cells. For example... Figure 4FAs shown, PS-positive cells stained with Alexa 647 poly-T oligonucleotide project 7 and propidium iodide simultaneously showed a high correlation. Furthermore, PS-positive cells stained with Alexa 647 poly-T oligonucleotide project 7 and FITC project 5 simultaneously also showed a high correlation. This experiment was repeated three times. (Source: [Original Source Name]) Figure 4D , Figure 4E and Figure 4F Data shows that oligonucleotide project 7 can bind to PS-positive cells.

[0187] Figure 4G The flowchart of single-cell sequencing is shown. Item 7, labeled with oligonucleotides, is mixed with different oligonucleotide-conjugated antibodies containing unique barcodes. This mixture is then incubated with a heterogeneous cell population followed by a wash. Individual cells are then captured into a microfluidic device, each cell co-encapsulated in a tiny droplet with a different barcoded microparticle by hybridizing a polyA capture sequence with complementary polyT beads. Once the cells are isolated into the droplets, they are lysed, releasing mRNA and hybridizing with primers. STAMPS are generated, amplified, and sequenced. Bioinformaticians can then perform biological data analysis, such as RNA sequencing, by using the STAMPS barcodes to infer the source cell of each transcript. PS-positive dying and dead cells are identified by unique barcodes and filtered out using pattern / sequence matching tools before complex biological data analysis. In this way, bioinformaticians will analyze only live cells, which effectively saves time and improves data quality.

Claims

1. A phosphatidylserine (PS) conjugated compound wherein the residue Trp (BODIPY) at position 6 of the amino acid sequence of SEQ ID NO:1 has been replaced by the natural amino acid Trp to remove fluorescence, and wherein the residue Gly at position 7 has been replaced by the amino acid Cys or the synthetic amino acid propargylglycine (Pra).

2. The phosphatidylserine (PS) binding compound according to claim 1, comprising the sequence of SEQ ID NO:2, wherein the amino acid Cys at position 7 is used to provide a thiol group for coupling with a maleimide-containing tag by means of a thiol-maleimide reaction.

3. The phosphatidylserine (PS) binding compound according to claim 2, wherein the maleimide-containing tag is used as a detectable unit, wherein the tag is selected from: fluorophores, solid-phase carriers, oligonucleotides, biotin, avidin, proteins, enzymes, and radionuclides.

4. The phosphatidylserine (PS) conjugated compound according to claim 2, wherein the maleimide-containing label is a maleimide-containing bifunctional Pol, wherein the bifunctional Pol is selected from: maleimide-Pol-maleimide, maleimide-Pol-thiol, maleimide-Pol-azide, maleimide-Pol-alkyne, maleimide-Pol-NHSter, maleimide-Pol-amine, and maleimide-Pol-COOH, wherein Pol is a polymer.

5. The phosphatidylserine (PS) conjugated compound according to claim 4, wherein Pol is selected from: (PEG) n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is an integer from 1 to 60, preferably from 1 to 24, and more preferably from 1 to 12.

6. The phosphatidylserine (PS) binding compound according to claim 1, comprising the amino acid sequence of SEQ ID NO:3, wherein the synthesized amino acid propargylglycine (Pra) is used to provide an alkyne functional group for coupling with an azide-containing tag by means of an azide-alkyne reaction.

7. The phosphatidylserine (PS) binding compound according to claim 6, wherein the azide-containing tag is used as a detectable unit, wherein the tag is selected from: fluorophores, solid-phase supports, oligonucleotides, biotin, avidin, proteins, enzymes, and radionuclides.

8. The phosphatidylserine (PS) conjugated compound of claim 6, wherein the azide-containing tag is an amino acid having an azide functional group, wherein the amino acid having an azide functional group is selected from: azido-lysine, azido-propargylglycine, azido-L-propargylglycine, azido-histidine, azido-tryptophan, azido-phenylalanine, azido-arginine, azido-glutamine, azido-glycine, azido-valine, and azido-alanine.

9. The phosphatidylserine (PS) binding compound according to claim 6, wherein the azide-containing tag serves as an azide-containing bifunctional connector selected from: azido-Pol-maleimide, azido-Pol-thiol, azido-Pol-alkyne, azido-Pol-azide, azido-Pol-amine, azido-Pol-NHSter, and azido-Pol-COOH, wherein Pol is a polymer.

10. The phosphatidylserine (PS) conjugated compound according to claim 9, wherein Pol is selected from: (PEG) n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is an integer from 1 to 60, preferably from 1 to 24, and more preferably from 1 to 12.

11. A fusion of a phosphatidylserine (PS) binding compound and an arm compound, wherein the PS binding compound is the PS binding compound according to any one of claims 2-10, and wherein the arm compound is capable of being linked to the Cys of the compound or the synthetic amino acid propargylglycine (Pra).

12. The fusion according to claim 11, wherein the PS binding compound is the PS binding compound according to any one of claims 6-10.

13. The fusion according to claim 11 or 12, wherein the arm compound comprises the amino acid sequence of SEQ ID NO:4, wherein the sequence of SEQ ID NO:4 provides a functional group for coupling with at least one tag containing a functional group.

14. The fusion according to claim 13, wherein Xa1 (azido) in the sequence of SEQ ID NO:4 is an amino acid having an azide functional group, wherein the amino acid having an azide functional group is selected from: azido-lysine, azido-propargylglycine, azido-L-propargylglycine, azido-histidine, azido-tryptophan, azido-phenylalanine, azido-arginine, azido-glutamine, azido-glycine, azido-valine, and azido-alanine.

15. The fusion according to claim 13 or 14, wherein Xa2 is an amino acid having an amine functional group and a carboxylic acid group, wherein the amino acid is selected from: Lys, Arg, His, Ser, Thr, Cys, Asn, Gln, Pra and Tyr.

16. The fusion according to any one of claims 13-15, wherein (Pol-Xa3) m These are independent variables, where Pol is selected from: (PEG) n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is an integer from 1 to 60, preferably from 1 to 24, and more preferably from 1 to 12.

17. The fusion according to claim 16, wherein the PEG is Fmoc-NH-PEG-COOH.

18. The fusion according to any one of claims 13-17, wherein Xa3 is an amino acid having a functional group for chemical labeling, wherein the functional group is selected from amines, thiols and alkynes, and wherein the amino acid is selected from Lys, Arg, His, Met, Cys and Pra, and wherein m is an integer from 1 to 10.

19. The fusion according to any one of claims 13-18, wherein the functional group-containing tag is used as a detectable unit, wherein the functional group is selected from: NHSter, maleimide and azide, and wherein the tag is selected from: fluorophore, oligonucleotide, solid support, biotin, avidin, protein, enzyme and radionuclide.

20. The fusion according to claim 11 or 12, wherein an arm compound comprising the sequence of SEQ ID NO:6 is added, wherein the sequence of SEQ ID NO:6 provides a functional group for coupling with a tag containing the functional group.

21. The fusion according to claim 20, wherein Xa1 (azido) in the sequence of SEQ ID NO:6 is an amino acid having an azide functional group, wherein the amino acid having an azide functional group is selected from: azido-lysine, azido-propargylglycine, azido-L-propargylglycine, azido-histidine, azido-tryptophan, azido-phenylalanine, azido-arginine, azido-glutamine, azido-glycine, azido-valine, and azido-alanine.

22. The fusion according to claim 19 or 20, wherein Xa2 is an amino acid having amine and carboxylic acid functional groups, wherein the amino acid is selected from: Lys, Arg, His, Ser, Thr, Cys, Asn, Gln, Pra and Tyr.

23. The fusion according to any one of claims 20-22, wherein Pol is selected from: (PEG) n (PEO) n (ε-caprolactam) n (CH2) n and [(PEG)] n (CH2) n ], where n is an integer from 1 to 60, preferably from 1 to 24, and more preferably from 1 to 12.

24. The fusion according to claim 23, wherein the PEG is Fmoc-NH-PEG-COOH.

25. The fusion according to any one of claims 20-24, wherein Xa3 is a functional group-containing amino acid, wherein the functional group is selected from: amines, thiols, alkynes, and wherein the amino acid is selected from: Cys, Pra, Met, Lys, Arg, and His.

26. The fusion according to any one of claims 20-25, wherein the tag containing a functional group serves as a detectable unit, wherein the functional group is selected from: NHSter, maleimide and azide, and wherein the tag is selected from: fluorophores, oligonucleotides, solid-phase carriers, biotin, avidin, enzymes and radionuclides.

27. The phosphatidylserine (PS) binding compound according to any one of claims 2-10, or the fusion compound according to any one of claims 11-26, wherein the tag is an oligonucleotide, the oligonucleotide being coupled to the PS binding compound to bind PS-positive cells, thereby distinguishing PS-positive cells from live cells by recognizing a unique DNA barcode sequence in single-cell sequencing (scRNA-seq), wherein the oligonucleotide comprises a PCR operation sequence for PCR amplification following the formation of a single-cell transcriptome (STAMP) attached to the microparticle, a unique DNA barcode sequence for PS-positive cells, and a capture sequence for binding its complementary sequence on a cell capture bead, thereby encapsulating each cell in a microdroplet, respectively, with a different barcode microparticle.

Images