Method for recognizing intracellular and extracellular G-quadruplex by using bacterium-derived aggregation-induced emission agent

Abstract

The invention relates to a method for recognizing a G-quadruplex (G4) in a cell by utilizing a G4 fluorescent probe 2-(2-hydroxy-6-methoxy-3-propionyl phenyl) quinazoline-4 (3H)-ketone (HMPQ). The method comprises the following steps: firstly, adding a fluorescent probe 2-(2-hydroxy-6-methoxy-3-propionyl phenyl) quinazoline-4 (3H)-ketone (HMPQ) into a cell; the probe has specific binding affinity to G-quadruplex, and can illuminate the non-standard nucleic acid structures without changing the conformation of the probe. G4 participates in chromatin tissue, gene regulation, and genomic stability, and appears to contribute to the growth and progression of cancer. By utilizing the unique fluorescence mechanism of HMPQ (i.e., excited state intramolecular proton transfer), the method can achieve label-free accurate detection of the G4 structure in the cell nucleus. The invention also relates to an unmarked kit for selectively detecting the G-quadruplex (G4) in the cell.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 550,624, filed February 7, 2024, which is incorporated herein by reference in its entirety, including any tables, figures, or images therein.

[0003] Description of Sequence Listing

[0004] The sequence listing for this application is labeled "SequenceListing.xml," was created on February 6, 2025, and is 19,598 bytes in size. The entire contents of the sequence listing are incorporated herein by reference in their entirety. Technical Field

[0005] The present application relates to the field of biomedicine. More specifically, the present application relates to a label-free method for identifying G4 in cells or cell nuclei. Background Art

[0006] G-quadruplexes (G4) have become a potential therapeutic target of great interest due to their potential roles in chromatin organization, gene regulation and genome stability, as well as their potential contribution to the treatment of various human diseases (see Ruggiero, E., & Richter, SN (2018). G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy. Nucleic acids research, 46(7), 3270-3283; Chambers, VS, Marsico, G., Boutell, JM, Di Antonio, M., Smith, GP, & Balasubramanian, S. (2015). High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nature biotechnology, 33(8), 877-881; -Hertsch, R., Di Antonio, M., & Balasubramanian, S. (2017). DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential. Nature reviews Molecular cell biology, 18 (5), 279-284). Although many G4 ligands have been designed to regulate these biological switches (see Li, Q., Xiang, JF, Yang, QF, Sun, HX, Guan, AJ, & Tang, YL (2013). G4LDB: a database for discovering and studying G-quadruplex ligands. Nucleic acids research, 41 (D1), D1115-D1123), there is still a lack of comprehensive understanding of the exact nature of G4. This challenge is mainly due to the lack of effective probes, so G4 in cells, especially in the nucleus, is difficult to detect and analyze.

[0007] The discovery of aggregation-induced emission (AIE) has prompted the identification and design of many small molecules and polymer materials with AIE properties, which are usually called AIE luminescent agents (AIEgen) (see Mei, J., Leung, NL, Kwok, RT, Lam, JW, & Tang, BZ (2015). Aggregation-induced emission: together we shine, united we soar!. Chemical reviews, 115(21), 11718-11940; Hong, Y., Lam, JW, & Tang, BZ (2011). Aggregation-induced emission. Chemical Society Reviews, 40(11), 5361-5388; Mei, J., Hong, Y., Lam, JW, Qin, A., Tang, Y., & Tang, BZ (2014). Aggregation-induced emission: the whole is more brilliant than the parts. Advanced materials, 26(31),5429-5479).With their excellent optical properties, these materials have been widely used in many fields such as bioimaging, optoelectronic devices and biochemical sensors (see Ding, D., Li, K., Liu, B., & Tang, BZ (2013). Bioprobes based on AIE fluorogens. Accounts of chemical research, 46(11), 2441-2453; Wang, H., Li, Q., Alam, P., Bai, H., Bhalla, V., Bryce, MR, ... & Tang, BZ (2023). Aggregation-induced emission (AIE), life and health. ACS nano, 17(15), 14347-14405; Hu, F., & Liu, B. (2016). Organelle-specific bioprobes based on fluorogens with aggregation-induced emission (AIE) characteristics. Organic & biomolecular chemistry, 14(42),9931-9944; Long, R., Tang, C., Xu, J., Li, T., Tong, C., Guo, Y.,… & Wang, D. (2019). Novel natural myricetin with AIE and ESIPT characteristics for selective detection and imaging of superoxide anions in vitro and in vivo. Chemical Communications, 55(73),10912-10915). However, the chemical synthesis of AIEgens faces many challenges, such as limited structural diversity, restricted physicochemical properties, environmental issues, high cost, and uncertain biocompatibility (Cai, XM, Lin, Y., Li, Y., Chen, X., Wang, Z., Zhao, X.,...& Tang, BZ (2021). BioAIEgens derived from rosin: how does molecular motion affect their photophysical processes in solid state?. Nature communications, 12(1), 1773).On the contrary, natural products have become an attractive alternative due to their strong biocompatibility and rich bioactive properties. In recent decades, the complex structure and chemical diversity of natural products have brought many inspirations to chemists (Nicolaou, KC, Hale, CR, Nilewski, C., & Ioannidou, HA (2012). Constructing molecular complexity and diversity: total synthesis of natural products of biological and medicinal importance. Chemical Society Reviews, 41(15), 5185-5238).It is worth noting that some AIEgens (such as curcumin, quercetin, berberine and coumarin) have been isolated from plants (see Shen, Y., Nie, C., Zhu, C., Zheng, Z., & Wu, Y. (2022). Aggregation-induced emission fluorophore-incorporated curcumin-based ratiometric nanoprobe for hypochlorite detection in food matrices. Journal of Agricultural and Food Chemistry, 70(30), 9577-9583; He, T., Niu, N., Chen, Z., Li, S., Liu, S., & Li, J. (2018). Novel quercetin aggregation-induced emission luminogen (AIEgen) with excited-state intramolecular proton transfer for in vivo bioimaging. Advanced Functional Nanotechnology, 2018). Materials, 28(11),1706196; Gu, Y., Zhao, Z., Su, H., Zhang, P., Liu, J., Niu, G.,... & Tang, BZ (2018). Exploration of biocompatible AIEgens from natural resources. Chemical science, 9(31), 6497-6502; Chen, SS, Wang, H., Wu, B., Li, Q., Gong, J., Zhao, YL,... & Tang, BZ (2023). Natural Coumarin Isomers with Dramatically Different AIE Properties: Mechanism and Application. ACS Central Science, 9(5), 883-891).

[0008] In addition, small molecule secondary metabolites produced by bacteria have many advantages, including short growth cycle, suitability for large-scale fermentation, easy analysis and engineering of biosynthetic pathways, and relatively simple separation and purification processes. However, in the field of optically active and luminescent materials, the exploration of bacterial metabolites as another abundant natural resource is still relatively limited (see Nguyen, PQ, Courchesne, NMD, Duraj-Thatte, A., Praveschotinunt, P., & Joshi, NS (2018). Engineered living materials: prospects and challenges for using biological systems to direct the assembly of smart materials. Advanced Materials, 30(19), 1704847; Lane, AL, & Moore, BS (2011). A sea of biosynthesis: marine natural products meet the molecular age. Natural product reports, 28(2), 411-428; Berdy, J. (2005). Bioactive microbial metabolites. The Journal of antibiotics, 58(1), 1-26). However, bacterial metabolites from the unique high-salt, high-pressure, and low-temperature marine environment are attractive sources of drug leads and biochemicals (see Montaser, R., & Luesch, H. (2011). Marine natural products: a new wave of drugs?. Future medicinal chemistry, 3(12), 1475-1489). Therefore, given the importance of G4 in biology and medicine, there is an urgent need to develop an efficient method to explore G4 in cells using highly specific and readily available AIEgen probes. Summary of the Invention

[0009] This application discloses a label-free method for identifying intracellular or intranuclear G4s. In some embodiments, the method includes obtaining a fluorescent probe that selectively binds to intracellular G4 structures containing nucleic acids. In some embodiments, the fluorescent probe is contacted with the nucleic acid within the cell. When the fluorescent probe binds to the G-quadruplex structure, the probe emits a fluorescent signal that can be detected and analyzed, thereby enabling the precise identification of intracellular G4s.

[0010] In some embodiments, fluorescent probes include but are not limited to 2-(2-hydroxy-6-methoxy-3-propionylphenyl)quinazolin-4(3H)-one (HMPQ). In a preferred embodiment, the fluorescent probe is HMPQ. In a more preferred embodiment, the probe used comprises high-purity HMPQ. In some embodiments, the purity grade of HMPQ includes at least DNA grade, proteomics grade, molecular biology grade, or ultrapure grade, depending on the specific technology or experimental type using HMPQ. Selecting the purity grade of HMPQ is within the ability of one of ordinary skill in the art. In a most preferred embodiment, the HMPQ used is in the form of a Type 2 conformational isomer polymorph.

[0011] In some embodiments, HMPQ selectively binds to G4 structures within cells with a peak emission wavelength of about 497 nm to about 500 nm. In some embodiments, HMPQ is able to bind to G4 without altering its conformation. Furthermore, in certain embodiments, HMPQ is non-cytotoxic to mammalian cells and cell lines.

[0012] In some embodiments, HMPQ can be used in aqueous media.In some embodiments, binding of HMPQ to G4 can be detected in subcellular compartments including, but not limited to, the nucleus, cytoplasm, and mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figures 1A-1E : Figures 1A-1C The fermentation, separation and structure elucidation process of the natural bacterial metabolite AIE compound HMPQ are demonstrated. Figure 1A The fermentation process of AIE compounds, which are natural bacterial metabolites, was demonstrated; Figure 1B The isolation process of this natural bacterial metabolite AIE compound is demonstrated; Figure 1C The structural elucidation of the natural bacterial metabolite AIE compound is shown. Figure 1D The fluorescence image of HMPQ crystal sample under 365nm UV light is shown; Figure 1E The normalized absorption spectrum of HMPQ in solid thin film and the photoluminescence spectrum of HMPQ in crystalline state are shown with an excitation wavelength of 320 nm.

[0014] Figures 2A-2C : Figure 2A Shows photos of HMPQ in different solvent systems taken under 365 nm UV light from a handheld UV lamp; Figure 2B demonstrated the enol-keto conversion during excited-state intramolecular proton transfer (ESIPT); Figure 2CThe normalized photoluminescence (PL) spectra of HMPQ in different solvent systems are shown with an excitation wavelength of 340 nm and a solution concentration of 10 μM.

[0015] Figures 3A-3D : Figure 3A Photoluminescence (PL) spectra are shown; Figure 3B The relative PL intensity and emission peak wavelength are shown; and Figure 3C The quantum yield of films made from polymethyl methacrylate (PMMA) mixtures with different mass percentages of HMPQ is shown; Figure 3D The quantum yield of HMPQ in different sample states is shown. These films are made of a mixture of HMPQ and PMMA, where wt.% represents the weight percentage of HMPQ, and the excitation wavelength is 320 nm.

[0016] Figures 4A-4D : Figure 4A and Figure 4C Space-filling models of distorted and planar polymorphs and their molecular packing are shown; Figure 4B It shows that under ultraviolet light (excitation wavelength: 385 nm), the crystal morphologies of HMPQ with different conformations exhibit different photoluminescence quantum yields; Figure 4C The space-filling model of the planar polymorph and its molecular packing are shown; Figure 4D Density functional theory (DFT) analysis of the Gibbs free energies of two conformations of HMPQ calculated at the M062x / TZVP level of theory in the IEFPCM model (methanol) is shown.

[0017] Figures 5A-5E : Figure 5A Shown are the photoluminescence (PL) spectra of HMPQ mixed with different G4 solutions (50 μM, 400 μL) in 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0); Figure 5B shows the PL spectra of HMPQ in buffers containing different amounts of c-kit2; Figure 5C The Stern-Volmer plot of relative intensity (I / I0) versus c-kit2 concentration is shown, where I0 is the PL intensity in the absence of c-kit2; Figure 5D shows the fluorescence images taken under 365 nm UV illumination; Figure 5E Schematic representation of the working mechanism by which HMPQ lights up G4 but not other oligonucleotides.

[0018] Figures 6A-6C : Figure 6AShown are nuclear magnetic resonance (NMR) titrations of HMPQ with c-kit2 G4 at different molar ratios: 0.05 mM c-kit2 and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0); Figure 6B The circular dichroism (CD) spectra of HMPQ and c-kit2 G4 in the same buffer are shown, wherein the CD spectra confirm its non-destructive effect on the native conformation of c-kit2 G4; Figure 6C Shown are confocal images of HeLa cells treated with HMPQ, where G4 was detected by the G4 structure-specific antibody BG4 with an overlap coefficient of 0.943.

[0019] Figure 7 The high resolution mass spectrum (HRMS) of HMPQ is shown (C 18 H 15 N2O4 - , calculated value: 323.1037, measured value: 323.1035, error 0.62ppm).

[0020] Figure 8 Shown are the high performance liquid chromatography (HPLC) spectrum (30% acetonitrile-water isocratic elution, observation at 254 nm) and UV spectrum of HMPQ.

[0021] Figure 9 Showing HMPQ 1 H NMR spectrum (500 MHz, DMSO-d6). 1 H NMR (500MHz, DMSO-d6) δ12.97 (s, 1H), 12.41 (s, 1H), 8.16 (dd, J=7.9, 1.6Hz, 1H), 8.14 (d, J=9.1Hz, 1H), 7.83 (ddd, J=8.5, 7.2, 1.6Hz, 1H ), 7.70-7.63 (m, 1H), 7.55 (ddd, J=8.1, 7.1, 1.2Hz, 1H), 6.81 (d, J=9.1Hz, 1H), 3.86 (s, 3H), 3.12 (q, J=7.2Hz, 2H), 1.12 (t, J=7.2Hz, 3H).

[0022] Figure 10 Showing HMPQ 13 C NMR spectrum (126 MHz, DMSO-d6). 13C NMR (126MHz, DMSO-d6) δ206.26, 162.82, 161.79, 160.94, 149.06, 148.84, 134.46, 13 4.28, 127.19, 126.84, 125.81, 121.25, 113.60, 111.80, 103.15, 56.46, 31.11, 8.29.

[0023] Figure 11 Correlation spectra (COSY) of HMPQ (500 MHz, DMSO-d6) are shown.

[0024] Figure 12 Heteronuclear single quantum coherence (HSQC) spectroscopy of HMPQ (500 MHz, DMSO-d6) is shown.

[0025] Figure 13 The heteronuclear multiple bond correlation (HMBC) spectrum of HMPQ (500 MHz, DMSO-d6) is shown.

[0026] Figure 14 The normalized absorption spectra of HMPQ in solution and thin film states are shown, with the solution concentration being 10 μM.

[0027] Figure 15 The molecular packing of the type 1 conformer is shown.

[0028] Figure 16 The inter- and intramolecular interactions of the type 1 conformer are demonstrated.

[0029] Figure 17 The molecular packing of the type 2 conformer is shown.

[0030] Figure 18 The inter- and intramolecular interactions of type 2 conformers are demonstrated.

[0031] Figure 19 Shown are NMR titrations of HMPQ with c-myc (SEQ ID NO: 1) at different molar ratios: 0.05 mM c-myc and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0032] Figure 20 Shown are NMR titrations of HMPQ with c-kit1 (SEQ ID NO: 2) at different molar ratios: 0.05 mM c-kit1 and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0033] Figure 21Shown are NMR titrations of HMPQ with c-kit2 (SEQ ID NO: 3) at different molar ratios: 0.05 mM c-kit2 and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0034] Figure 22 Shown are NMR titrations of HMPQ with htel23 (SEQ ID NO: 4) at different molar ratios: 0.05 mM htel23 and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0035] Figure 23 Shown are NMR titrations of HMPQ with htel21_T18 (SEQ ID NO: 5) at different molar ratios: 0.05 mM htel21_T18 and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0036] Figure 24 Shown are NMR titrations of HMPQ with (G4C2)4 (SEQ ID NO: 6) at different molar ratios: 0.05 mM (G4C2)4 and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0037] Figure 25 Shown are NMR titrations of HMPQ with r(G4C2)2 (SEQ ID NO:7) at various molar ratios: 0.05 mM r(G4C2)2, 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0038] Figure 26 Shown are NMR titrations of HMPQ with rTerra (SEQ ID NO: 8) at different molar ratios: 0.05 mM rTerra and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0039] Figure 27 Shown are NMR titrations of HMPQ with double-stranded (ds)DNA G4 (SEQ ID NO: 9) at different molar ratios: 0.05 mM dsDNA and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0040] Figure 28Shown are NMR titrations of HMPQ with single-stranded (ds)DNA G4 (SEQ ID NO: 10) at different molar ratios: 0.05 mM dsDNA and 20 mM KH2PO4 buffer (70 mM KCl, 10% D2O, pH 7.0).

[0041] Figure 29 CD spectra of c-kit1 G4 before and after HMPQ addition are shown, which confirm that HMPQ does not destroy the native conformation of c-kit1 G4.

[0042] Figure 30 Circular dichroism (CD) spectra of c-myc G4 before and after HMPQ addition are shown, and the CD spectra confirm that HMPQ does not destroy the original conformation of c-myc G4.

[0043] Sequence Description

[0044] SEQ ID NO:1: TGA GGG TGG GTA GGG TGG GTA A(c-myc)DNA G4

[0045] SEQ ID NO:2: AGG GAG GGC GCT GGG AGG AGG G(c-kit1)DNA G4

[0046] SEQ ID NO:3: CGG GCG GGC GCG AGG GAG GGG(c-kit2)DNA G4

[0047] SEQ ID NO:4: TAG GGT TAG GGT TAG GGT TGG GG(htel23)DNA G4

[0048] SEQ ID NO:5: GGG AGG CGT GGC CTG GGC GGG ACT GGG G(LTR-III)DNA G4

[0049] SEQ ID NO:6: GGG TTA GGG TTA GGG TTT GGG(htel21 T18)DNA G4

[0050] SEQ ID NO:7: GGG GCC GGG GCC GGG GCC GGG GCC(G4C2)4DNA G4

[0051] SEQ ID NO:8: GGG GCC GGG GCC(G4C2)2RNA G4

[0052] SEQ ID NO:9: UAG GGU UAG GGU (Terra)RNA G4

[0053] SEQ ID NO:10: GCT TTA AAA AGT AAG TT (AT-rich) dsDNA

[0054] SEQ ID NO:11: TTC GCG CGC GTT TTC GCG CGC G (ds22) dsDNA SEQ ID NO:12: GCG CGC GCG CGC GCG C (d(GC)8) dsDNA

[0055] SEQ ID NO:13: CTA GGG CCT AG (ds11) dsDNA

[0056] SEQ ID NO:14: GGC CCT TTT TTT TCT AG (T-rich) ssDNA

[0057] SEQ ID NO:15: CCT TCC CCA CCC TCC CCA CCC TCC CCA (s-myc) ssDNA

[0058] SEQ ID NO:16: CCC TAA CCC TAA CCC TAA CCC T (s-Tel) ssDNA SEQ ID NO:17: GCG CGC GCG CGC GCG C (Z-DNA) ssDNA

[0059] SEQ ID NO:18: CGG GCG GGC GCG AGT GAG GGG (c-kit2_T15) ssDNA

[0060] SEQ ID NO:19: CGC GGT GTC CGC G (DNA hairpin) ssDNA

[0061] SEQ ID NO:20: GGA GAU CGC ACU CCA (RNA hairpin) ssRNA Detailed implementation manners

[0062] Selected definitions

[0063] As used herein, the singular forms "a," "an," "said," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, when the terms "including," "includes," "having," "have," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The transition words / phrases (and any grammatical variations thereof) "comprising," "comprises," "consisting essentially of," "consisting essentially of," "consisting of," and "consisting of" are used interchangeably.

[0064] The phrase "consisting essentially of" or "composed essentially of" indicates that the claimed aspect encompasses embodiments including the specified materials or steps and those embodiments that do not materially affect the basic and novel characteristics of the claimed aspect.

[0065] The term "about" refers to an acceptable error range for a particular value determined by one of ordinary skill in the art, which error range depends in part on the manner in which the value is measured, i.e., the limitations of the measurement system. In the context of compositions involving amounts of ingredients and using the term "about," these compositions contain a specified amount of ingredients with a variation (error range) within the range of 0-10% of that value (X ± 10%). In other contexts, the term "about" means a variation (error range) of 0-10% (X ± 10%) centered around a given value. Obviously, this variation represents a range of up to 10% above or below a given value, such as X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0066] In the present disclosure, in order to avoid lengthily listing and describing each value in the scope, the range notation of abbreviation is adopted. Any appropriate value in the range can be selected as needed as the upper limit, lower limit or endpoint value of the scope. For example, the scope 0.1-1.0 represents endpoint values 0.1 and 1.0, and intermediate values 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and all intermediate ranges included in 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0 etc. It is envisaged that there are values of at least two significant figures in the scope, for example, the scope 5-10 represents all values between 5.0 and 10.00 and between 5.00 and 10.00, including endpoint values. When using scope in this article, the combination and sub-combination (for example, the sub-range in the disclosed range) and specific embodiments thereof of the scope are clearly included.

[0067] As used herein, the term "nucleic acid" or "polynucleotide" refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and its single-stranded or double-stranded form. Unless otherwise specified, the term encompasses those nucleic acids comprising known analogs of natural nucleotides, which have a binding performance similar to the nucleic acid mentioned and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), complementary sequences, and sequences specifically indicated. The term "nucleic acid" is used interchangeably with gene, cDNA, and gene-encoded mRNA.

[0068] As used herein, the terms "oligonucleotide" and "oligo" are used interchangeably to describe a short single strand of synthetic DNA or RNA, for example, a sequence of about 5 to about 500 nucleic acid bases.

[0069] As used herein, the term "gene" refers to a DNA segment involved in producing a polypeptide chain; it includes regions before and after the coding region (leader and trailer regions) involved in the transcription / translation and regulation of the gene product, as well as intervening sequences (introns) between individual coding segments (exons).

[0070] In this application, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0071] The term "label" and similar terms refer to compositions that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, labels include fluorescent dyes (fluorophores), luminescent agents, electron-dense reagents, enzymes (e.g., enzymes commonly used in enzyme-linked immunosorbent assays (ELISA)), biotin, enzymes acting on substrates (e.g., horseradish peroxidase), digoxigenin, 32P and other isotopes, haptens, and proteins that can be made detectable by, for example, incorporating fluorescent labels into peptides or for detecting antibodies that specifically react with peptides. The term includes combinations of single labeling agents, for example, combinations of fluorophores that provide unique detectable characteristics at a specific wavelength or wavelength combination. In the context of detecting nucleic acids (e.g., target sequences), the probes of the present application are fluorescent and therefore unlabeled, i.e., do not require secondary labeling.

[0072] As used herein, an "isolated" or "purified" compound is substantially free of other compounds. In certain embodiments, the purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the compound of interest. For example, the purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) by weight of the desired compound. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis.

[0073] "Decrease" refers to a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0074] By "increase" is meant a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0075] As used herein, the term "organism" includes viruses, bacteria, fungi, plants, and animals. Examples of other organisms are known to those of ordinary skill in the art, and such examples are within the scope of the materials and methods disclosed herein. The assays described herein can be used to analyze any genetic material obtained from any organism.

[0076] As used herein, the terms "genome," "genomic," "genetic material," or other grammatical variations thereof refer to genetic material from any organism. The genetic material can be viral genomic DNA or RNA, nuclear genetic material (such as genomic DNA), or genetic material present in organelles (such as mitochondrial DNA or chloroplast DNA). It can also represent genetic material from a natural or artificial mixture or a mixture of genetic material from several organisms.

[0077] As used herein, a "target nucleic acid" or "target sequence" is a G4 or G4 structure in the genetic material of an organism.

[0078] When used to describe two sequences, the term "hybridizes to..." means that the two sequences are sufficiently complementary to each other to allow nucleotide base pairing between the two sequences. Sequences that hybridize to each other can be completely complementary, but there can also be mismatches to some extent. Therefore, the sequences at the 5' and 3' ends of the extension and ligation probes described herein can have some mismatches with the corresponding target sequences at the 5' and 3' ends of the target genomic region, as long as the extension and ligation probes are able to hybridize with the target sequence to promote capture of the target genomic region. Depending on the stringency of the hybridization, up to about 5% to 20% mismatch between the two complementary sequences will allow hybridization between the two sequences. Generally, high stringency conditions have higher temperatures and lower salt concentrations, while low stringency conditions have lower temperatures and higher salt concentrations. High stringency conditions for hybridization are preferred, and therefore, the sequences at the 3' and 5' ends of the extension and ligation probes are preferably completely complementary to the corresponding target sequences at the 3' and 5' ends of the target genomic region.

[0079] In addition, two sequences corresponding to each other (e.g., target sequence and primer sequence) have at least 90% sequence identity, preferably at least 95% sequence identity, even more preferably at least 97% sequence identity, and most preferably at least 99% sequence identity over at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the sequence. Alternatively, the two sequences corresponding to each other are reverse complementary to each other and have at least 90% perfect match, more preferably at least 95% perfect match, even more preferably at least 97% perfect match, and most preferably at least 99% perfect match over at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the sequence. Thus, the two sequences corresponding to each other can hybridize to each other or to a common reference sequence over at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the sequence. Preferably, the two sequences corresponding to each other are 100% identical over the entire length of the two sequences or are 100% reverse complementary over the entire length of the two sequences.

[0080] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0081] Other features and advantages of the present application will be apparent from the following description of its preferred embodiments, and from the claims.

[0082] All references cited herein are incorporated by reference in their entirety.

[0083] The present application relates to a novel label-free method for detecting G-quadruplexes (G4) in cells using an AIEgen. In some embodiments, the method utilizes an AIEgen fluorescent probe that can selectively bind to G4. In some embodiments, the method utilizes an AIEgen fluorescent probe that can detect G4 without the need for secondary labeling. In a preferred embodiment, the AIEgen used as a probe is 2-(2-hydroxy-6-methoxy-3-propionylphenyl)quinazolin-4(3H)-one (HMPQ), which has formula (I):

[0084]

[0085] wherein R1-R6 are independently selected from the group consisting of:

[0086] (a) an alkyl group;

[0087] (b) alkenyl;

[0088] (c) alkynyl;

[0089] (d) halogen-substituted alkyl groups;

[0090] (e) hydroxyl group (-OH);

[0091] (f) ether bond (-O-);

[0092] (g) carbonyl group;

[0093] (h) carboxyl group (-COOH);

[0094] (i) Ester (-COOR);

[0095] (j) amino group (-NH2);

[0096] (k) nitro (-NO2);

[0097] (1) sulfonic acid group (-SO3H); and

[0098] (m) Mercapto (-SH).

[0099] In certain embodiments, the alkyl group includes at least one of a methyl group (—CH3) (e.g., toluene (C6H5-CH3)), an ethyl group (—C2H5 or —CH2CH3) (e.g., ethylbenzene (C6H5-CH2CH3)), a propyl group (—C3H7) (e.g., n-propyl (—CH2CH2CH3) and isopropyl (—CH(CH3)2)), or a butyl group (—C4H9) (e.g., n-butyl (—CH2CH2CH2CH3), sec-butyl (—CH2CH(CH3)2), isobutyl (—CH2CH2(CH3)), and tert-butyl (—C(CH3)3).

[0100] In certain embodiments, the alkenyl group includes at least one of a vinyl group (-CH=CH2) (e.g., vinyl chloride (CH2=CHCl) of the monomer polyvinyl chloride (PVC)), a propenyl group (-CH2-CH=CH2), or an allyl group (-CH=CH-CH3) (e.g., allyl alcohol (CH2=CH-CH2OH)).

[0101] In certain embodiments, alkynyl groups include ethynyl (—C≡CH) (eg, acetylene (C 2 H 2 )).

[0102] In certain embodiments, the halogen-substituted alkyl group includes at least one of fluorine-substituted (-CH2F, -CF3) (e.g., trifluoromethane (CHF3)), chlorine-substituted (-CH2Cl, -CCl3, etc.) (e.g., chloroform (CHCl3)), bromine-substituted (e.g., -CH2Br, -CBr3), or iodine-substituted (e.g., -CH2I, -CI3).

[0103] In certain embodiments, the hydroxyl group (-OH) comprises at least one of an alcohol (eg, CH3OH, ethanol C2H5OH) or a phenol (eg, C6H5OH).

[0104] In certain embodiments, the ether linkage group (—O—) includes at least one of CH 3 —O—CH 3 or diethyl ether (C 2 H 5 —O—C 2 H 5 ).

[0105] In certain embodiments, the carbonyl group includes an aldehyde (-CHO) (eg, formaldehyde (HCHO) and acetaldehyde (CH3CHO)).

[0106] In certain embodiments, a keto group (—CO—) includes, for example, acetone (CH 3 —CO—CH 3 ).

[0107] In certain embodiments, the carboxyl group (-COOH) includes, for example, formic acid (HCOOH), acetic acid (CH3COOH).

[0108] In certain embodiments, the ester group (-COOR) includes, for example, ethyl acetate (CH3COOC2H5).

[0109] In certain embodiments, the amino group (-NH2) includes an amine compound (eg, methylamine (CH3NH2)).

[0110] In certain embodiments, the nitro group (-NO2) includes a nitro compound (eg, nitrobenzene (C6H5NO2)).

[0111] In certain embodiments, the sulfonic acid group (-SO3H) includes a sulfonic acid compound.

[0112] In certain embodiments, the sulfhydryl group (—SH) includes a thiol compound (eg, ethanethiol (C 2 H 5 SH)).

[0113] In a preferred embodiment, a synthetic HMPQ homologue 310 is provided, wherein R1 and R3-R6 are hydrogen atoms, and R2 is a hydroxyl group (-OH). In some embodiments, the HMPQ homologue 310 emits blue light under ultraviolet light.

[0114] Traditional fluorescent probes rely on hydrophobic aromatic rings and π-conjugated chromophores (see Hong, Y., M., Lam, JW, Li, Z., Sin, KK, Dong, Y., ... & Tang, BZ (2008). Label-free fluorescent probing of G-quadruplex formation and real-time monitoring of DNA folding by a quaternized tetraphenylethene salt with aggregation-induced emission characteristics. Chemistry-A European Journal, 14(21), 6428-6437). However, they tend to aggregate in hydrophobic pockets when binding to nucleic acid chains in an aqueous environment, often leading to quenching of emission light. In contrast, AIEgens perform well in aqueous media and can avoid quenching caused by aggregation. In a preferred embodiment, photoluminescence and NMR titration experiments clearly show that HMPQ molecules can selectively bind to specific G4, while having no interaction with conventional double-stranded, single-stranded or hairpin structured nucleotides. In some embodiments, binding to G4 limits the mobility of HMPQ molecules, resulting in a significant enhancement of fluorescence intensity. CD spectroscopy confirmed that HMPQ maintained the original conformation of G4, and its high biocompatibility enabled G4 to be observed in the cell nucleus. In some embodiments, the method of the present application significantly expands the application of AIE active molecules in G4 staining, which is a rarely reported AIEgen application field (see Wang, KN, Liu, LY, Mao, D., Hou, MX, Tan, CP, Mao, ZW, & Liu, B. (2022). A Nuclear-Targeted AIE Photosensitizer for Enzyme Inhibition and Photosensitization in Cancer Cell Ablation. Angewandte Chemie International Edition, 61 (15), e202114600), and can observe the illuminated G4 in the cell without the need for secondary labeling.

[0115] In certain embodiments, HMPQ is suitable for use in living cells, as previously described (see Qin, W., Alifu, N., Lam, JWY, Cui, Y., SU.H., LG, Qian, J., & Tang BZ (2020) Facile Synthesis of Efficient Luminogens with AIE Features for Three-Photon Fluorescence Imaging of the Brain through the Intact Skull. Advanced Materials, 32(23), e202000364). In some embodiments, nanoparticles containing HMPQ can be used to detect G4 in living cells by confocal microscopy.

[0116] In certain embodiments, HMPQ is the first bio-AIE luminescent molecule (BioAIEgen) isolated from bacterial metabolites and subjected to careful single crystal structure analysis and rigorous photophysical testing. The fluorescence of HMPQ originates from the restriction of intramolecular and intermolecular motion (see Mei, J., Hong, Y., Lam, JW, Qin, A., Tang, Y., & Tang, BZ (2014). Aggregation-induced emission: the whole is more brilliant than the parts. Advanced materials, 26 (31), 5429-5479), and HMPQ exhibits an intrinsic feature called excited state intramolecular proton transfer (ESIPT) (see Lipps, HJ, & Rhodes, D. (2009). G-quadruplex structures: in vivo evidence and function. Trends in cell biology, 19 (8), 414-422). ESIPT causes a significant Stokes shift, which makes HMPQ an excellent candidate for a small molecule fluorescent probe. In preferred embodiments, the high biocompatibility of HMPQ further facilitates the visualization of G4 within the cell nucleus.

[0117] In a preferred embodiment, HMPQ exhibits specific binding affinity for G4, which is a non-canonical secondary structure found in guanine-rich regions of DNA or RNA, such as telomeres, promoters, and untranslated regions (UTRs) (see Balasubramanian, S., & Neidle, S. (2009). G-quadruplex nucleic acids as therapeutic targets. Current opinion in chemical biology, 13(3), 345-353; Cheng, A., Liu, C., Ye, W., Huang, D., She, W., Liu, X., & Qian, PY (2022). Selective c9orf72g-quadruplex-binding small molecules ameliorate pathological signatures of als / ftd models. Journal of Medicinal Chemistry, 65(19), 12825-12837). These structures are of great significance as potential therapeutic targets.

[0118] In one example, the term "quencher" refers to a substance that is capable of reducing the emission of a fluorescence donor when placed in proximity to the fluorescence donor. In preferred embodiments, the quencher is within 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide bases of the fluorescent label. Fluorescence is quenched when the fluorescence emitted by the fluorophore is detectably reduced, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more.

[0119] In certain embodiments, the concentration of the fluorescent probe in the compositions and methods of use is about 0.01 μM to about 1000 μM, about 0.1 μM to about 100 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 10 μM, or about 0.5 μM to about 3 μM. In certain embodiments, the concentration of the fluorescent probe is about 0.01 μM, about 0.1 μM, 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, or about 3 μM.

[0120] In certain embodiments, HMPQ exhibits fluorescence at approximately 500 nm, which has the advantage of being within the visible spectrum and can be detected using existing photomultiplier tubes. In some embodiments, the visible fluorescence signal can be seen with the naked eye or photographed with a smartphone.

[0121] In certain embodiments, the compositions of the present application may include other compounds, such as antibodies, fluorophores, oligonucleotides, preservatives, buffers, and any combination thereof. These compounds may be added to the composition in an amount of 0.01% to 99.9%, 0.1% to 90%, 0.5% to 80%, 0.75% to 70%, 1.0% to 50%, 1.5% to 25%, or 2.0% to 15% by weight relative to the total composition.

[0122] Materials and methods

[0123] method

[0124] General information

[0125] Streptomyces chrestomyceticus strain BCC24770 was purchased from the Thailand Bioresource Research Center. All oligonucleotides were purchased from Integrated DNA Technologies (Singapore). NMR spectra were recorded at 25°C on a 500M or 800M Varian NMR spectrometer. HRMS data were recorded on a Waters Xevo G2-XS QTOF mass spectrometer. HPLC analysis or preparation was performed using a Waters HPLC system (Waters 2695 Separations Module; Milford, MD, USA) and monitored under a Waters 2998 photodiode array detector. Semi-preparative HPLC was performed using a Phenomenex Luna C18 column (250 mm × 10 mm, particle size 5 μm). Single crystal XRD data were collected on a Rigaku-Oxford Diffraction SupernovaDual Atlas diffractometer. Crystal images were taken under a Nikon ECLIPSE Ts2R fluorescence microscope. CD spectra were collected using a Chirascan circular dichroism (CD) spectrometer. UV-visible absorption spectra were obtained using a Shimadzu UV-2600 spectrophotometer (medium scan rate, 2 cm pathlength quartz cuvette). Photoluminescence spectra were recorded on an Edinburgh FS5 fluorescence spectrometer. Absolute quantum yields were collected using an integrating sphere on a Hamamatsu Quantum Yield Spectrometer C11347 Quantaurus. All digital photographs were recorded on a Canon EOS 7D camera. A Leica SP8 confocal microscope was used for cell imaging.

[0126] Preparation procedure

[0127] Similar to our previous process (see Ye, W., Lui, ST, Zhao, Q., Wong, YM, Cheng, A., Sung, HHY, & Huang, P. (2023). Novel marine natural products as effective TRPV1 channel blockers. International Journal of Biological Macromolecules, 253, 127136), Streptomyces strain BCC24770 was cultured in ten 2.5L Erlenmeyer flasks, each containing 1L GYM medium (4g / L glucose, 4g / L yeast extract and 10g / L malt extract; pH 7.2-7.4) and about 100 glass beads with a diameter of 3mm, and cultured at 30°C with shaking at 180rpm for 8 days. The bacterial culture was extracted three times with an equal volume of ethyl acetate to obtain 2.1g of crude extract, which was then separated by column chromatography using reverse phase silica gel (Phenomenex, C18-T, 50μm, ) and eluted with different gradients of acetonitrile-water (1:9 to 10:0) to yield different fractions. After rotary evaporation, HMPQ accumulated in the 40% acetonitrile-water fraction. The dried powder was observed to emit green fluorescence and was therefore further purified by semi-preparative HPLC using an isocratic flow of 37% acetonitrile-water at a flow rate of 3 mL / min, with 0.05% trifluoroacetic acid in the mobile phase. Approximately 15 mg of HMPQ was obtained from 10 L of culture broth.

[0128] Structural analysis

[0129] The obtained high-purity HMPQ was a white amorphous powder. High-resolution mass spectrometry showed a [MH]-peak at m / z 323.1037, indicating that its molecular formula was C 18 H 16N2O4, with an unsaturation degree of 12. NMR analysis using DMSO-d6 detected all proton and carbon signals. 1H, 13C, COSY, HSQC, and HMBC NMR analysis of HMPQ showed the presence of a methoxy group, a methyl group, and a methylene group connected to the carbonyl carbon to form a propionyl group. Six aromatic protons are located on two non-adjacent ring systems, two of which are adjacent protons on the benzene ring, and the other four are continuously connected to the quinazolinone, as confirmed by COSY signals. Of the two active protons, one is the hydroxyl group connected to the benzene ring to form the 1-(2-hydroxy-4-methoxyphenyl)propan-1-one structure; the other is the amide proton on the quinazolinone. The amide proton associated with C14 of the 1-(2-hydroxy-4-methoxyphenyl)propan-1-one structure and the aromatic proton H12 associated with C2 from the HMBC signal connect the two parts together.

[0130] X-ray crystallographic analysis of HMPQ

[0131] The sample was dissolved in a glass vial containing only methanol, sealed with parafilm, and slowly evaporated. After one week, two single crystal samples were obtained: a colorless plate-like crystal at the bottom and a yellow needle-like crystal on the vial film. The collection size was approximately 0.13 × 0.12 × 0.03 mm. 3 and 0.3×0.02×0.02mm 3 Crystal samples were immersed in Paratone-N and processed using MiTeGen TM The crystal samples were picked out by cryostat. HRMS confirmed that they had the same molecular weight. The diffractometer was Rigaku OD Supernova (Cu-Kα radiation, )Data were collected at 100K. The structure was solved using the Olex2 software (see reference Dolomanov, OV, Bourhis, LJ, Gildea, RJ, Howard, JA, & Puschmann, H. (2009). OLEX2: a complete structure solution, refinement and analysis program. Journal of applied crystallography, 42(2), 339-341), the SHELXT (see reference Sheldrick, GM (2015). SHELXT–Integrated space-group and crystal-structure determination. Acta Crystallographica Section A: Foundations and Advances, 71(1), 3-8) structure solution program using the intrinsic phase method, and the olex2.refine (see reference Bourhis, LJ, Dolomanov, OV, Gildea, RJ, Howard, JA, & Puschmann, H. (2015). The anatomy of a comprehensive constrained, restrained refinement program for the modern Refinement was performed using the Gauss-Newton minimization package (in the computing environment–Olex2 dissected

[15] . Acta Crystallographica Section A: Foundations and Advances, 71(1), 59-75). The resulting monoclinic crystal structures, with space groups P21 / c and P21 / n, were successfully refined to R1 = 5.32% and 8.12%, respectively. The CIF files have been uploaded to the Cambridge Crystallographic Data Centre (CCDC) and are numbered #2311188 and #2311189, respectively.

[0132] Cell culture and cytotoxicity assay

[0133] HEK293 and HeLa cells were purchased from the American Type Culture Collection (ATCC), and HT22 cells were purchased from Sigma-Aldrich. They were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, 11965092) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, 26140079) and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO₂. After one day of culture, cells were seeded at a density of 5,000 cells per well in 96-well plates and cultured for an additional 24 hours. HMPQ was then added at various concentrations for 24 hours. After incubation with 20 μL of thiazolyl blue tetrazolium bromide (5 mg / mL, PBS buffer; Sigma-Aldrich) for 4 hours, the supernatant was removed, and 100 μL of DMSO was added to dissolve the formazan. After shaking in the dark for 5 minutes, the absorbance at 570 nm was measured using a Multiskan FC microplate reader.

[0134] G4 DNA sample preparation

[0135] Single-stranded DNA nucleotides were purchased from Integrated DNA Technologies. A 100 μM DNA sample (single-stranded) was heated to 95°C for 15 minutes in 70 mM KCl and 20 mM potassium phosphate annealing buffer (pH 7.0), then slowly cooled to room temperature overnight for reannealing. The final NMR sample contained 0.1 mM DNA or RNA in 20 mM potassium phosphate buffer (pH 7.0) and 70 mM KCl.

[0136] One-dimensional (1D) 1 H-NMR titration experiments

[0137] To verify whether the compounds interacted with G4 DNA, we collected 1D 1 H NMR spectra were used to perform NMR titration experiments. All compounds were dissolved in isotope-labeled d6-DMSO (purchased from Sigma-Aldrich) at a concentration of approximately 50 mM as stock solutions. To avoid chemical shift changes in the G4 sample due to the addition of d6-DMSO, 10 μL d6-DMSO was added to 500 μL of 0.1 mM G4 solution in NMR buffer (20 mM potassium phosphate buffer and 70 mM KCl, pH 7.0, 10% D2O), and then 1D 1 H-NMR spectra were used as reference. In the NMR titration experiments, each compound was added to the G4 sample solution, and a maximum volume of 10 μL of each compound in d6-DMSO solution was used as the final data point.

[0138] CD spectrum test

[0139] CD spectra of G4 containing HMPQ were recorded at room temperature using a Chirascan CD spectrometer in liquid mode from 220 nm to 320 nm. A quartz cuvette with a 1 mm pathlength was used, and the sample volume was 400 μL. Each G4 sample was prepared at the same concentration of 15 μM. Compounds were mixed with G4 at a 1:10 molar ratio. Each measurement was scanned in triplicate, and the buffer background was subtracted.

[0140] Cell imaging

[0141] HeLa cells were seeded into 12-well plates with coverslips and cultured at 37°C in 5% CO₂ for one day. They were then incubated with 10 μM HMPQ overnight. The next day, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. G4-specific antibody BG4 (Sigma-Aldrich, Cat. No. MABE 1126) was added at a 1:500 dilution for staining and incubated overnight at 4°C. Cells were washed three times with PBS and then incubated with a secondary antibody (Alexa Fluor 647 anti-rabbit for G4) at a 1:1000 dilution for one hour at room temperature. After washing the cells three times with PBS to remove excess secondary antibody, DAPI was added and incubated for 5 minutes, followed by another wash with PBS. The cells on the slides were air-dried, mounted in Hydromount medium, and observed using a Leica SP8 confocal microscope.

[0142] Live cell imaging

[0143] HMPQ was dissolved with polymer Pluronic F-127 and organic solvent tetrahydrofuran (THF) and water, and the mixture was then sonicated. After removing THF and filtering, synthesized nanoparticles were obtained (see document Qin, W., Alifu, N., Lam, JWY, Cui, Y., SU.H., LG, Qian, J., & Tang BZ (2020) Facile Synthesis of Efficient Luminogens with AIE Features for Three-Photon Fluorescence Imaging of the Brain through the Intact Skull. Advanced Materials, 32 (23), e202000364). Nanoparticles were incubated with living cells overnight. Living cells containing HMPQ nanoparticles were observed using a Leica SP8 confocal microscope.

[0144] Statistical analysis

[0145] All data were obtained from at least three independent preparations. Quantitative analysis was performed in a blinded manner. Statistical analysis was performed using Origin 2018.

[0146] Data availability

[0147] The crystallographic data for the reported structures (Form 1 and Form 2 conformers) have been submitted to the Cambridge Crystallographic Data Centre (CCDC) under CCDC accession numbers #2311188 and #2311189, respectively. These data are available free of charge via the CCDC website: ccdc.cam.ac.uk / structures / . All data supporting the findings of this study are provided in the article and its supplementary information.

[0148] All cited patents, patent applications, provisional applications, and publications are incorporated herein by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0149] The following are examples illustrating the procedures for implementing the present application. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are calculated by weight and all solvent mixture ratios are calculated by volume.

[0150] result

[0151] Example 1 - Discovery of Natural Bacterial Metabolite AIEgen

[0152] In the process of searching for novel bioactive compounds from marine bacteria, our previous studies have successfully isolated several complex compounds, such as chrexanthomycins, by large-scale fermentation (see Cheng, A., Liu, C., Ye, W., Huang, D., She, W., Liu, X., & Qian, PY (2022). Selective c9orf72 g-quadruplex-binding small molecules ameliorate pathological signatures of als / ftdmodels. Journal of Medicinal Chemistry, 65(19), 12825-12837; Ye, W., Lui, ST, Zhao, Q., Wong, YM, Cheng, A., Sung, HHY, & Huang, P. (2023). Novel marine natural products as effective TRPV1 channel blockers. International Journal of Biological Macromolecules, 253, 127136) and albofungins (see Ye, W., She, W., Sung, HY, Qian, P., & Williams, ID (2020). Albofungin and chloroalbofungin: antibioticcrystals with 2D but not 3D isostructurality. Acta Crystallographica Section C: Structural Chemistry,76(12),1100-1107; She, W., Ye, W., Cheng, A., Liu, Microbiology,12,635268)( Figure 1AThis effort resulted in the accumulation and identification of previously unknown secondary metabolites, albeit in limited quantities. Serendipitously, during the HPLC preparation process, a novel small molecule derived from the marine bacterium Streptomyces strain BCC24770 was discovered. This molecule exhibited strong green luminescence in its powder form under ultraviolet (UV) excitation, indicating its potential as an AIEgen.

[0153] The structure of the compound was determined by various organic spectroscopic analyses ( Figure 1B ), including high-resolution mass spectrometry (HRMS), one- and two-dimensional nuclear magnetic resonance (NMR), and single-crystal X-ray diffraction (SXRD) ( Figure 7-13 , Table 1). The compound was identified as 2-(2-hydroxy-6-methoxy-3-propionylphenyl)quinazolin-4(3H)-one ( Figure 1C ). It contains a 4(3H)-quinazolinone skeleton, which is commonly found in plant, fungal and bacterial metabolites. 4(3H)-quinazolinone is also known for its diverse pharmacological activities (see KSHIRSAGAR, UA Recent developments in the chemistry of quinazolinone alkaloids. Organic & biomolecular chemistry, 2015, 13.36: 9336-9352; Chen, XW, Rao, L., Chen, JL, & Zou, Y. (2022). Unexpected assembly machinery for 4(3H)-quinazolinone scaffold synthesis. Nature Communications, 13(1), 6522). Figure 1D As shown, the marine AIEgen HMPQ can be easily crystallized into sufficient amount. The obtained crystal sample emits bright luminescence under UV irradiation with a peak emission wavelength of 500 nm ( Figure 1E ).

[0154] Atomic labeling of HMPQ and its 1H-1H COSY and key HMBC correlations.

[0155]

[0156] Table 1. HMPQ 1 H NMR (500 MHz, DMSO-d6) and 13 C NMR (126 MHz) data.

[0157]

[0158] a HMBC correlations are from protons to designated carbons.

[0159] Example 2 - Photophysical properties of HMPQ

[0160] After the successful isolation of HMPQ in large quantities, a systematic characterization was carried out to evaluate its photophysical properties. ESIPT is a reversible phototautomerism process that occurs in the excited state and is promoted by intramolecular hydrogen bonds. Due to its fascinating emission phenomena and potential applications, this process has attracted widespread research interest (see Sedgwick, AC, Wu, L., Han, HH, Bull, SD, He, XP, James, TD, ... & Yoon, J. (2018). Excited-state intramolecular proton-transfer (ESIPT) based fluorescence sensors and imaging agents. Chemical Society Reviews, 47 (23), 8842-8880; Wang, Z., Zhou, F., Wang, J., Zhao, Z., Qin, A., Yu, Z., & Tang, BZ (2018). Electronic effect on the optical properties and sensingability of AIEgens with ESIPT process based on salicylaldehyde azine. Science China Chemistry, 61, 76-87; Song, Z., Kwok, RT, Zhao, E., He, Z., Hong, Y., Lam, JW, ... & Tang, BZ (2014). A ratiometric fluorescent probe based on ESIPT and AIE processes for alkaline phosphatase activity assay and visualization in living cells. ACS Applied Materials & Interfaces, 6(19), 17245-17254). The ESIPT property of HMPQ makes its fluorescence emission extremely sensitive to the polarity of the surrounding microenvironment ( Figure 2A ).

[0161] In polar aprotic solvents such as dimethyl sulfoxide and dimethylformamide, the ESIPT tautomerism from the enol to the keto form is mainly suppressed. The result is a bright blue fluorescence with a peak at 457 nm under photoexcitation. As the polarity of the solvent decreases, the ESIPT mechanism is activated because the less polar solvent is better able to stabilize the intramolecular hydrogen bonds in the excited state. This phenomenon is manifested as a significant red shift in emission to 497 nm in moderately polar solvents such as chloroform, and a peak green fluorescence in non-polar toluene ( Figure 2B This ESIPT-induced emission modulation spans approximately 60 nm in the transition from methanol (MeOH) to toluene ( Figure 2C ), demonstrating an excellent solvatochromic range afforded by excited-state tautomerism (keto-enol).

[0162] After observing the strong emission of crystalline HMPQ, we studied its photophysical properties in a polymer matrix. HMPQ was dissolved and mixed with the common polymer polymethyl methacrylate (PMMA) (see Zeng, Q., Li, Z., Dong, Y., Qin, A., Hong, Y., Ji, L., ... & Tang, BZ (2007). Fluorescence enhancements of benzene-cored luminophors by restricted intramolecular rotations: AIE and AIEE effects. Chemical communications, (1), 70-72). A series of thin films were prepared by coating the mixtures containing different weight percentages (wt%) of HMPQ on a quartz substrate. The obtained thin films containing different wt% HMPQ were analyzed to measure their photoluminescence (PL) and quantum yield (QY) ( Figures 3A-3D and Figure 14 , Table 2).

[0163] Figure 3A and Figure 3B The PL intensity increases with increasing HMPQ wt%. The intensity peaks at approximately 20 wt%, exhibiting a maximum intensity 4.8 times that observed at 1 wt%. Increasing the HMPQ concentration to 20 wt% does not shift the emission maximum, but further increases result in a gradual redshift, indicating stronger π-π interactions.

[0164] This discovery and Figure 3A The red shift observed between 1wt% and 100wt% samples is consistent. Figure 3C In the figure, the QY of the sample varies with the PL intensity. Figure 3DThe QY of the film is significantly improved compared to the solution state (the QY of the THF solution is 2.4%). The 30wt% film reaches a QY of 30.5%. The significantly improved QY of the film compared to the solution indicates the AIE property of the molecule.

[0165] Figure 3D The QY of the solid-state emission is significantly improved compared to that of the solution state. The QY of the above samples is summarized in Table 2.

[0166] Table 2. Quantum yields of HMPQ in different states. a

[0167]

[0168] a Excitation wavelength: 320nm. b Quantum yield of films prepared with different mass percentages of HMPQ in PMMA mixtures.

[0169] Example 3 - Polymorphs of HMPQ

[0170] HMPQ crystals exhibit strong blue-green fluorescence under ultraviolet inspection, but the pattern is uneven. This phenomenon is due to the production of two different polymorphic forms during the crystallization of HMPQ, namely, the majority are yellow needle-shaped crystals and a few colorless plate-shaped crystals. The luminescence of the yellow needle-shaped crystals is significantly higher than that of the colorless plate-shaped crystals. Crystallographic analysis of these two polymorphic forms shows that the colorless plate-shaped crystals exhibit a distorted conformation (type 1 conformer), while the yellow needle-shaped crystals have a planar structure (type 2 conformer) (see the above-mentioned literature Ye, W., Lui et al., Dolomanov et al., Sheldrick et al.) (Tables 3 and 4). This structural difference has a significant impact on their photophysical properties ( Figures 4A-4C ).

[0171] Specifically, in the type 1 conformer, the 4(3H)-quinazolinone skeleton in the crystal is oriented perpendicularly to the methoxyphenyl ring. There is only one intramolecular hydrogen bond within the methoxyphenyl ring, resulting in an extremely low quantum yield of 2.2% under ultraviolet excitation. In contrast, the type 2 conformer shows the presence of two intramolecular hydrogen bonds connecting the quinazolinone skeleton and the methoxyphenyl ring. As a result, crystal samples of this polymorph emit bright fluorescence under ultraviolet light, with a quantum yield of an astonishing 17.4%.

[0172] The difference in quantum yield can be attributed to the difference in conformation and molecular packing within the lattice (see Yang, J., Ren, Z., Chen, B., Fang, M., Zhao, Z., Tang, BZ, ... & Li, Z. (2017). Three polymorphs of one luminogen: how the molecular packing affects the RTP and AIE properties?. Journal of Materials Chemistry C, 5(36), 9242-9246; Liu, H., Lu, Z., Tang, B., Zhang, Z., Wang, Y., & Zhang, H. (2018). AIE-active organic polymorphs displaying molecular conformation-dependent amplified spontaneous emissions (ASE). Dyes and Pigments, 149, 284-289; Huang, B., Chen, WC, Li, Z., Zhang, J., Zhao, W., Feng, Y., ... & Lee, CS (2018). Manipulation of molecular aggregation states to realize polymorphism, AIE, MCL, and TADF in a single molecule. Angewandte Chemie, 130 (38), 12653-12657). Type 2 conformers exhibit an ordered and tightly packed molecular configuration. In contrast, type 1 conformers exhibit lower packing efficiency, which can be seen from their larger specific molecular volume and lower rigidity ( Figure 15-18). This structural flexibility enables the methoxyphenyl ring in the type 1 conformer to rotate. This stabilizes the excited state molecular conformation by electron delocalization and resonance, which is conducive to excited state molecular motion. Therefore, non-radiative deactivation of the excited state occurs (see Cai, XM, Lin, Y., Li, Y., Chen, X., Wang, Z., Zhao, X., ... & Tang, BZ (2021). BioAIEgens derived from rosin: how does molecular motion affect their photophysicalprocesses in solid state?. Nature communications, 12 (1), 1773). Density functional theory (DFT) calculations illustrate the energy difference between the two conformers (see Parr, RG, & Yang, W. (1995). Density-functional theory of the electronic structure of molecules. Annual review of physical chemistry, 46 (1), 701-728). When the two conformers were subjected to restricted geometry optimization and frequency analysis at the M062x / TZVP level of theory in the IEFPCM model (methanol) (see Frisch, MJ, Trucks, GW, Schlegel, HB, Scuseria, GE, Robb, MA, Cheeseman, JR, ... & Fox, DJ (2009). Gaussian 09, Revision D.01, Gaussian, Inc., Wallingford CT. See also: URL: http: / / www.gaussian.com), the Gibbs free energy of the type 2 conformer (5.81 kcal / mol) was much lower than that of the type 1 conformer. This result indicates that the type 2 conformer is more stable than the type 1 conformer, providing additional support for our experimental results ( Figure 4D ). Therefore, it is easy to establish the connection between the molecular structure of HMPQ and its AIE properties. The key factors regulating the quantum yield in different states include conformation, intramolecular hydrogen bonding, molecular stacking, and stacking efficiency.

[0173] Table 3. Crystallographic data and structure refinement of ye67CuLT_auto (type 1 conformer)

[0174]

[0175] Table 4. Crystallographic data and structure refinement of YE72-1 CuLT_auto (type 2 conformers)

[0176]

[0177] Example 4—HMPQ as a G4 fluorescent probe

[0178] The interesting conformational changes observed in HMPQ lead to excellent emission properties, prompting us to explore its application as a fluorescent probe. The two inherent planar aromatic rings of HMPQ offer the potential to interact with guanine quadruplexes or conjugate with the side chains of novel nucleic acid secondary structures G4 (see references Duarte, AR, Cadoni, E., AS, Moreira, R., & Paulo, A. (2018). Design of Modular G-quadruplex Ligands. ChemMedChem, 13 (9), 869-893; Murat, P., Singh, Y., & Defrancq, E. (2011). Methods for investigating G-quadruplex DNA / ligand interactions. Chemical Society Reviews, 40 (11), 5293-5307). In order to evaluate the binding affinity of HMPQ, we introduced various G4 samples and HMPQ to conduct a series of photoluminescence tests (Table 5). After mixing and filtration, almost no fluorescence emission was observed in the background solution containing only G4 or HMPQ, as shown in Table 5. Figure 5A and Figure 5D In contrast, the addition of HMPQ to specific G4s triggered a significantly enhanced fluorescence emission response (turn-on effect). The corresponding PL spectra showed that the binding of HMPQ to specific G4s caused a new red shift centered at 497 nm.

[0179] Among the G4s tested, the "turn-on" effect of c-kit2 (SEQ ID NO: 3) was most pronounced when interacting with HMPQ; the fluorescence at 497 nm was enhanced by approximately 300-fold. It is noteworthy that the c-kit2 sequence, derived from the KIT proto-oncogene, has been shown to regulate cancer pathogenesis (see Peterková, K., Durník, I., Marek, R., Plavec, J., & P.(2021).c-kit2 G-quadruplex stabilized via a covalent probe:exploring G-quartet asymmetry.Nucleic Acids Research,49(15),8947-8960;Gregory-Bryson,E.,Bartlett,E.,Kiupel,M.,Hayes,S.,&Yuzbasiyan-Gurkan,V.(2010).Canine and human gastrointestinal stromal tumors display similar mutationsin c-KIT exon 11.BMC cancer,10(1),1-9). In order to further study the sensitivity of HMPQ as a fluorescent G4 probe, we obtained the PL spectra of HMPQ (0.15mM) at different concentrations of c-kit2, as shown in Figure 2. Figure 5B As the concentration of c-kit2 increased from 0 μM to 50 μM, a significant and gradual increase in PL intensity was observed. Figure 5CThe Stern-Volmer diagram in the figure shows the fluorescence turn-on process in more detail, from which the Stern-Volmer constant m is calculated to be 1.94×106M-1. According to the limit of detection (LOD) equation, LOD = 3SB / m (SB = standard deviation of 10 blank measurements, m = slope of relative intensity versus HMPQ concentration), the LOD was calculated to be 28.7 nM (see Liu, Y., Tang, Y., Barashkov, NN, Irgibaeva, IS, Lam, JW, Hu, R., ... & Tang, BZ (2010). Fluorescent chemosensor for detection and quantitation of carbon dioxide gas. Journal of the American Chemical Society, 132(40), 13951-13953; Wang, X., Han, T., Gong, J., Alam, P., Zhang, H., Lam, JW, & Tang, BZ (2022). Diversity-Oriented Synthesis of Functional Polymers with Multisubstituted Small Heterocycles by Facile Stereoselective Multicomponent Polymerizations. Macromolecules, 55(11), 4389-4401). This result highlights the excellent sensitivity of HMPQ as a specific G4 probe. Figure 5E The illustration of HMPQ in Figure 4 illustrates its ability to selectively recognize and illuminate G4, thus indicating its great potential to facilitate the detection and detailed study of G4 structure.

[0180] In addition to fluorescence spectroscopy, we also performed NMR titration, another common method for evaluating binding affinity. NMR titration relies on the perturbation of chemical shifts when small molecules bind to G4 imino protons (see Liu, C., Zhou, B., Geng, Y., Tam, DY, Feng, R., Miao, H., ... & Zhu, G. (2019). A chair-type G-quadruplex structure formed by a human telomeric variant DNA in K+ solution. Chemical science, 10 (1), 218-226). Our NMR titration results are consistent with the PL results ( Figure 6A and Figure 19-28 ), indicating that the binding of the compound illuminates the G4 structure. In addition, circular dichroism (CD) spectroscopy confirmed its non-destructive effect on the original conformation of G4 ( Figure 6B 、 Figure 29 and Figure 30 ).

[0181] HMPQ showed no cytotoxicity to human cell lines including HEK293 and HeLa cells, as well as immortalized mouse hippocampal neuronal cells HT22, even at a concentration of 100 μM. This extremely high biosafety prompted us to explore its potential for precise visualization of subcellular structures and the spatial distribution of G4 within cells. Using the well-established G4 structure-specific antibody BG4 as a control, HMPQ was able to effectively penetrate the cell nucleus. Comparison with BG4 produced very interesting results, such as Figure 6C The overlap coefficient between HMPQ and BG4 was as high as 94.3%. This finding indicates that HMPQ and BG4 colocalize to a large extent, effectively mapping the presence of G4 structures in cells.

[0182] Table 5. Oligonucleotide samples used in this experiment.

[0183]

[0184]

[0185] It should be understood that the embodiments and methods described herein are for illustrative purposes only, and that those skilled in the art will make various modifications or changes based on these embodiments and embodiments, which modifications or changes should be included in the spirit and scope of this application and within the scope of the appended claims. In addition, any element or limitation of any invention or embodiment disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, and all such combinations are within the scope of the present invention without limitation.

[0186] Selected implementation plans

[0187] Embodiment 1 A method for identifying a G-quadruplex (G4) in a cell, the method comprising:

[0188] (a) obtaining a fluorescent probe that can selectively bind to the G4 structure;

[0189] (b) obtaining cells containing the nucleic acid;

[0190] (c) contacting the fluorescent probe with the nucleic acid in the cell;

[0191] (d) detecting a fluorescent signal emitted when the fluorescent probe selectively binds to the G-quadruplex structure in the cell;

[0192] (e) analyzing the fluorescence signal; and

[0193] (f) Recognize G4 structures in cells,

[0194] The fluorescent probe is 2-(2-hydroxy-6-methoxy-3-propionylphenyl)quinazolin-4(3H)-one (HMPQ).

[0195] Embodiment 2. The method according to embodiment 1, wherein the HMPQ used is selected from the group consisting of DNA grade, proteomics grade, molecular biology grade and ultrapure grade HMPQ.

[0196] Embodiment 3. The method according to embodiment 1, wherein the polymorphic form of HMPQ used is the Type 2 conformer.

[0197] Embodiment 4. The method according to any one of the preceding embodiments, wherein the peak emission wavelength of HMPQ when bound to the G4 structure in the cell is about 497 to about 500 nm.

[0198] Embodiment 5. The method according to any one of the preceding embodiments, wherein the peak emission wavelength of HMPQ when bound to the G4 structure in the cell is 500 nm.

[0199] Embodiment 6. The method according to any one of the preceding embodiments, wherein the nucleic acid is selected from the group consisting of DNA and RNA.

[0200] Embodiment 7. The method according to any one of the preceding embodiments, wherein HMPQ does not bind to nucleic acid structures that do not form G4.

[0201] Embodiment 8. The method according to any one of the preceding embodiments, wherein HMPQ is substantially non-cytotoxic to mammalian cells and cell lines.

[0202] Embodiment 9. The method according to any one of the preceding embodiments, wherein HMPQ binds to G4 without changing its conformation.

[0203] Embodiment 10. The method according to any one of the preceding embodiments, wherein HMPQ can be used in an aqueous medium.

[0204] Embodiment 11. The method according to any one of the preceding embodiments, wherein the limit of detection (LOD) of HMPQ is about 25 nM.

[0205] Embodiment 12. The method according to any one of the preceding embodiments, wherein binding of HMPQ to the G4 structure causes an increase in fluorescence of up to about 300-fold at about 497 nm to 500 nm.

[0206] Embodiment 13. The method according to any one of the preceding embodiments, wherein the binding of HMPQ to G4 is detectable in a subcellular compartment.

[0207] Embodiment 14. The method according to embodiment 13, wherein the binding of HMPQ to G4 is detectable in the cell nucleus.

[0208] Embodiment 15. The method according to any one of the preceding embodiments, wherein HMPQ is applied to the cells at a concentration of about 0.1 μM to about 1,000 μM.

[0209] Embodiment 16. The method according to any one of the preceding embodiments, wherein the method is label-free.

[0210] Embodiment 17. A label-free kit for selectively detecting G-quadruplexes (G4) in cells, the kit comprising HMPQ of at least molecular biology grade and at least one of the following additional components: a cell fixative, a cell permeabilizing agent, a cell fixation buffer, a cell permeabilization buffer, a wash buffer, or any combination thereof.

[0211] Embodiment 18. The kit according to embodiment 17, wherein the cell fixative is 4% paraformaldehyde in PBS buffer, the cell permeabilizing agent is 0.2% Triton-X100 in PBS buffer, and the washing buffer is PBS buffer.

Claims

1. A method for identifying a G-quadruplex (G4) in a cell, the method comprising: (a) obtaining a fluorescent probe capable of selectively binding to G4; (b) obtaining cells containing the nucleic acid; (c) contacting the fluorescent probe with the nucleic acid in the cell; (d) detecting a fluorescent signal emitted when the fluorescent probe selectively binds to the G4 structure in the cell; (e) analyzing the fluorescence signal; as well as (f) identifying said G4 structure in said cell, The fluorescent probe is 2-(2-hydroxy-6-methoxy-3-propionylphenyl)quinazolin-4(3H)-one (HMPQ).

2. The method according to claim 1, wherein the HMPQ used is selected from the group consisting of DNA grade, proteomics grade, molecular biology grade and ultrapure grade HMPQ.

3. The method according to claim 1, wherein the polymorphic form of HMPQ used is conformational isomer Type 2.

4. The method of claim 1, wherein the peak emission wavelength of the HMPQ when bound to the G4 structure in the cell is about 497 nm to about 500 nm. The method according to claim 1 , wherein the peak emission wavelength of the HMPQ when it binds to the G4 structure in the cell is 500 nm. The method of claim 1 , wherein the nucleic acid is selected from the group consisting of DNA and RNA. The method of claim 1 , wherein the HMPQ does not bind to nucleic acid structures that do not form G4.

8. The method of claim 1, wherein the HMPQ is substantially non-cytotoxic to mammalian cells and cell lines.

9. The method of claim 1, wherein the HMPQ binds to G4 without changing its conformation.

10. The method of claim 1, wherein the HMPQ is usable in an aqueous medium.

11. The method of claim 1, wherein the limit of detection (LOD) of HMPQ is about 25 nM.

12. The method of claim 1, wherein the binding of HMPQ to the G4 structure causes a fluorescence enhancement of up to about 300 times at about 497 nm to 500 nm.

13. The method of claim 1, wherein the binding of HMPQ to G4 is detectable in a subcellular compartment. The method according to claim 13 , wherein the binding of HMPQ to G4 is detectable in the cell nucleus.

15. The method of claim 1, wherein the HMPQ is applied to the cells at a concentration of about 0.1 μM to about 1,000 μM.

16. The method of claim 1, wherein the method is label-free.

17. A label-free kit for selectively detecting G-quadruplex (G4) in cells, the kit comprising HMPQ of at least molecular biology grade and at least one of the following additional components: a cell fixative, a cell permeabilizing agent, a cell fixation buffer, a cell permeabilization buffer, a washing buffer, or any combination thereof. 18 . The kit according to claim 17 , wherein the cell fixative is 4% paraformaldehyde in PBS buffer, the cell permeabilizing agent is 0.2% Triton-X100 in PBS buffer, and the washing buffer is PBS buffer.

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