Molecular probes for nucleic acid detection, their preparation and use

Abstract

The present disclosure relates to the molecular probe for detecting nucleic acid, and its preparation and use.In particular, the molecular probe for detecting nucleic acid described herein comprises (1) a molecular probe carrier comprising a cell-penetrating peptide and a detectable label coupled to the cell-penetrating peptide, and (2) a targeting oligonucleotide, and the targeting oligonucleotide is coupled to the molecular probe carrier.The method for preparing the molecular probe, the method for detecting nucleic acid, and the kit for detecting nucleic acid are also described herein.

Description

[Technical Field]

[0001] This disclosure relates to the field of biomolecular detection, and more particularly to oligonucleotide molecular probes for nucleic acid detection in vivo or in vitro, as well as methods for preparing and using the same. [Background technology]

[0002] Nucleic acid detection technology is widely used in the fields of medical diagnostics, food safety, and environmental monitoring. This technique aims to detect target nucleic acids derived from pathogens or diseases, for example, and determines the presence of pathogens or diseases (e.g., pathogens, diseased cells or tissues) by identifying the presence of the target nucleic acid or detecting the level of the target nucleic acid.

[0003] Existing nucleic acid detection methods include nucleic acid blotting, microarrays, and nucleic acid amplification (e.g., RT-PCR, RT-LAMP, SAT). However, these methods require nucleic acid extraction, amplification, and detection, involving redundant operational steps and expensive equipment and materials, making detection costly, time-consuming, and cumbersome. Furthermore, variations in working conditions and reagent selection, as well as differences in operator performance, can interfere with nucleic acids derived from samples (e.g., causing their loss or blockage), preventing the successful detection of target nucleic acids from multiple samples, which easily leads to false negative results.

[0004] Nucleic acid probes used for in situ hybridization are increasingly being used for the detection of extracellular and intracellular biochemical substances. If the structure of the cell or tissue remains unchanged, a labeled nucleotide fragment can, based on the principle of base pairing, specifically bind (e.g., hybridize) to the corresponding target nucleic acid in the cell or tissue being tested, and be identified by optical detection or imaging techniques to enable detection of the target nucleic acid (e.g., visual localization and / or quantification). Probes commonly used for such detection include those labeled with radionuclides or fluorescence. However, these probes still have limitations in terms of detection accuracy and safety.

[0005] Therefore, there remains a need for a new type of molecular probe that is easy to prepare and capable of providing safe and accurate nucleic acid detection. [Overview of the project]

[0006] In one aspect, (1) A molecular probe carrier comprising a cell-permeable peptide and a detectable label coupled to the cell-permeable peptide, and (2) Targeting Oligonucleotides, Includes, Targeting The oligonucleotide is bound to the molecular probe support. Molecular probes for the detection of nucleic acids are provided herein.

[0007] In another embodiment, a method for preparing a molecular probe, To obtain a molecular probe carrier by coupling a detectable label to a cell-permeable peptide, and Molecular probe support, Targeting Along with oligonucleotides, molecular probe carriers and Targeting To obtain molecular probes, incubate for a sufficient duration to allow binding between oligonucleotides. Methods including the above are provided herein.

[0008] In another embodiment, Applying the molecular probe described in claim 1 to a sample of the target or a sample derived from the target, Determining the presence and / or level of a target nucleic acid by detecting detectable markers in a sample of the subject or a sample derived from the subject. Methods for detecting nucleic acids, including the above, are provided herein.

[0009] In another embodiment, A container comprising the molecular probe carrier described in claim 1, Claim 1 Targeting A container containing an oligonucleotide, and Optional: Instructions for use A kit for the detection of nucleic acids, including the above, is provided herein.

[0010] The molecular probes described herein include a molecular probe carrier (i.e., a conjugate of a detectable label and a cell-permeable peptide) and Targeting It has been found that the probes can be prepared from oligonucleotides by a one-step method. Compared with existing multi-step probe synthesis methods, the method for preparing molecular probes described herein is simple, efficient, raw material-efficient, and environmentally friendly, and the prepared probes can provide safe and accurate nucleic acid detection.

[0011] The Disclosure will be further described below with reference to the drawings. The drawings and descriptions provided are for illustrative purposes only and are not intended to limit the scope of the Disclosure. [Brief explanation of the drawing]

[0012] [Figure 1] This scheme demonstrates the preparation of a cell-permeable peptide-VSP molecular probe carrier. [Figure 2]This is a figure showing the experimental verification of the effect of TTR-targeted siRNA targeting TTR mRNA. Panels 2a, 2b, and 2c show the results of fluorescence imaging of rat hepatocytes BRL3A 12 hours after transfection in vitro. Panel 2a shows the results of transfection with TTR-targeted siRNA. Panel 2b shows the results of transfection with NC-siRNA as a negative control. Panel 2c shows the results of non-transfected cells as a blank control. Figure 2d shows the fluorescence concentration curves of the group transfected with TTR-targeted siRNA, the group transfected with NC-siRNA as a negative control, and the non-transfected group as a blank control at different time points. [Figure 3] This is a figure showing the effect of TTR-targeted siRNA targeting TTR mRNA analyzed by Western blot. [Figure 4] This is a figure showing the abnormal tissue state and abnormal expression of TTR protein in the case of liver fibrosis disorder compared with normal liver. Panels 4a - 4c show the results of HE, Masson, and TTR protein immunohistochemical staining in healthy rats. Panels 4d - 4f show the results of HE, Masson, and TTR protein immunohistochemical staining in liver fibrosis model rats. [Figure 5] This is a figure showing the uptake of 18F-TTR-targeted siRNA molecular probe by the livers of healthy rats and liver fibrosis model rats. Panels 5a and 5c are pseudo-color and grayscale PET images of the molecular probe accumulated in the liver fibrosis model group 60 minutes after probe injection. Panels 5b and 5d are pseudo-color and grayscale PET images of the molecular probe accumulated in healthy rats 60 minutes after probe injection. Panel 5e shows the radioactivity of the molecular probe in the liver of the liver fibrosis model group and healthy rats as a function of time.

Mode for Carrying Out the Invention

[0013] The meanings of scientific and technical terms in this disclosure are consistent with the general understanding in the technical field unless otherwise specified. In this disclosure, "a / an" or its combinations with various quantifiers include both the singular and plural meanings unless specifically specified otherwise. In this disclosure, when multiple values, value ranges, or combinations thereof are given for the same parameter or variable, it is equivalent to specifically disclose these values, the end values of the ranges, and the ranges formed by any two of these values. Whether any numerical value is modified by a modifier such as "about" or not, it covers an approximate range that can be understood by those skilled in the art, for example, plus or minus 10%, 5%, etc. In this disclosure, each "embodiment" refers to and covers the implementation of the methods and systems of this application to the same extent. One or more technical features in any one embodiment can be freely combined with one or more technical features in any one or more other embodiments, and the embodiments obtained thereby also belong to the content disclosed in this application.

[0014] In one aspect, (1) a molecular probe carrier comprising a cell-penetrating peptide and a detectable label coupled to the cell-penetrating peptide, and (2) Targeting an oligonucleotide, comprising, Targeting where the oligonucleotide is bound to the molecular probe carrier, a molecular probe for the detection of nucleic acids is provided herein.

[0015] In some embodiments, the molecular probes described herein can be oligonucleotide molecular probes.

[0016] In some embodiments, nucleic acid detection can be in vitro nucleic acid detection. In some embodiments, nucleic acid detection can be in vitro nucleic acid detection in a cell-containing sample. In other embodiments, nucleic acid detection can be in vivo detection.

[0017] The molecular probes described herein include cell-permeable peptides. The term “cell-permeable peptide” (also referred to herein as “CP”), as used herein, refers to a class of short peptides capable of facilitating the uptake of various molecular cargoes (from nanoparticles to small chemical molecules and large DNA fragments). They have the function of delivering the cargo into the cell.

[0018] In some embodiments, the cell-permeable peptide may include the amino acid sequence shown in SEQ ID NO: 1.

[0019] In one embodiment, the cell-permeable peptide may include an amino acid sequence containing one or two amino acid mutations at any one or two positions in the amino acid sequence shown in SEQ ID NO: 1. In a particular embodiment, the cell-permeable peptide may include an amino acid sequence containing one or two amino acid mutations at any one or two positions in the amino acid sequence shown in SEQ ID NO: 1, while the lysine residue in SEQ ID NO: 1 remains unchanged. The amino acid mutations are selected from the group consisting of additions, substitutions, deletions, and combinations thereof. The (one or more) amino acid mutations may occur at either end, both ends, or at the same or different positions within the amino acid sequence shown in SEQ ID NO: 1.

[0020] In some embodiments, the length of the cell-permeable peptide can be in the range of 8 to 12 amino acids. In some alternative embodiments, the length of the cell-permeable peptide can be within the range formed by taking any two points among 8, 9, 10, 11, and 12 as endpoints.

[0021] In some embodiments, the cell-permeable peptide may include an amino acid sequence having at least 80%, at least 90%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the cell-permeable peptide consists of the amino acid sequence shown in SEQ ID NO: 1.

[0022] In some embodiments, the cell-permeable peptide can be a cyclic peptide. A cyclic peptide can be formed by linking the first and last amino acid residues of the amino acid sequence of the cell-permeable peptide.

[0023] The cell-permeable peptides described herein can be obtained according to polypeptide synthesis and / or polypeptide modification methods known in the art.

[0024] The molecular probes described herein further include detectable labels. The term "detectable label," as used herein, refers to a substance capable of generating or enhancing a signal for detection.

[0025] In some embodiments, the detectable label may be a contrast agent. The term “contrast agent,” as used herein, refers to a substance introduced into a site being measured (such as within an cell, tissue, or organ) to form an image or enhance the imaging effect. In some cases, “contrast agent” may be referred to as “imaging agent.”

[0026] In some embodiments, the contrast agent may include a CT contrast agent. The CT contrast agent may be used as a detectable label for electron computed tomography (CT) detection. In some embodiments, the CT contrast agent may include one or more selected from the group consisting of iodixanol, iohexol, iopamidol, iopromide, and ioversol. In some embodiments, the contrast agent may be iodixanol. The term “iodixanol” in this disclosure may be used interchangeably with its trade name “Visipaque” (abbreviated as VSP), which may be used as a nonionic contrast agent containing six iodine disomes in CT detection.

[0027] Generally, iodixanol has a structure represented by the following formula (I).

[0028] [ka]

[0029] In some embodiments, the detectable marker may also include one or more contrast agents selected from other contrast agents, such as MRI contrast agents (e.g., USPIO), fluorescent contrast agents (e.g., 5-FAM SE), and radioactive contrast agents (e.g., positron-mediated isotopes 18F or 68Ga).

[0030] The detectable labels described herein can be directly or indirectly coupled to cell-permeable peptides via linkers.

[0031] In some embodiments, the detectable labels described herein may be coupled to cell-permeable peptides in a covalent manner. In some embodiments, the detectable labels may be coupled to cell-permeable peptides via amide bonds. In some embodiments, the detectable labels may be coupled to amino groups in amino acid residues contained in cell-permeable peptides. In certain embodiments, the detectable labels are coupled to lysine residues (e.g., amino groups in lysine residues) contained in cell-permeable peptides. In some embodiments, the detectable labels may be indirectly coupled to cell-permeable peptides via linkers. In some embodiments, the linkers may include alkylene carbonyl groups. In some embodiments, the alkylene carbonyl groups may be C1-C 12 Alkylene carbonyl groups, for example, C1-C 10 It can be an alkylene carbonyl group, a C1-C8 alkylene carbonyl group, a C1-C6 alkylene carbonyl group, or a C1-C4 alkylene carbonyl group. When used herein, "alkylene" refers to a linear alkylene, a branched alkylene, or a cycloalkylene, preferably a linear alkylene. For example, "C1-C" as described herein. 12 "Alkylene" refers to an alkylene group containing the indicated number (1 to 12) carbon atoms.

[0032] In exemplary embodiments where the detectable label is VSP, the VSP can be coupled to the cell-permeable peptide via an amide bond. In some embodiments, the VSP can be coupled to an amino group in an amino acid residue contained in the cell-permeable peptide. In some embodiments, the VSP can be coupled to an amino group in an amino acid residue contained in the cell-permeable peptide. In certain embodiments, the VSP is coupled to a lysine residue (e.g., an amino group in a lysine residue) contained in the cell-permeable peptide. In some embodiments, the VSP can be indirectly coupled to the cell-permeable peptide via a linker. In some embodiments, the linker may include an alkylene carbonyl group. In some embodiments, the alkylene carbonyl group may be C1-C 12 Alkylene carbonyl groups, for example, C1-C 10 The group is an alkylene carbonyl group, a C1-C8 alkylene carbonyl group, a C1-C6 alkylene carbonyl group, or a C1-C4 alkylene carbonyl group. In exemplary embodiments, VSP is -(CH2) n The cell-permeable peptide is coupled via a linker containing CO- (wherein n is an integer from 1 to 10). Furthermore, in certain embodiments, VSP and the cell-permeable peptide are coupled by the following method, namely, [VSP]-(CH2) n It can be coupled in CO-[cell-permeable peptide] (wherein n is an integer from 1 to 10). In some alternative embodiments, n can be an integer from 1 to 8. In some alternative embodiments, n can be an integer from 1 to 6, specifically from 1 to 4, and more specifically from 2, 3, or 4.

[0033] In this disclosure, a detectable label coupled to a cell-permeable peptide may be referred to herein as a "probe carrier" or "molecular probe carrier." Targeting It is used for further bonding of alkyl groups.

[0034] The molecular probes described herein are TargetingIncludes oligonucleotides. Targeting As used herein, the term “oligonucleotide” refers to a short nucleic acid sequence that targets (e.g., is complementary to) a target gene (e.g., within a target nucleic acid or a target site within a target gene), and has a length of up to 100 bases (e.g., 10–80, 10–50, 10–40). Targeting Oligonucleotides include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). Targeting Oligonucleotides can be single-stranded or double-stranded.

[0035] In some embodiments, Targeting Oligonucleotides are oligonucleotides that can specifically bind to specific target sites of pathogenic microorganisms (e.g., the genome of the pathogenic microorganism), for example, oligonucleotides that are specifically complementary to the target sites of the genome of the pathogenic microorganism. Examples of pathogenic microorganisms include, but are not limited to, viruses, mycoplasmas, chlamydia, bacteria, and fungi. In further embodiments, Targeting Oligonucleotides are oligonucleotides that target disease-specific or disorder-specific target sites. Diseases or disorders include, but are not limited to, precancerous disorders, cancers, histofibrosis, inflammatory diseases, genetic disorders, and malformations, and are typically characterized by the abnormal activity of one or more biomolecules (e.g., overexpression) or the abnormal activity of one or more variants of biomolecules.

[0036] In some embodiments, Targeting The oligonucleotide can be siRNA or shRNA. In some embodiments, Targeting The oligonucleotide can be siRNA.

[0037] In an exemplary embodiment, TargetingThe oligonucleotide may be an siRNA that targets a specific site in the trans tiretin (TTR) gene, which may be referred to herein as “TTR-targeted siRNA”. In certain embodiments, the TTR-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 2. In even more certain embodiments, the TTR-targeted siRNA may include a sense strand containing the methylated nucleotide sequence shown in SEQ ID NO: 2. For example, the methylated nucleotide sequence shown in SEQ ID NO: 2 may be 5'-mCAGmUGmUmUmCmUmUGmCmUmCmUAmUAAdTdT-3' (where m represents the 2' hydroxymethylation of the corresponding base). In certain embodiments, the TTR-targeted siRNA may include an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 3. In even more certain embodiments, the TTR-targeted siRNA may include an antisense strand containing the methylated nucleotide sequence shown in SEQ ID NO: 3. For example, the methylated nucleotide sequence shown in Sequence ID No. 3 can be 5'-UmUAmUAGAGmCAAGAAmCACUGdTdT-3' (where m represents the 2' hydroxymethylation of the corresponding base).

[0038] In certain embodiments, the TTR-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 2 and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 3.

[0039] In an exemplary embodiment, TargetingOligonucleotides are siRNAs that target specific sites in the genes of the novel coronavirus (SARS-CoV-2) (also known as "novel coronavirus" or "2019-nCoV"), which may also be referred to herein as "novel coronavirus-targeted siRNA" or "SARS-CoV-2-targeted siRNA." In certain embodiments, the SARS-CoV-2-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 4. In another particular embodiment, the SARS-CoV-2-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 5.

[0040] In an exemplary embodiment, Targeting Oligonucleotides, also referred to herein as “IAV-targeted siRNAs,” are siRNAs that target specific sites in the genes of influenza A virus (IAV). In certain embodiments, the IAV-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 6. In another specific embodiment, the IAV-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 7. The exemplified IAV-targeted siRNAs are siRNAs that have broad-spectrum antiviral activity against specific sites designed for highly conserved sequences in the influenza A virus gene.

[0041] In some embodiments, Targeting Oligonucleotides can be bound to molecular probe carriers via covalent or non-covalent interactions. In some embodiments, target oligonucleotides can be bound to molecular probe carriers via non-covalent interactions.

[0042] In another embodiment, a method for preparing a molecular probe, To obtain a molecular probe carrier by coupling a detectable label to a cell-permeable peptide, and Molecular probe support, Targeting Along with oligonucleotides, molecular probe carriers and Targeting To obtain molecular probes, incubate for a sufficient duration to allow binding between oligonucleotides. Methods including the above are provided herein.

[0043] The methods for preparing molecular probes described herein may include providing cell-permeable peptides.

[0044] In some embodiments, the cell-permeable peptide may include the amino acid sequence shown in SEQ ID NO: 1.

[0045] In one embodiment, the cell-permeable peptide may include an amino acid sequence containing one or two amino acid mutations at any one or two positions in the amino acid sequence shown in SEQ ID NO: 1. In a particular embodiment, the cell-permeable peptide may include an amino acid sequence containing one or two amino acid mutations at any one or two positions in the amino acid sequence shown in SEQ ID NO: 1, while the lysine residue in SEQ ID NO: 1 remains unchanged. The amino acid mutations are selected from the group consisting of additions, substitutions, deletions, and combinations thereof. The (one or more) amino acid mutations may occur at either end, both ends, or at the same or different positions within the amino acid sequence shown in SEQ ID NO: 1.

[0046] In some embodiments, the length of the cell-permeable peptide can be in the range of 8 to 12 amino acids. In some alternative embodiments, the length of the cell-permeable peptide can be within the range formed by taking any two of 8, 9, 10, 11, and 12 as endpoints.

[0047] In some embodiments, the cell-permeable peptide may include an amino acid sequence having at least 80%, at least 90%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the cell-permeable peptide consists of the amino acid sequence shown in SEQ ID NO: 1.

[0048] In some embodiments, the cell-permeable peptide can be a cyclic peptide. A cyclic peptide can be formed by linking the first and last amino acid residues of the amino acid sequence of the cell-permeable peptide.

[0049] The cell-permeable peptides described herein can be obtained according to polypeptide synthesis and / or polypeptide modification methods known in the art.

[0050] Methods for preparing molecular probes described herein may include providing a detectable label. In one embodiment, the detectable label may be a contrast agent.

[0051] In some embodiments, the contrast agent may include a CT contrast agent. In some embodiments, the CT contrast agent may include one or more selected from the group consisting of iodixanol, iohexol, iopamidol, iopromide, and ioversol. In some embodiments, the contrast agent may be iodixanol (VSP).

[0052] In some embodiments, the detectable marker may also include one or more contrast agents selected from other contrast agents, such as MRI contrast agents (e.g., USPIO), fluorescent contrast agents (e.g., 5-FAM SE), and radioactive contrast agents (e.g., positron-mediated isotopes 18F or 68Ga).

[0053] In some cases, the methods for preparing molecular probes described herein may further include modifying the detectable label such that the modified detectable label has a portion for coupling to a cell-permeable peptide. In some embodiments, the coupling may be covalent coupling.

[0054] In some embodiments, the moiety for coupling to the cell-penetrating peptide can comprise a carboxyl group. In some embodiments, the moiety for coupling to the cell-penetrating peptide can comprise an alkylene carboxyl group. In some embodiments, the alkylene carbonyl group is C1-C 12 An alkylene carbonyl group, such as C1-C 10 The alkylene carbonyl group can be a C1-C8 alkylene carbonyl group, a C1-C6 alkylene carbonyl group or a C1-C4 alkylene carbonyl group.

[0055] In an exemplary embodiment where the detectable label is a VSP, the VSP can be modified to include a moiety for coupling to the cell-penetrating peptide. In some embodiments, the moiety for coupling to the cell-penetrating peptide can comprise a carboxyl group. In some embodiments, the moiety for coupling to the cell-penetrating peptide can comprise an alkylene carboxyl group. In some embodiments, the alkylene carbonyl group is C1-C 12 An alkylene carbonyl group, such as C1-C 10 The alkylene carbonyl group can be a C1-C8 alkylene carbonyl group, a C1-C6 alkylene carbonyl group or a C1-C4 alkylene carbonyl group. In some embodiments, the VSP can be modified to include a moiety for coupling to the cell-penetrating peptide that comprises -(CH2) n COOH (where n is an integer from 1 to 10). In some alternative embodiments, n can be an integer from 1 to 8. In some alternative embodiments, n can be an integer from 1 to 6, specifically from 1 to 4, more specifically 2, 3 or 4.

[0056] In an exemplary embodiment, the modified VSP has a structure represented by the following formula (II).

[0057] [Chemical formula]

[0058] The methods for preparing molecular probes described herein further include coupling a detectable label to a cell-permeable peptide to obtain a molecular probe carrier.

[0059] In some embodiments, the detectable labels described herein can be coupled to cell-permeable peptides in a covalent manner. In some embodiments, the detectable labels can be coupled to cell-permeable peptides via amide bonds. In some embodiments, the detectable labels can be coupled to cell-permeable peptides by forming an amide bond between the carboxyl group in the detectable label and the amino group in the amino acid residue contained in the cell-permeable peptide. In certain embodiments, the detectable labels are coupled to lysine residues contained in the cell-permeable peptide.

[0060] In exemplary embodiments, the VSP is modified to include a moiety for coupling to a cell-permeable peptide (e.g., a carboxyl-containing moiety), the modified VSP can be coupled to the cell-permeable peptide via an amide bond. In some embodiments, the modified VSP can be coupled to the cell-permeable peptide by forming an amide bond between the carboxyl group in the VSP and the amino group in the amino acid residue contained in the cell-permeable peptide. In some embodiments, the modified VSP can be coupled to a lysine residue contained in the cell-permeable peptide. In exemplary embodiments, the modified VSP can be coupled to the amino group in the lysine residue contained in the cell-permeable peptide via an amide bond of -(CH2) n It can be coupled to a cell-permeable peptide by a moiety for coupling to the cell-permeable peptide, such as COOH (wherein n is an integer from 1 to 10). In some alternative embodiments, n can be an integer from 1 to 8. In some alternative embodiments, n can be an integer from 1 to 6, specifically from 1 to 4, and more specifically from 2, 3, or 4.

[0061] In some cases, the methods described herein may further include activating the terminal carboxyl group of the detectable label before coupling the detectable label to a cell-permeable peptide. In some exemplary embodiments, activation can be performed using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (sulfo-NHS).

[0062] In some embodiments, the detectable label and the cell-permeable peptide may be coupled by mixing or incubating the two. In some embodiments, the mixing or incubation may be carried out for a duration of, for example, 1 to 4 hours, for example, 1 to 3 hours, or for example, 2 to 3 hours.

[0063] In some embodiments, mixing or incubation may be carried out at room temperature. In some embodiments, the detectable marker and the cell-permeable peptide may be mixed in a molar ratio of 3:1 to 1:3, for example, 2.5:1 to 1:2.5, 2:1 to 1:2, 1:1.5 to 1.5:1, or 1:1.

[0064] In some cases, the methods for preparing molecular probes described herein may further include separating the detectable label coupled to the cell-permeable peptide from a mixed reaction of the detectable label and the cell-permeable peptide, as a molecular probe carrier. In some embodiments, the separation may be performed by a separation method selected from centrifugation and HPLC separation.

[0065] The method for preparing molecular probes described herein involves using a molecular probe carrier Targeting Along with oligonucleotides, molecular probe carriers and Targeting The process further includes incubating for a sufficient duration to allow binding between oligonucleotides to obtain molecular probes.

[0066] In some embodiments, Targeting Oligonucleotides can be bound to molecular probe carriers via covalent or non-covalent interactions. In some embodiments, target oligonucleotides can be bound to molecular probe carriers via non-covalent interactions.

[0067] Targeting Oligonucleotides can be single-stranded or double-stranded.

[0068] In some embodiments, Targeting Oligonucleotides are oligonucleotides that can specifically bind to specific target sites of pathogenic microorganisms (e.g., the genome of the pathogenic microorganism), for example, oligonucleotides that are specifically complementary to the target sites of the genome of the pathogenic microorganism. Examples of pathogenic microorganisms include, but are not limited to, viruses, mycoplasmas, chlamydia, bacteria, and fungi. In further embodiments, Targeting Oligonucleotides are oligonucleotides that target disease-specific or disorder-specific target sites. Diseases or disorders include, but are not limited to, precancerous disorders, cancers, histofibrosis, inflammatory diseases, genetic disorders, and malformations, and are typically characterized by the abnormal activity of one or more biomolecules (e.g., overexpression) or the abnormal activity of one or more variants of biomolecules.

[0069] In some embodiments, Targeting The oligonucleotide can be siRNA or shRNA. In some embodiments, Targeting The oligonucleotide can be siRNA.

[0070] In an exemplary embodiment, TargetingThe oligonucleotide can be a TTR-targeted siRNA. In certain embodiments, the TTR-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 2. In even more specific embodiments, the TTR-targeted siRNA may include a sense strand containing the methylated nucleotide sequence shown in SEQ ID NO: 2. For example, the methylated nucleotide sequence shown in SEQ ID NO: 2 may be 5'-mCAGmUGmUmUmCmUmUGmCmUmCmUAmUAAdTdT-3' (where m represents the 2' hydroxymethylation of the corresponding base). In certain embodiments, the TTR-targeted siRNA may include an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 3. In even more specific embodiments, the TTR-targeted siRNA may include an antisense strand containing the methylated nucleotide sequence shown in SEQ ID NO: 3. For example, the methylated nucleotide sequence shown in SEQ ID NO: 3 may be 5'-UmUAmUAGAGmCAAGAAmCACUGdTdT-3' (where m represents the 2' hydroxymethylation of the corresponding base).

[0071] In certain embodiments, the TTR-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 2 and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 3.

[0072] In an exemplary embodiment, Targeting The oligonucleotide can be a SARS-CoV-2 targeted siRNA. In certain embodiments, the SARS-CoV-2 targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 4. In another specific embodiment, the SARS-CoV-2 targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 5.

[0073] In an exemplary embodiment, TargetingThe oligonucleotide can be an IAV-targeted siRNA. In certain embodiments, the IAV-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 6. In another specific embodiment, the IAV-targeted siRNA may include a sense strand containing the nucleotide sequence shown in SEQ ID NO: 7.

[0074] In some embodiments, molecular probe carrier and Targeting Oligonucleotides may be mixed for incubation in molar ratios of 5:1 to 30:1, for example, 10:1 to 25:1, or 15:1 to 20:1. In some embodiments, the mixed medium may be an aqueous liquid, such as pure water or an aqueous solution (e.g., cell medium). In some embodiments, incubation is carried out at ambient temperature (e.g., room temperature conditions, e.g., 15°C to 25°C). In some embodiments, incubation is sustained for 10 to 30 minutes, for example, 10 to 20 minutes, or e.g., 15 to 20 minutes.

[0075] In some embodiments, molecular probes obtained after incubation can be used directly without further purification.

[0076] In some embodiments, molecular probes may be prepared for nucleic acid detection in vivo or in vitro.

[0077] In some embodiments, molecular probe carriers (e.g., detectable labels coupled to cell-permeable peptides described herein) may be prepared using a one-step method.

[0078] In some embodiments, Targeting Incubation of molecular probe carriers with oligonucleotides may be performed immediately before detection. In some embodiments, the molecular probes described herein may be prepared by a one-step method from a molecular probe carrier (i.e., a detectable label coupled to a cell-permeable peptide described herein) and a targeted nucleotide.

[0079] Compared with existing multi-stage probe synthesis methods, the method for preparing molecular probes described herein is simple, efficient, material-efficient, and environmentally friendly. The method described in this application has good practicality and is suitable for mass production of molecular probes.

[0080] In another embodiment, Applying the molecular probe described in claim 1 to a sample of a subject or a sample derived from a subject, and determining the presence and / or level of a target nucleic acid by detecting a detectable marker in the sample of a subject or a sample derived from a subject, Methods for detecting nucleic acids, including the above, are provided herein.

[0081] In some embodiments, the subject is an animal such as a mammal, such as a human.

[0082] In some embodiments, nucleic acid detection may be in vivo nucleic acid detection. In some embodiments, the molecular probe may be administered by one or more methods selected from injection (e.g., intramuscular injection, intravenous injection, etc.), oral administration, and inhalation (e.g., aerosol inhalation). In some embodiments, the molecular probe may be administered by aerosol inhalation. In some embodiments, the molecular probe may be administered in an amount ranging from 6.5 to 32.5 mg / kg of the subject's body weight. In some embodiments, detection may be performed 1 to 4 hours, preferably 2 to 3 hours, after administration of the molecular probe.

[0083] In some embodiments, detection methods include, but are not limited to, CT detection, MRI detection, fluorescence detection, radionuclide detection, and similar methods. In some embodiments, detection is CT detection.

[0084] In some embodiments, detection may be in vitro nucleic acid detection. In some embodiments, the sample derived from the subject is an ex vivo cell-containing sample obtained from the subject, e.g., cell-containing tissue or cell population (e.g., biopsy material, tissue specimen, cell suspension, etc.). In some embodiments, the ex vivo cell-containing sample may be a single cell suspension or a culture of adherent cells. In some embodiments, administration of the molecular probe may include contacting the sample derived from the subject with the molecular probe described herein (e.g., incubation). In embodiments where the ex vivo cell-containing sample is a culture of adherent cells, the molecular probe may be added when the cells are 30% to 80%, preferably 40% to 70%, confluent. In some embodiments, after adding the molecular probe, the sample is incubated with the molecular probe under conditions suitable for cell culture (e.g., 37°C, 5% CO2). In some embodiments, the incubation process may be carried out for 12 to 36 hours, e.g., 12 to 24 hours, e.g., 18 to 24 hours.

[0085] In some embodiments, the method may optionally further include halving the cell medium for the cells (e.g., cell medium containing 20% ​​fetal bovine serum) after incubation is complete. “Half-volume replacement” or “half-volume replacement” of the cell medium, as used herein, means replacing half of the old medium with fresh medium for cell culture. In some embodiments, detection may be performed 80 to 72 hours after the completion of incubation, for example, 4 to 48 hours, for example, 4 to 24 hours, for example, 8 to 12 hours.

[0086] In some embodiments, the method may optionally further include washing the cells before detection to remove molecular probes that do not bind to the target gene and are excreted by the cells. In some embodiments, washing may be performed two to three times with PBS or cell medium (e.g., cell medium containing 20% ​​fetal bovine serum).

[0087] In some embodiments, the method involves administering the molecular probe before using the molecular probe carrier and the method described herein. Targeting This may further include preparing molecular probes from oligonucleotides.

[0088] In some embodiments, molecular probe carriers and as described herein Targeting The preparation of molecular probes from oligonucleotides can be achieved by a one-step method, i.e., by using a molecular probe support. Targeting Molecular probes can be prepared by mixing them with oligonucleotides and incubating them, and these resulting molecular probes can be used for detection without further processing.

[0089] In some embodiments, the molecular probe carrier may include a detectable label coupled to the cell-permeable peptide described above. In some embodiments, the method may use the molecular probe carrier and for incubation in a molar ratio of 5:1 to 30:1, for example, 10:1 to 25:1, or 15:1 to 20:1. Targeting This may include mixing oligonucleotides. In some embodiments, the mixed medium may be an aqueous liquid, such as pure water or an aqueous solution (e.g., cell medium). In some embodiments, incubation may be carried out at ambient temperature (e.g., room temperature conditions, e.g., 15°C to 25°C). In some embodiments, incubation may last for 10 to 30 minutes, e.g., 10 to 20 minutes, e.g., 15 to 20 minutes.

[0090] The method for detecting nucleic acids provided herein involves, immediately before detection, deploying a molecular probe to a molecular probe carrier and using a one-step method. Targeting This allows for the preparation of molecular probes from oligonucleotides. The preparation of molecular probes is convenient and safe, and the prepared molecular probes can be used for detection without further processing.

[0091] Furthermore, compared to probes currently used for nucleic acid detection (probes with radionuclides or fluorescent labels), the probes provided herein cause little radioactive contamination, less background disturbance, and therefore can provide improved detection accuracy.

[0092] In another embodiment, A container comprising the molecular probe carrier described in claim 1, Claim 1 Targeting A container containing an oligonucleotide, and Optional: Instructions for use A kit for the detection of nucleic acids, including the above, is provided herein.

[0093] In some embodiments, the kit may be used for in vivo detection. In other embodiments, the kit may be used for in vitro detection.

[0094] In some embodiments, Targeting Oligonucleotides can be in ready-to-use solution (which can be used directly without further dilution), concentrated solution, or lyophilized form.

[0095] In some embodiments, the molecular probe support may be in the form of a ready-to-use solution (which can be used directly without further dilution), a concentrated solution, or a lyophilized form.

[0096] In some alternative embodiments, the kit includes a molecular probe carrier and Targeting The kit may further include a culture medium for mixing oligonucleotides, such as an aqueous liquid, such as pure water or aqueous solution (e.g., cell culture medium). In some alternative embodiments, the kit may include a molecular probe carrier and / or Targeting The apparatus may further include one or more devices (e.g., one or more containers) for diluting and / or mixing oligonucleotides. [Examples]

[0097] The technical solutions described herein, along with specific examples, are described below. It should be understood that these examples are used solely to illustrate the principles and effects of the disclosure and should not be construed as limiting the disclosure. All of the above examples can be modified and altered by those skilled in the art without departing from the spirit and scope of the disclosure as defined in the appended claims below.

[0098] Unless otherwise specified, percentages and portions are calculated by weight. Unless otherwise defined, all technical and scientific terms used herein may have the same meaning as those well known to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the technical solutions of this disclosure. Preferred embodiments and examples described herein are for illustrative purposes only.

[0099] [Example 1] Preparation of cell-permeable peptide-VSP molecular probe carriers Studies have shown that cyclic cell-permeable peptides can provide better cell transfection compared to linear cell-permeable peptides. In the following exemplary examples, a cyclic peptide having the amino acid sequence shown in SEQ ID NO: 1 (hereinafter abbreviated as "CP") was used as the cell-permeable peptide for the following experiments.

[0100] In this embodiment, the contrast agent VSP-320 (GE Healthcare, catalog number: 1181607CHN) was used as the detectable label.

[0101] A) VSP structure and modification

[0102] The VSP-320 used in this embodiment has the structure shown in the following formula (I).

[0103] [ka]

[0104] The VSP-320 of formula (I) was modified to include a carbon chain with a carboxyl group at its terminus to form a modified visipaque (cVSP) having the structure shown by formula (II) below.

[0105] [ka]

[0106] B) The terminal carboxyl groups of cVSP were activated using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, SIGMA, catalog number: E7750) and N-hydroxythiosuccinimide (sulfo-NHS, SIGMA, catalog number: 56485) by the following process.

[0107] a. To 1 mL of cVSP solution (10 mg / mL), 11.93 mg of DEC (final concentration 0.0623 mM / mL) was added, followed by 32.58 mg of sulfo-NHS (final concentration 0.156 mM / mL).

[0108] b. The mixture was thoroughly mixed and allowed to react at room temperature for 15 minutes.

[0109] c. Concentrated PBS (10×) was added to the reaction solution to increase the pH of the buffer above 7.0 and stop the above reaction.

[0110] C) A probe support was obtained by reacting the amino group of the lysine in the cyclic peptide with terminally activated cVSP using the following process.

[0111] a. 6.85 mg of the cyclic peptide was added to the reaction solution of B)c. above, the solution was thoroughly mixed, and the reaction was allowed to proceed at room temperature for 2 hours.

[0112] b. After the reaction was complete, 0.33 mg of hydroxylamine was added to the above solution to bring the final concentration to 10 mM and inhibit the reaction.

[0113] D) The final product VSP-CP was separated from the reaction by HPLC.

[0114] Figure 1 provides a scheme illustrating the preparation of a cell-permeable peptide-VSP molecular probe carrier.

[0115] [Example 2] Preparation of cell-permeable peptide-VSP-TTR target siRNA molecular probes We used an siRNA (customized and synthesized by GenePharma Inc.) targeting the liver fibrosis-specific marker gene TTR, which has the following sequence. Sense chain: 5'-mCAGmUGmUmUmCmUmUGmCmUmCmUAmUAAdTdT-3' Antisense chain: 5'-UmUAmUAGAGmCAAGAAmCACUGdTdT-3' In the formula, m represents the 2' hydroxymethylation of the corresponding base.

[0116] Molecular probes were prepared using the following process.

[0117] A) siRNA was added to DEPC water to obtain a final concentration of 5 μM / mL for subsequent use.

[0118] B) The molecular probe carrier VSP-CP and siRNA were mixed in a molar ratio of 20:1 and incubated at room temperature for 15-20 minutes.

[0119] C) The target molecular probe was obtained by binding siRNA to the molecular probe carrier VSP-CP via non-covalent interactions.

[0120] The molecular probes obtained in this way can be immediately used for in vitro cell detection or aerosol inhalation detection.

[0121] [Example 3] Detection of Targeted Nucleic Acids Using Cell-Permeable Peptide-VSP-TTR Targeted siRNA Molecular Probes

[0122] A) Rat BRL3A cells (purchased from ATCC) were divided into 1.2 × 10⁻⁶ cells. 5 Cells were seeded in a 6-well plate at a cell density of 1 cell / well. 1.5 mL of serum-containing MEM medium was added, and the cells were incubated at 37°C for 24 hours under 5% CO2 to achieve 40%–70% cell confluence.

[0123] B) Preparation of molecular probes for transfection

[0124] a. Solution A was siRNA diluted in serum-free medium with a final volume of 600 μL and a concentration of 0.4 nm / μL.

[0125] b. Solution B was VSP-CP diluted in serum-free medium with a final volume of 600 μL and a concentration of 8 nm / μL.

[0126] c. Solutions A and B were mixed, gently shaken, and left at room temperature for 15-20 minutes to form the VSP-CP-siRNA molecular probe.

[0127] C) Pre-treatment for transfection: Cells were washed twice with serum-free medium, and serum-free medium was added to the cells at a rate of 1.3 mL / well.

[0128] D) 200 μL / well of the VSP-CP-siRNA molecular probe was added to a 6-well plate, gently shaken, and incubated at 37°C for 24 hours under 5% CO2.

[0129] E) Cells were washed 2-3 times with PBS 80 minutes, 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours after incubation, and then detected by CT.

[0130] [Example 4] Verification of the efficacy of TTR-targeted siRNAs that target TTR mRNA. In this example, the effectiveness of TTR-targeted siRNA targeting TTR mRNA was experimentally verified.

[0131] The results are shown in Figure 2. Panels 2a, 2b, and 2c show the fluorescence imaging results of rat hepatocytes BRL3A 12 hours after transfection in vitro. Panel 2a shows the results of transfection with TTR-targeted siRNA. Panel 2b shows the results of transfection with NC-siRNA as a negative control. Panel 2c shows the results of untransfected cells as a blank control. Figure 2d shows the fluorescence intensity curves of the group transfected with TTR-targeted siRNA, the group transfected with NC-siRNA as a negative control, and the untransfected group as a blank control at different time points.

[0132] The results show that the fluorescence intensity shown in panel 2a is significantly higher than that in panels 2b and 2c, indicating that the TTR-targeted siRNA successfully entered the cells and specifically bound to the TTR target site. From Figure 2d, it can be seen that BRL3A cells gradually began to fluoresce 80 minutes after transfection, and then the fluorescence gradually accumulated within the cells. The fluorescence density in the cells increased sharply 8 hours after transfection and peaked at 12 hours. The fluorescence density in the cells began to decline and gradually stabilized at 24 hours. The best detection time was estimated to be approximately 12 hours after probe incubation.

[0133] TTR-targeted siRNA Targeting The effects were further analyzed by Western blotting. BRL3A cells were transfected with 40 nM TTR-targeted siRNA or NC-siRNA using N-TER as the vector. After 48 hours, cell proteins were extracted and Western blotting was performed to detect TTR protein expression in the cells. Untreated BRL3A cells served as a blank control, and β-actin served as an internal control. The results are shown in Figure 3.

[0134] The results in Figure 3 suggest that TTR-targeted siRNA may inhibit TTR protein expression by targeting and interacting with TTR mRNA.

[0135] [Example 5] Verification of abnormal TTR expression in a rat model of liver fibrosis. In this example, the abnormal tissue state and abnormal expression of TTR protein in liver fibrosis were examined in comparison with that of a normal liver.

[0136] Experimental procedure:

[0137] Immediately after the imaging scan was completed, rats from both the healthy control group and the liver fibrosis model group were sacrificed to obtain liver specimens. After fixing the liver specimens in 10% formaldehyde for 14 days, 5 μm paraffin sections of the tissue were obtained. All liver specimens from rats in both the healthy control group and the liver fibrosis model group were treated with HE staining, Masson staining, and pathological staining for TTR molecules for comparison.

[0138] HE staining was performed according to the following procedure. 1. The paraffin slices were placed in an oven at 60°C overnight. 2. Remove the sections from the oven and immediately place them in xylene. The sections were then treated sequentially with xylene for 20 minutes each (twice), 100% ethanol for 5 minutes each (twice), and 95% ethanol for 5 minutes each (twice), after which they were dewaxed and placed in water. 3. The sections were washed three times with TPBS (PBS containing 0.05% Tween-20) for 2 minutes each time, and then placed in distilled water. 4. The sections were stained with hematoxylin for 3-5 minutes. 5. Separation of hydrochloric acid and ethanol. 6. Treat with ammonium hydroxide for 20 seconds (until the color returns to blue). 7. Eosin staining for 2-3 minutes. 8. After spinning to dry and dehydrate, the sections were sequentially treated with 95% ethanol twice for 5 minutes each time, 100% ethanol twice for 5 minutes each time, and xylene twice for 20 minutes each time, and then placed in a neutral resin.

[0139] Masson staining was performed according to the following procedure. 1. The paraffin slices were placed in an oven at 60°C overnight. 2. Remove the sections from the oven and immediately place them in xylene. The sections were then treated sequentially with xylene for 20 minutes each (twice), 100% ethanol for 5 minutes each (twice), and 95% ethanol for 5 minutes each (twice), after which they were dewaxed and placed in water. 3. Oxidation with 1% potassium permanganate for 5 minutes. 4. Rinse with distilled water and decolorize with oxalic acid for 1 minute. 5. Rinse with distilled water and stain with celestin blue for 5 minutes. 6. Direct staining with Regaud hematoxylin for 3-5 minutes for core staining, without washing. 7. Thorough rinsing in distilled water. 8. Treatment with Ponceau Red / acid fuchsin solution for 5-10 minutes. 9. Wash with a 2% glacial acetic acid solution for 1 minute. 10. Separation using a 1% phosphomolybdic acid solution for 3-5 minutes. 11. Direct staining with aniline blue for 5 minutes without washing. 12. Wash with a 0.2% glacial acetic acid solution for 1 minute. 13. After spinning to dry and dehydrate, the sections were sequentially treated with 95% ethanol twice for 5 minutes each time, 100% ethanol twice for 5 minutes each time, and xylene twice for 20 minutes each time, and then placed in a neutral resin.

[0140] Pathological staining for TTR molecules was performed according to the following procedure. 1. The paraffin slices were placed in an oven at 60°C overnight. 2. Remove the sections from the oven and immediately place them in xylene. The sections were then treated sequentially with xylene for 20 minutes each (twice), 100% ethanol for 5 minutes each (twice), and 95% ethanol for 5 minutes each (twice), after which they were dewaxed and placed in water. 3. The sections were washed three times with TPBS (PBS containing 0.05% Tween-20) for 2 minutes each time, and then placed in distilled water. 4. The sections were placed in citrate buffer (pH 6.0) and treated in a microwave oven at a temperature above 95°C under medium and high heat for 4-5 minutes (until boiling), maintained under low and medium heat for 15 minutes, and allowed to cool naturally at room temperature. 5. The sections were immersed in distilled water for 3 minutes, and then washed three times with TPBS for 2 minutes each. 6. Endogenous HRP activity was eliminated by adding a horseradish enzyme inhibitor (3% H2O2) dropwise and incubating at room temperature in a humid chamber for 15 minutes. 7. Wash with TPBS three times, each time for 2 minutes. 8. Add goat serum dilution standard solution (10% goat serum standard stock solution diluted in PBS) dropwise and incubate in a humid chamber at room temperature for 30 minutes. Remove the serum without washing. 9. Add anti-TTR (dilution of sheep serum dilution standard solution) dropwise (in a ratio of 1:200). Leave overnight in a humid chamber at 4°C. 10. The following day, the sections were allowed to reach room temperature and washed three times with PBS for 2 minutes each time. 11. Reagent 1 from the PV-9001 Kit (Zsbio) was added dropwise, and the sections were incubated in a humid chamber for 20 minutes. 12. Wash with TPBS three times, each time for two minutes. 13. Reagent 2 from the PV-9001 Kit (Zsbio) was added dropwise, and the sections were incubated in a humid chamber for 30 minutes. 14. Wash with TPBS three times, each time for two minutes. 15. A diluted standard solution of DAB was prepared immediately before use. 16. The prepared diluted standard solution was added to each section in an amount of 30-40 μL or enough to cover the test specimen, and color development was observed under a microscope. 17. Rinse with distilled water to stop the reaction. 18. Counterstain with hematoxylin for 2 minutes, then wash with distilled water for 5 minutes. 19. Immerse in PBS for 3 minutes (until blue again), then wash with tap water. 20. After spinning to dry and dehydrate, the sections were sequentially treated with 95% ethanol for 5 minutes twice, 100% ethanol for 5 minutes twice, and xylene for 20 minutes twice, and then placed in a neutral resin.

[0141] The results are shown in Figure 4. Panels 4a-4c show the results of HE, Masson, and TTR protein immunohistochemical staining in healthy rats, and panels 4d-4f show the results of HE, Masson, and TTR protein immunohistochemical staining in liver fibrosis model rats.

[0142] The study showed that liver TTR protein expression in liver fibrosis model rats was significantly higher than in normal healthy rats. Therefore, liver TTR protein can be used as a marker for detecting liver fibrosis.

[0143] [Example 6] In vivo detection of 18F-TTR-targeted siRNA probes in healthy rats and liver fibrosis model rats. In this example, an 18F-TTR-targeted siRNA probe was used for in vivo detection in healthy rats and liver fibrosis model rats.

[0144] Experimental procedure:

[0145] PET / CT imaging was performed in healthy and liver fibrosis model rats at week 4 after treatment with thioamide solution. Prior to imaging, rats were fasted for 6 hours without water. Prior to imaging, 0.3 mL / 100g of 10% chlorohydrate was intraperitoneally injected into the rats for anesthesia. 0.5 mCi 18F-TTR targeted siRNA probe was injected into the tail vein of rats in the liver fibrosis model and control groups. The rats were dynamically scanned. The PET scan thickness was 3.75 mm. The CT scan parameters were 80 kV, 50 mA. Thickness: 3.75 mm. After the CT scan, a PET dynamic scan was continued for 60 minutes, followed by a normal scan at 80 minutes. The retrieved images were pre-processed using SharpIR, VUE Point HD image reconstruction techniques, and OSEM iterative reconstruction, and processed using GE AW4.5 and Xeleris3.0 workstations. Figure 5 shows 3D PET and CT fusion images of cross-sections, sagittal planes, and frontal planes of rat liver, as well as radioactivity as a function of time.

[0146] Figure 5 shows the uptake of the 18F-TTR-targeted siRNA molecular probe by the livers of healthy rats and liver fibrosis model rats. Panels 5a and 5c are pseudocolor and grayscale PET images of the molecular probe accumulated in the liver fibrosis model group 60 minutes after probe injection. Panels 5b and 5d are pseudocolor and grayscale PET images of the molecular probe accumulated in healthy rats 60 minutes after probe injection. Panel 5e shows the radioactivity of the molecular probe in the livers of the liver fibrosis model group and healthy rats as a function of time.

[0147] The study showed that the radioactivity of the liver decreased over time in rats with hepatic fibrosis and in healthy rats. The difference in radioactivity between the two groups began to appear 24 minutes after probe injection, and the tracking rate of the liver in healthy rats became significant at the end of the scan (80 minutes after injection).

[0148] The results shown in Figures 4 and 5 indicate that molecular probes targeting TTR mRNA may be useful for the non-invasive detection of liver fibrosis.

[0149] [Example 7] Preparation of alternative cell-permeable peptide-VSP-siRNA molecular probes In this example, CP-VSP-siRNA molecular probes carrying other disease-specific siRNA molecules were prepared for nucleic acid detection of the corresponding disorder or disease.

[0150] (1) Preparation of cell-permeable peptide-SARS-CoV-2 target siRNA molecular probes

[0151] We used an siRNA targeting the genome of the novel coronavirus (customized and synthesized by GenePharma Inc.) with the following sequence. Sense strand: 5'-GCGAAAUACCAGUGGCUUAdTdT-3' (SEQ ID NO: 4), or 5'-GCUACUAAUGGACCACUUAdTdT-3'(Sequence ID 5)

[0152] Molecular probes were prepared according to the following procedure.

[0153] A) For subsequent use, siRNA was added to DEPC water to a final concentration of 5 μM / mL.

[0154] B) The molecular probe carrier VSP-CP (prepared according to Example 1) and siRNA were mixed in a molar ratio of 20:1 and incubated at room temperature for 15-20 minutes.

[0155] C) The target molecular probe was obtained by binding siRNA to the molecular probe carrier VSP-CP via non-covalent interactions.

[0156] The molecular probes obtained in this way can be immediately used for in vitro cell detection or aerosol inhalation detection.

[0157] (2) Preparation of cell-permeable peptide-IAV target siRNA molecular probes

[0158] An siRNA targeting the genome of influenza A virus (customized and synthesized by GenePharma Inc.) having the following sequence was used: Sense strand: 5'-CAAGCAGUGUGUACAUUGAdTdT-3' (SEQ ID NO: 6), or 5'-GGAGACGUGGUGUUGGUAAdTdT-3'(Sequence ID 7)

[0159] Molecular probes were prepared according to the following procedure.

[0160] A) For subsequent use, siRNA was added to DEPC water to a final concentration of 5 μM / mL.

[0161] B) The molecular probe carrier VSP-CP (prepared according to Example 1) and siRNA were mixed in a molar ratio of 20:1 and incubated at room temperature for 15-20 minutes.

[0162] C) The target molecular probe was obtained by binding siRNA to the molecular probe carrier VSP-CP via non-covalent interactions.

[0163] The molecular probes obtained in this way can be immediately used for in vitro cell detection or aerosol inhalation detection.

[0164] [Example 8] In vitro nucleic acid detection of molecular probe according to Example 7

[0165] A) Cells (attached) obtained from the detected target, 1.2 × 10 5 Cells were seeded in a 6-well plate at a cell density of 1 cell / well. 1.5 mL of serum-containing MEM medium was added, and the cells were incubated at 37°C for 24 hours under 5% CO2 to achieve 40%–70% cell confluence.

[0166] B) Preparation of molecular probes immediately before transfection

[0167] a. Solution A was siRNA (siRNA described in Example 7) diluted in serum-free medium with a final volume of 600 μL and a concentration of 0.4 nm / μL.

[0168] b. Solution B was VSP-CP diluted in serum-free medium with a final volume of 600 μL and a concentration of 8 nm / μL.

[0169] c. Solutions A and B were mixed, gently shaken, and left at room temperature for 15-20 minutes to form the VSP-CP-siRNA molecular probe.

[0170] C) Pre-treatment for transfection: Cells were washed twice with serum-free medium, and fresh serum-free medium was added to the cells at a rate of 1.3 mL / well.

[0171] D) 200 μL / well of the VSP-CP-siRNA molecular probe was added to a 6-well plate, gently shaken, and incubated at 37°C for 12 hours under 5% CO2.

[0172] E) After incubation, the cell medium was halved and replaced with cell medium containing 20% ​​fetal bovine serum, and the cells were placed at 37°C under 5% CO2 for a duration of 80 minutes to 72 hours. The cells were then washed with the medium and observed using CT for detection of the presence and / or level of the target.

[0173] After reading the above teachings of this disclosure, a person skilled in the art will understand that various modifications or alterations can be made to the technical solutions disclosed herein, and that these equivalent forms also fall within the scope defined by the claims appended to this disclosure. The present invention includes the following embodiments. <1> (1) A molecular probe carrier comprising a cell-permeable peptide and a detectable label coupled to the cell-permeable peptide, and (2) Targeting oligonucleotides, Includes, The targeting oligonucleotide is bound to the molecular probe carrier. Molecular probes for nucleic acid detection. <2> The cell-permeable peptide comprises an amino acid sequence having at least 80%, at least 90%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and more specifically, the cell-permeable peptide comprises an amino acid sequence shown in SEQ ID NO: 1, and more specifically, the cell-permeable peptide is a cyclic peptide. <1> Molecular probes as described above. <3> The detectable label is a contrast agent, and specifically, the contrast agent includes a CT contrast agent. <1> or <2> Molecular probes as described above. <4> The CT contrast agent is iodixanol. <3> Molecular probes as described above. <5> The detectable label is directly or indirectly coupled to the cell-permeable peptide via a linker. <1> ~ <4> A molecular probe as described in any of the following. <6> The detectable label is coupled to an amino group in an amino acid residue contained in the cell-permeable peptide. <5> Molecular probes as described above. <7> The detectable label is coupled to a lysine residue contained in the cell-permeable peptide. <6> Molecular probes as described above. <8> A method for preparing molecular probes, To obtain a molecular probe carrier by coupling a detectable label to a cell-permeable peptide, and A method comprising incubating the molecular probe carrier together with a targeting oligonucleotide for a sufficient duration to enable binding between the molecular probe carrier and the targeting oligonucleotide to obtain the molecular probe. <9> The further step includes modifying the detectable label prior to the coupling step such that the modified detectable label has a portion for binding to the cell-permeable peptide. <8> Methods used. <10> The cell-permeable peptide comprises an amino acid sequence having at least 80%, at least 90%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and more specifically, the cell-permeable peptide comprises an amino acid sequence shown in SEQ ID NO: 1, and more specifically, the cell-permeable peptide is a cyclic peptide. <8> Methods used. <11> <1> ~ <7> Applying a molecular probe described in any of the above to a target or a sample derived from the said target, A method for detecting nucleic acids, comprising determining the presence and / or level of a target nucleic acid by detecting a detectable marker in the subject or the sample derived from the subject. <12> The process further includes preparing the molecular probe from the molecular probe carrier and the targeting oligonucleotide by a one-step process before applying the molecular probe, <11> Methods used. <13> <1> ~ <7> A container containing a molecular probe carrier as described in any of the following: <1> A container containing the targeting oligonucleotide described above, and Optional: Instructions for use A kit for nucleic acid detection, including [specific component].

Claims

1. (1) A molecular probe carrier comprising a cell-permeable peptide and a detectable label coupled to the cell-permeable peptide, and (2) Targeting oligonucleotides, Includes, The targeting oligonucleotide is bound to the molecular probe carrier. A molecular probe for the detection of nucleic acids, The cell-permeable peptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in Sequence ID No.

1. A molecular probe in which the detectable label is coupled to a lysine residue contained in the cell-permeable peptide.

2. The molecular probe according to claim 1, wherein the cell-permeable peptide comprises the amino acid sequence shown in Sequence ID No.

1.

3. The molecular probe according to claim 1 or 2, wherein the cell-permeable peptide is a cyclic peptide.

4. The molecular probe according to any one of claims 1 to 3, wherein the detectable label is a contrast agent, and specifically, the contrast agent includes a CT contrast agent.

5. The molecular probe according to claim 4, wherein the CT contrast agent is iodixanol.

6. The molecular probe according to any one of claims 1 to 5, wherein the detectable label is directly or indirectly coupled to the cell-permeable peptide via a linker.

7. A method for preparing a molecular probe according to claim 1, To obtain a molecular probe carrier by coupling a detectable label to a cell-permeable peptide, and A method comprising incubating the molecular probe carrier together with a targeting oligonucleotide for a sufficient duration to enable binding between the molecular probe carrier and the targeting oligonucleotide, thereby obtaining the molecular probe, The cell-permeable peptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in Sequence ID No.

1. A method comprising coupling the detectable label to a lysine residue contained in the cell-permeable peptide.

8. The method according to claim 7, further comprising modifying the detectable label prior to the coupling step such that the modified detectable label has a portion for binding to the cell-permeable peptide.

9. The method according to claim 7, wherein the cell-permeable peptide comprises the amino acid sequence shown in SEQ ID NO:

1.

10. The method according to claim 7, wherein the cell-permeable peptide is a cyclic peptide.

11. Applying a molecular probe according to any one of claims 1 to 6 to a sample derived from the target, A method for detecting nucleic acids, comprising determining the presence and / or level of a target nucleic acid by detecting a detectable marker in the sample derived from the subject.

12. The method according to claim 11, further comprising preparing the molecular probe from the molecular probe carrier and the targeting oligonucleotide by a one-step process before applying the molecular probe.

13. A molecular probe according to any one of claims 1 to 6 for use in a method for diagnosing a disease or disorder, wherein the method is The application of the molecular probe described in any one of claims 1 to 6, and A molecular probe comprising determining the presence and / or level of a target nucleic acid by detecting a detectable marker in the subject.

14. The molecular probe according to claim 13, wherein the disease or disorder is selected from precancerous disorders, cancer, histofibrosis, inflammatory diseases, genetic disorders, and malformations.

15. A container comprising a molecular probe carrier according to any one of claims 1 to 6, A container containing the targeting oligonucleotide described in claim 1, and Optional: Instructions for use A kit for nucleic acid detection, including [specific component].

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