WO2024204691A1 - Method for detecting target molecule using peptide aptamer - Google Patents

Abstract

The present invention addresses the problem of providing a method for detecting a target molecule more simply and safely than a conventional detection method by using a peptide aptamer specifically binding to a virus including influenza and other target molecules. The problem is solved by providing a detecting agent of a target molecule, comprising the following (1) and (2): (1) an alcohol-resistant peptide aptamer for the target molecule, containing an amino acid linked with a fluorescent molecule, and (2) an alcohol.

Description

Method for detecting target molecules using peptide aptamers

The present invention relates to a method for detecting a target molecule using a peptide aptamer.

As society develops, the paradigm of human health has continued to strive to deal with the emergence of new diseases. To manage the disease caused by the pandemic coronavirus that emerged at the end of 2020, it is necessary to simultaneously conduct research such as prescribing antibiotics for prevention, laboratory investigations for diagnosis, isolating and caring for infected individuals, and investigating the cause of the spread of infection. Until now, research directions such as the development of antiviral drugs and diagnostic methods have required antibodies that can strongly bind to specific antigens.

Influenza virus, one of the causes of infectious diseases, is transmitted through the respiratory tract, and it has been reported that 5-10% of adults and 20-30% of children worldwide are infected every year. Symptoms of influenza virus infection range from simple high fever, cough, abdominal pain, and muscle pain to severe death, and 250,000 to 500,000 people die every year. Influenza virus is composed of eight single-stranded RNAs and envelope proteins. Influenza virus infection begins when the hemagglutinin (HA) protein expressed on the surface of the virus binds to the sialic acid receptor expressed on the surface of the host. After invading the cell, it is replicated and released outside the cell by binding to the host cell protein, and continues to grow. At that time, it has been reported that the nucleoprotein (NP), which is the main structural protein of the influenza virus, is involved in transcription and replication. The NP, which contains the viral RNA, contains a nuclear transport sequence, and is known to play a role not only in transporting the viral RNA into the nucleus, but also in stabilizing the transcribed and replicated RNA.

Currently, real-time PCR, RT-PCR, enzyme-linked immunosorbent assay (ELISA), immunochromatography, and other methods are used to diagnose influenza virus infections. Although these diagnostic methods are highly specific and reliable, they all require some effort. For example, real-time PCR and RT-PCR require an amplification step using a thermal cycler with enzymes and primers. ELISA usually requires combining a primary antibody that recognizes the influenza virus itself with a secondary antibody that recognizes the primary antibody, and then adding a reagent so that the secondary antibody emits fluorescence to capture the influenza virus. In addition, because the diagnosis of influenza virus infections involves the risk of infection for medical personnel involved in the diagnosis, reducing this risk has also been a challenge.

Meanwhile, peptide aptamers have been attracting attention as molecules other than antibodies for capturing target molecules. Peptide aptamers are peptides that specifically bind to target molecules (proteins, sugar chains, cells, viruses, etc.), and can bind to target molecules via the three-dimensional structure formed by the peptide. Furthermore, by including a fluorescent or luminescent molecule in the peptide sequence, when the peptide aptamer binds to a target molecule, the solvent environment around the fluorescent or luminescent molecule changes, allowing it to emit fluorescence (Non-Patent Document 1). This eliminates the need for a process such as washing away antibodies that are not bound to the target molecule, as in ELISA, and for reagents that emit fluorescence.

Peptide aptamers are molecules with high binding specificity and have been developed for a variety of purposes, including medical diagnosis, treatment, and biosensors.

Wei Wang et al., Chem Commun., 50(22), 2962-2964, 2014

The objective of the present invention is to create peptide aptamers that specifically bind to viruses, including influenza, and other target molecules, and to provide a method for detecting target molecules using the peptide aptamers more simply and safely than conventional methods such as real-time PCR, RT-PCR, ELISA, and immunochromatography.

The present inventors used the ribosome display method to screen for peptide sequences containing amino acids linked to the fluorescent molecule 7-nitro-2,1,3-benzoxadiazole (NBD) or fluorescein, which have high affinity for influenza virus NP. Furthermore, when the peptide dissolved in an alcohol solvent was reacted with influenza virus NP spotted on a membrane filter, it was confirmed that the peptide bound to the influenza virus NP based on the change in fluorescence intensity. Furthermore, when the above method was performed using an antibody against influenza virus NP instead of the peptide, the antibody was unable to bind to influenza virus NP. Furthermore, when the peptide was reacted in an alcohol solvent with influenza virus NP contained in the alcohol solvent, it was confirmed that the peptide bound to influenza virus NP based on the change in fluorescence intensity. The present inventors conducted further research based on these findings, and as a result, they have completed the present invention.

That is, the present invention is as follows.
[1] A detection agent for a target molecule, comprising the following (1) and (2):
(1) An alcohol-tolerant peptide aptamer for a target molecule, comprising an amino acid linked to a fluorescent molecule;
(2) Alcohol.
[2] A fluorescent molecule having the following formula:

[In the formula, n is an integer from 0 to 6.]
or a compound represented by the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
The detection agent according to [1], which is a compound represented by the formula:
[3] The detection agent according to [1] or [2], wherein the target molecule is immobilized on a carrier.
[4] The detection agent according to [1] or [2], wherein the target molecule is contained in a liquid phase.
[5] The detection agent according to any one of [1] to [4], wherein the target molecule is a protein constituting an influenza virus.
[6] The detection agent according to [5], wherein the protein constituting the influenza virus is a nucleoprotein.
[7] An alcohol-tolerant peptide aptamer for a target molecule, comprising an amino acid linked to a fluorescent molecule, represented by the following formula:
XM(Xaa1)(Xaa2)VWL(Xaa3)F(Xaa4-Xaa10) (SEQ ID NO: 1)
[wherein X is the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
where Xaa1-Xaa10 are any amino acids.
The detection agent according to [6], which is a compound represented by the formula:
[8] Xaa1 is a basic amino acid selected from the group consisting of R, H and K, or A;
Xaa2 is a basic amino acid selected from the group consisting of R, H and K, or A;
Xaa3 is a hydrophobic amino acid selected from the group consisting of G, P, A, I, L, M, V, F and W;
Xaa4 is a basic amino acid selected from the group consisting of R, H and K, or A;
Xaa5 is a basic amino acid selected from the group consisting of R, H and K, or A;
Xaa6 is a neutral amino acid selected from the group consisting of N, C, Q, S and T, or A;
Xaa7 is a neutral amino acid selected from the group consisting of N, C, Q, S and T, or A;
Xaa8 is a hydrophobic amino acid selected from the group consisting of G, P, A, I, L, M, V, F and W, Xaa9 is a basic amino acid selected from the group consisting of R, H and K or A, and
Xaa10 is a neutral amino acid selected from the group consisting of N, C, Q, S and T, or A;
The detection agent according to claim 7.
[9] An alcohol-tolerant peptide aptamer for a target molecule, comprising an amino acid linked to a fluorescent molecule, represented by the following formula:
XMKHVWLGFKRSSWRC (SEQ ID NO: 2)
[wherein X is the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
The detection agent according to [7] or [8], which is a compound represented by the formula:
[10] The detection agent according to [9], wherein m is 1 and n is 2.
[11] An alcohol-tolerant peptide aptamer for a target molecule, comprising an amino acid linked to a fluorescent molecule, represented by the following formula:
(1) XMKHVFLRRRRRWGFWC (SEQ ID NO: 3),
(2) MTTCRFSVYBMRLGFC (SEQ ID NO: 4),
(3) MTTCBGWSTBWASLP (SEQ ID NO: 5),
(4) MTTCGBSSIBVWGLNC (SEQ ID NO: 6),
(5) XMKHVLGFWRRRGWC (SEQ ID NO: 7),
(6) MTTCFRRGNBRBVFSC (SEQ ID NO: 8),
(7) XMTNVWWGWRRRWRLG (SEQ ID NO: 9),
(8) XMTTCRRRRGWRWLGWC (SEQ ID NO: 10), and
(9) XMKHVGWFGRLRRWC (SEQ ID NO: 11)
[In each formula, X represents the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
B is a compound represented by the following formula:

[In the formula, -COOH of adjacent amino acids and _-NH2 in the formula form a peptide bond, and -COOH in the formula and _-NH2 of another adjacent amino acid form a peptide bond.]
It is a modified phenylalanine represented by the formula:
The detection agent according to [6], which is a compound selected from the group consisting of:
[12] The detection agent according to [11], wherein m is 1 and n is 2.
[13] The detection agent according to any one of [1] to [4], wherein the target molecule is ovalbumin.
[14] An alcohol-tolerant peptide aptamer for a target molecule, comprising an amino acid linked to a fluorescent molecule, represented by the following formula:
XMTTCTRRRSRWNWICSWD (SEQ ID NO: 12)
[wherein X is the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
It is a compound represented by the formula:
The detection agent according to [13], which is a compound represented by the formula:
[15] The detection agent according to [14], wherein m is 0.
[16] The detection agent according to any one of [1] to [4], wherein the target molecule is a nucleoprotein constituting norovirus.
[17] An alcohol-tolerant peptide aptamer for a target molecule, comprising an amino acid linked to a fluorescent molecule, represented by the following formula:
(1) XMKHVLFIFFRCGRSVLG (SEQ ID NO: 13), and
(2) XMTTCFYYRRSRTWVC (SEQ ID NO: 14)
[wherein X is the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
It is a compound represented by the formula:
The detection agent according to [16], which is a compound selected from the group consisting of:
[18] The detection agent according to [17], wherein (1) m is 1 and n is 2, or (2) m is 0.
[19] The detection agent according to any one of [1] to [18], wherein the peptide aptamer has a final concentration of 0.02 μM to 10 μM.
[20] The detection agent according to any one of [1] to [19], wherein the alcohol is methanol, ethanol or isopropanol.
[21] The detection agent according to any one of [1] to [20], wherein the final concentration of alcohol is 40% to 80%.
[22] A method for detecting a target molecule in a test sample, comprising the following steps:
(1) contacting a test sample with the detection agent according to any one of [1] to [21], (2) measuring the fluorescence intensity of a fluorescent molecule, and (3) determining the presence or absence of a target molecule in the test sample based on a change in the fluorescence intensity.
[23] The detection method according to [22], wherein the test sample is immobilized on a carrier.
[24] The detection method according to [22], wherein the test sample is contained in a liquid phase.
[25] A method for screening a peptide aptamer for a target molecule, comprising the steps of:
(1) preparing a random peptide library consisting of peptides containing amino acids linked to fluorescent molecules;
(2) evaluating the stability of the secondary structure of each peptide contained in the library in the presence and absence of alcohol;
(3) selecting a peptide whose secondary structure stability in the presence of alcohol is equal to or greater than the same level as that in the absence of alcohol;
(4) contacting the selected peptide with a target molecule in the presence of alcohol;
(5) measuring the fluorescence intensity of the fluorescent molecule; and (6) selecting a peptide aptamer for the target molecule based on the change in fluorescence intensity.
[26] The screening method according to [25], wherein in step (2), the stability of the secondary structure of the peptide is evaluated by circular dichroism measurement.

In the case of the dot blot method, after spotting a test sample suspected of containing a target molecule onto a hydrophilized filter, in the case of staining with an antibody, it is necessary to re-hydrophilize the filter. This is because antibodies often undergo a change in secondary structure in the presence of alcohol and lose their ability to bind to target molecules, so a step of removing the alcohol used to hydrophilize the filter from the filter is essential. On the other hand, the peptide aptamer for a target molecule found in the present invention, which contains amino acids linked to fluorescent molecules, can bind to the target molecule spotted on the filter and emit fluorescence even if it is mixed with alcohol in advance. Furthermore, even if the peptide aptamer of the present invention is mixed with a target molecule dissolved in a solvent containing alcohol, it can bind to the target molecule and emit fluorescence. This suggests that the binding activity of the peptide aptamer of the present invention is not lost due to alcohol. Therefore, when detecting a target molecule using the peptide aptamer of the present invention, there is no need to dealcoholize the solvent or filter, which was necessary in the dot blot method using conventional antibodies. Furthermore, if the target molecule is a virus or bacterium that causes an infectious disease, conventional detection methods cannot use alcohol because it inhibits detection, and medical personnel involved in the detection are at risk of infection. In this regard, even though the detection agent of the present invention contains high concentrations of alcohol, it is able to detect the target molecule without denaturing the peptide aptamer or inhibiting the fluorescence intensity. Therefore, the risk of infection during detection can be reduced by denaturing the virus or bacteria with the high concentrations of alcohol contained in the detection agent.

Figure 1 shows in vitro selection of peptide aptamers incorporating fluorescent amino acids capable of binding to influenza virus nucleoprotein by ribosome display. Figure 2 shows the specific binding ability of NPBP2 to influenza virus NP by dot blot assay obtained from NPBP2. A) 2 μg of NP (top) and BSA (bottom) were spotted onto a PVDF membrane filter. B) Background subtracted image. C, D, E) Fluorescence intensity along the line profile of the raw image. Figure 3 shows the specific binding ability of NPBP8 to influenza virus NP by dot blot assay obtained from NPBP8. A) 2 μg of NP (top) and BSA (bottom) were spotted onto a PVDF membrane filter. B) Background subtracted image. C, D, E) Fluorescence intensity along the line profile of the raw image. Figure 4 shows the specific binding ability of NPBP2 to influenza virus NP by dot blot assay obtained from NPBP2. A) Graded amounts (20, 15, 10, 7.5, 5, 2.5, 1, 0.5, 0.25 μg) of NP (top three rows) and BSA (bottom three rows) were spotted onto a membrane filter. B) Background subtracted images. C, D, E) Fluorescence intensity along the line profile of the raw images. Figure 5 shows the specific binding ability of NPBP8 to influenza virus NP by dot blot assay obtained from NPBP8. A) Graded amounts (20, 15, 10, 7.5, 5, 2.5, 1, 0.5, 0.25 μg) of NP (top three rows) and BSA (bottom three rows) were spotted onto a membrane filter. B) Background subtracted images. C, D, E) Fluorescence intensity along the line profile of the raw images. FIG. 6 shows that the fluorescence intensities of NPBP2 and NPBP8 are clearly dependent on the amount of NP spotted onto the membrane filter. FIG. 7 shows that binding of NPBP2, 6, and 11 to full-length NP was confirmed using biolayer interferometry (BLI). FIG. 8 shows the optimal concentrations of methanol, ethanol, and isopropanol for hydrophilizing a PVDF membrane filter. FIG. 9 shows the results of reacting influenza virus NP spotted on a PVDF membrane filter hydrophilized with ethanol with NPBP2 dissolved in 50%, 70% or 80% alcohol. FIG. 10 shows the results of reacting influenza virus NP spotted on a PVDF membrane filter hydrophilized with ethanol with NPBP6 dissolved in 50%, 70% or 80% alcohol. Figure 11 shows the simple dot blot method using antibodies. A) When the antibody was dissolved in high-concentration alcohol, influenza virus NP could not be detected. B) When the simple dot blot method was performed using antibodies without blocking treatment and incubation time, influenza virus NP could hardly be detected. Figure 12 shows the reaction of influenza virus NP, SARS2 NP, saliva 50%, MERS NP, NL63 NP and OC43NP spotted on a PVDF membrane filter hydrophilized with ethanol with NPBP6 dissolved in 50% ethanol. Figure 13 shows the reaction of ovomucoid spotted on a PVDF membrane filter hydrophilized with ethanol (from left: 2 μg ovomucoid, 2 μg ovomucoid (50% saliva/nasal solution), 100% saliva/nasal solution only) with the ovomucoid-binding peptide OvaBP6 dissolved in 50% ethanol. Figure 14 shows norovirus, which is available as a reagent, spotted onto a PVDF membrane filter that has been hydrophilized with ethanol (from left: 2 μg of norovirus, 2 μg of norovirus (50% saliva/nasal water), 100% saliva/nasal water only) and reacted with Noro1, which binds to norovirus dissolved in 50% ethanol. Fig. 15A is a diagram showing the fluorescence intensity in a liquid state when NPs of various concentrations are added to NPBP6 at a final concentration of 0.1 μM. Fig. 15B is a diagram showing the fluorescence intensity in a liquid state when NPs of various concentrations are added to NPBP6 at a final concentration of 0.1 μM containing 10% ethanol. FIG. 16 shows the binding ability of NP to mutant peptides in which the amino acids constituting NPBP6 are replaced one by one with alanine.

1. Target Molecule Detector The present invention provides a simple and safe target molecule detector (hereinafter, the detector of the present invention).

Target molecules detected by the detection agent of the present invention are not particularly limited, but include cells, microorganisms, vesicles secreted by the cells or microorganisms, viruses, nucleic acids, proteins, etc.

The cells are not particularly limited as long as they are eukaryotic cells, and are defined as including animal cells, plant cells, and fungi, with animal cells being preferred. Examples of animal cells include, but are not limited to, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells, stem cells, or cancer cells of these cells. Genetically modified versions of the above cells are also preferably used.

Microorganisms include eubacteria (hereinafter simply referred to as "bacteria") and archaebacteria, with bacteria being preferred among them. There is no particular limitation on the bacteria as long as they are prokaryotes having a cell membrane composed of a fatty acid ester of glycerol 3-phosphate, and they may be gram-negative or gram-positive bacteria. Gram-negative bacteria include, but are not limited to, the genus Neisseria, Branhamella, Haemophilus, Bordetella, Escherichia, Citrobacter, Salmonella, Shigella, Klebsiella, Enterobacter, Serratia, Hafnia, Proteus, Morganella, Providencia, and the like. Examples of bacteria include those belonging to the genera Providencia, Yersinia, Campylobacter, Vibrio, Aeromonas, Pseudomonas, Xanthomonas, Acinetobacter, Flavobacterium, Brucella, Legionella, Veillonella, Bacteroides, and Fusobacterium. Examples of gram-positive bacteria include, but are not limited to, bacteria belonging to the genera Staphylococcus, Streptococcus, Enterococcus, Corynebacterium, Bacillus, Listeria, Peptococcus, Peptostreptococcus, Clostridium, Eubacterium, Propionibacterium, and Lactobacillus.

Vesicles secreted by cells or microorganisms are not particularly limited as long as they have a lipid bilayer membrane as the outermost membrane, and examples of such vesicles include extracellular vesicles (e.g., exosomes, membrane vesicles, exosome-like vesicles, etc.).

Viruses include capsid-type viruses and envelope-type viruses. Capsid-type viruses include, but are not limited to, viruses belonging to the Papillomaviridae (e.g., human papillomavirus, etc.), Picornaviridae (e.g., poliovirus, hepatitis A virus, etc.), Caliciviridae (e.g., norovirus, etc.), and Reoviridae (e.g., rotavirus, etc.). Examples of enveloped viruses include, but are not limited to, viruses belonging to the Herpesviridae (e.g., chickenpox/varicella-zoster virus, etc.), Poxviridae (e.g., smallpox virus, etc.), Hepadnaviridae (e.g., hepatitis B virus, etc.), Flaviviridae (e.g., hepatitis C virus, Japanese encephalitis virus, Zika virus, etc.), Togaviridae (e.g., rubella virus, etc.), Coronaviridae (e.g., SARS coronavirus, MERS coronavirus, SARS coronavirus 2, etc.), Orthomyxoviridae (e.g., influenza virus, etc.), Paramyxoviridae (e.g., measles virus, human respiratory syncytial virus, etc.), Rhabdoviridae (e.g., rabies virus, etc.), Bunyaviridae (e.g., Crimean-Congo hemorrhagic fever virus, etc.), Filoviridae (e.g., Ebola virus, Marburg virus, etc.), and Retroviridae (e.g., human immunodeficiency virus, adult T-cell leukemia virus, etc.).
When the target molecule to be detected by the detection agent of the present invention is a virus, influenza virus is preferred among the above viruses in terms of practical demand.

The nucleic acid is not particularly limited, and examples thereof include nucleic acids derived from the above-mentioned cells, microorganisms, vesicles secreted by the cells or microorganisms, and viruses. The nucleic acid may be DNA or RNA, and may be single-stranded or double-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA/RNA hybrid.

The protein is not particularly limited, and examples thereof include the above-mentioned cells, microorganisms, vesicles secreted by the cells or microorganisms, virus-derived proteins, and allergens.
When the target molecule to be detected by the detection agent of the present invention is a protein derived from an influenza virus, nucleoprotein is preferred because it is abundant therein.

The form of the target molecule detected by the detection agent of the present invention is not particularly limited as long as it can be detected by the detection agent of the present invention, but it may be in a form immobilized on a carrier or contained in a liquid phase.

When the target molecule is immobilized on a carrier, examples of the carrier include synthetic resins such as polystyrene, polyacrylamide, and silicone, glass, thin metal films, PVDF membranes, and nitrocellulose membranes.

In the case where the target molecule is contained in a liquid phase, the liquid phase can be, for example, water, a buffer solution (e.g., phosphate buffer, citrate buffer, acetate buffer, Tris buffer, etc.), or an alcohol (e.g., methanol, ethanol, propanol, isopropanol, etc.).

The detection agent of the present invention includes the following (1) and (2).
(1) An alcohol-tolerant peptide aptamer for a target molecule, which comprises an amino acid linked to a fluorescent molecule (the peptide aptamer of the present invention).
(2) Alcohol.

The peptide aptamer of the present invention refers to a peptide molecule that has binding activity for a specific target molecule. The peptide aptamer of the present invention may be in the form of a linear, branched or dendrimer, and is preferably in the form of a linear aptamer.

When the peptide aptamer of the present invention is linear, its length is not particularly limited as long as it has binding activity to a predetermined target molecule, and can usually be about 50 amino acids or less, for example, about 30 amino acids or less, and preferably about 20 amino acids or less. A smaller total number of amino acids makes chemical synthesis and mass production easier, and also has a large cost advantage. It is also easier to chemically modify, has high in vivo stability, and is thought to have low toxicity. There is no particular limit to the lower limit of the length of the peptide aptamer of the present invention, but the length of the peptide aptamer can be, for example, about 5 amino acids or more, preferably about 10 amino acids or more. In a particularly preferred embodiment, the length of the peptide aptamer of the present invention is 14 to 19 amino acids.

The peptide aptamer of the present invention is a peptide aptamer having alcohol resistance. A peptide aptamer being alcohol resistant means that the stability of the secondary structure of the peptide in the presence of alcohol is at the same level or higher than that in the absence of alcohol. The stability of the secondary structure of the peptide can be examined by a method known in the prior art, for example, the stability of the secondary structure of the peptide can be evaluated by a circular dichroism measurement method. Specifically, when the absolute values of the circular dichroism values (mdeg) of the peptide at wavelengths of 208 nm and 222 nm measured by the circular dichroism measurement method in the presence of alcohol are the same or higher than those in the presence of non-alcohol, the peptide can be evaluated as forming a more stable α-helix structure in the presence of alcohol compared to that in the presence of non-alcohol. Similarly, when the absolute values of the circular dichroism values (mdeg) of the peptide at wavelengths of 216 nm measured by the circular dichroism measurement method in the presence of alcohol are the same or higher than those in the presence of non-alcohol, the peptide can be evaluated as forming a more stable β-sheet structure in the presence of alcohol compared to that in the presence of non-alcohol.

The amino acids constituting the peptide aptamer of the present invention are linked to fluorescent molecules. The amino acids constituting the peptide aptamer of the present invention may be either natural amino acids or non-natural amino acids. Furthermore, the amino acids may be either L- or D-amino acids.

The fluorescent molecule linked to the amino acid is not particularly limited as long as it is a molecule whose fluorescence intensity changes depending on the change in the solvent environment. For example, the following formula is one such fluorescent molecule:

[In the formula, n is an integer from 0 to 6.]
Examples of the compound include compounds represented by the following formula:

The amino acid constituting the peptide aptamer of the present invention to which the fluorescent molecule is linked includes, but is not limited to, 4-amino-phenylalanine, for example.

Another fluorescent molecule is, for example, the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6]
In one preferred embodiment, m is 0. In another preferred embodiment, when m is 1, n is 2 or 5.

The amino acid constituting the peptide aptamer of the present invention to which the fluorescent molecule is linked includes the N-terminal amino acid of the peptide aptamer of the present invention or lysine in the peptide chain.

The amino acid to which the fluorescent molecule represented by the above formula is linked is not particularly limited as long as it does not inhibit the binding activity to a specific target molecule, and may be linked to any amino acid constituting the peptide aptamer of the present invention. When linking, the functional groups of the side chains of the amino acids linked to -CO- in the fluorescent molecule represented by the above formula may be directly linked to each other.

In addition, in the peptide aptamer of the present invention, the number of amino acids to which the fluorescent molecule represented by the above formula is linked is not limited as long as a change in fluorescence intensity can be detected, and it may be linked to one to several (9, 8, 7, 6, 5, 4, 3, or 2) amino acids.

Further examples of fluorescent molecules that may be linked to the above amino acids include fluorescein isothiocyanate (FITC), 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) or tetraphenylethylene (TPE). Examples of amino acids that constitute the peptide aptamer of the present invention to which the fluorescent molecule is linked include the N-terminal amino acid of the peptide aptamer of the present invention or lysine in the peptide chain.

When the target molecule of the detection agent of the present invention is a nucleoprotein derived from influenza virus, one embodiment of the peptide aptamer of the present invention includes the following peptide (peptide I of the present invention).
XM(Xaa1)(Xaa2)VWL(Xaa3)F(Xaa4-Xaa10) (SEQ ID NO: 1)
[wherein X is the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
where Xaa1-Xaa10 are any amino acids.

In one preferred embodiment, m is 0. In another preferred embodiment, when m is 1, n is 2 or 5.

In peptide I of the present invention, Xaa1 may be any amino acid as long as peptide I of the present invention has binding activity to influenza virus-derived nucleoprotein, but is preferably a basic amino acid selected from the group consisting of R, H, and K, or A, and more preferably K.

In peptide I of the present invention, Xaa2 may be any amino acid as long as peptide I of the present invention has binding activity to the influenza virus-derived nucleoprotein, but is preferably a basic amino acid selected from the group consisting of R, H, and K, or A, and more preferably H.

In peptide I of the present invention, Xaa3 may be any amino acid as long as peptide I of the present invention has binding activity to the influenza virus-derived nucleoprotein, but is preferably a hydrophobic amino acid selected from the group consisting of G, P, A, I, L, M, V, F and W, and more preferably G.

In peptide I of the present invention, Xaa4 may be any amino acid as long as peptide I of the present invention has binding activity to influenza virus-derived nucleoprotein, but is preferably a basic amino acid selected from the group consisting of R, H, and K, or A, and more preferably K.

In peptide I of the present invention, Xaa5 may be any amino acid as long as peptide I of the present invention has binding activity to influenza virus-derived nucleoprotein, but is preferably a basic amino acid selected from the group consisting of R, H, and K, and more preferably R.

In peptide I of the present invention, Xaa6 may be any amino acid as long as peptide I of the present invention has binding activity to the influenza virus-derived nucleoprotein, but is preferably a neutral amino acid selected from the group consisting of N, C, Q, S, and T, or A, and more preferably S.

In peptide I of the present invention, Xaa7 may be any amino acid as long as peptide I of the present invention has binding activity to influenza virus-derived nucleoprotein, but is preferably a neutral amino acid selected from the group consisting of N, C, Q, S and T, and more preferably S.

In peptide I of the present invention, Xaa8 may be any amino acid as long as peptide I of the present invention has binding activity to the influenza virus-derived nucleoprotein, but is preferably a hydrophobic amino acid selected from the group consisting of G, P, A, I, L, M, V, F and W, and more preferably W.

In peptide I of the present invention, Xaa9 may be any amino acid as long as peptide I of the present invention has binding activity to influenza virus-derived nucleoprotein, but is preferably a basic amino acid selected from the group consisting of R, H, and K, or A, and more preferably R.

In peptide I of the present invention, Xaa10 may be any amino acid as long as peptide I of the present invention has binding activity to influenza virus-derived nucleoprotein, but is preferably a neutral amino acid selected from the group consisting of N, C, Q, S, and T, or A, and more preferably C.

As a specific embodiment of peptide I of the present invention,
[wherein X is the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6]
In one preferred embodiment, when m is 1, n is 2.

When the target molecule of the detection agent of the present invention is a nucleoprotein derived from influenza virus, further embodiments of the peptide aptamer of the present invention include the following peptides.
(1) XMKHVFLRRRRRWGFWC (SEQ ID NO: 3),
(2) MTTCRFSVYBMRLGFC (SEQ ID NO: 4),
(3) MTTCBGWSTBWASLP (SEQ ID NO: 5),
(4) MTTCGBSSIBVWGLNC (SEQ ID NO: 6),
(5) XMKHVLGFWRRRGWC (SEQ ID NO: 7),
(6) MTTCFRRGNBRBVFSC (SEQ ID NO: 8),
(7) XMTNVWWGWRRRWRLG (SEQ ID NO: 9),
(8) XMTTCRRRRGWRWLGWC (SEQ ID NO: 10), and
(9) XMKHVGWFGRLRRWC (SEQ ID NO: 11)
[In each formula, X represents the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6]
B is a compound represented by the following formula:

[In the formula, -COOH of adjacent amino acids and _-NH2 in the formula form a peptide bond, and -COOH in the formula and _-NH2 of another adjacent amino acid form a peptide bond.]
It is a modified phenylalanine represented by the formula:
In a preferred embodiment, when m is 1, n is 2. Among these, (2) MTTCRFSVYBMRLGFC (SEQ ID NO: 4) and (9) XMKHVGWFGRLRRWC (SEQ ID NO: 11) are preferred.

When the target molecule of the detection agent of the present invention is ovalbumin, the peptide aptamer of the present invention includes the following peptide (peptide II of the present invention).
XMTTCTRRRSRWNWICSWD (SEQ ID NO: 12)
[wherein X is the following formula:

[In the formula, m is 0 or 1, and n is an integer from 0 to 6]
It is a compound represented by the formula:
In one preferred embodiment, m is 0.

When the target molecule of the detection agent of the present invention is a protein constituting a norovirus, the peptide aptamer of the present invention includes the following peptide (peptide III of the present invention).
(1) XMKHVLFIFFRCGRSVLG (SEQ ID NO: 13), and
(2) XMTTCFYYRRSRTWVC (SEQ ID NO: 14)
[In each formula, X is

[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
It is a compound represented by the formula:
In one preferred embodiment, (1) m is 1 and n is 2, or (2) m is 0.

The lower limit of the final concentration of the peptide aptamer of the present invention contained in the detection agent of the present invention is not particularly limited, but is, for example, usually 0.02 μM or more, preferably 0.25 μM or more, more preferably 0.5 μM or more, and even more preferably 1 μM or more. If the final concentration of the peptide aptamer is lower than this, it is not preferred because it falls below the detection sensitivity limit. The upper limit of the final concentration of the peptide aptamer of the present invention contained in the detection agent of the present invention is not particularly limited, but is, for example, usually 10 μM or less, preferably 7.5 μM or less, and more preferably 5 μM or less. If the final concentration of the peptide aptamer is higher than this, it is not preferred because there is a concern that non-specific binding may occur.

The detection agent of the present invention also contains an alcohol. There are no limitations on the alcohol contained in the detection agent of the present invention, so long as it can dissolve the peptide aptamer of the present invention, does not denature the aptamer, promotes the formation of its secondary structure, does not inhibit the binding of the aptamer to the target molecule, and does not inhibit the change in the fluorescence intensity of the fluorescent molecule that accompanies the binding. Examples of such alcohols include methanol, ethanol, propanol, and isopropanol, and ethanol is preferred for general use.

The final concentration of alcohol contained in the detection agent of the present invention is not limited as long as it can dissolve the peptide aptamer of the present invention, does not denature the aptamer, promotes the formation of its secondary structure, does not inhibit the binding of the aptamer to the target molecule, and does not inhibit the change in fluorescence intensity of the fluorescent molecule that accompanies binding.
When the alcohol is methanol, the lower limit of the final concentration of methanol contained in the detection agent of the present invention is usually 40% or more, preferably 45% or more, more preferably 50% or more. The upper limit of the methanol contained in the detection agent of the present invention is usually 80% or less, preferably 60% or less, more preferably 55% or less.
When the alcohol is ethanol, the lower limit of the final concentration of ethanol contained in the detection agent of the present invention is usually 40% or more, preferably 45% or more, more preferably 50% or more. The upper limit of the ethanol contained in the detection agent of the present invention is usually 80% or less, preferably 60% or less, more preferably 55% or less.
When the alcohol is isopropanol, the lower limit of the final concentration of isopropanol contained in the detection agent of the present invention is usually 40% or more, preferably 45% or more, more preferably 50% or more. The upper limit of isopropanol contained in the detection agent of the present invention is usually 80% or less, preferably 60% or less, more preferably 55% or less.

2. Method for detecting target molecules As described above, by using the detection agent of the present invention, the presence or absence of a target molecule in a test sample can be detected simply by measuring the fluorescence intensity of the fluorescent molecule, without the use of a secondary antibody or staining of the secondary antibody as in an antigen-antibody reaction. Furthermore, although the detection agent of the present invention contains a high concentration of alcohol, it is unexpectedly not affected by the denaturation of the peptide aptamer or the inhibition of the fluorescence intensity, so that the alcohol can denature viruses and bacteria at the same time as detecting them, reducing the risk of infection for the tester.
Therefore, the present invention also provides a method for detecting a target molecule in a test sample using the detection agent of the present invention (the detection method of the present invention).

The test sample used in the detection method of the present invention is not particularly limited as long as it is a sample that may contain the target molecule of interest. The test sample may be liquid or solid. An example of a liquid sample is a biological sample. The biological sample may be tissue, cells, etc., but examples include those that can be easily collected from a living body, such as blood, plasma, serum, urine, and saliva. When serum or plasma is used, they can be prepared by collecting blood according to a conventional method and separating the liquid components. Other examples of liquid samples include water from rivers, ponds, swamps, etc., industrial wastewater, domestic wastewater, water from waterworks, drinking water, etc. Examples of solid samples include dried solids of the above liquid samples.

The detection method of the present invention comprises the following steps.
(1) A step of contacting the detection agent of the present invention with a test sample (step (1) of the detection method of the present invention).
(2) A step of measuring the fluorescence intensity of the fluorescent molecule (step (2) of the detection method of the present invention).
(3) A step of determining the presence or absence of a target molecule in a test sample based on the change in fluorescence intensity (step (3) of the detection method of the present invention).

In step (1) of the detection method of the present invention, the detection agent of the present invention is brought into contact with the test sample. The manner of contact between the detection agent of the present invention and the test sample may vary depending on the test sample. When the test sample is a liquid sample, the test sample and the detection agent of the present invention can be mixed to bring them into contact with each other. When the test sample is a solid sample, the detection agent of the present invention can be brought into direct contact with the solid sample, or the solid sample can be dissolved in a solvent and then mixed with the detection agent of the present invention to bring them into contact with each other. Examples of the solvent include water, buffer solutions (e.g., phosphate buffer, citrate buffer, acetate buffer, Tris buffer, etc.), and alcohols (e.g., methanol, ethanol, propanol, isopropanol, etc.).

The contact time and temperature between the detection agent of the present invention and the test sample can be appropriately selected by those skilled in the art, but for example, contact at room temperature for one to several minutes may be sufficient.

In step (2) of the detection method of the present invention, the fluorescence intensity of the fluorescent molecules of the detection agent of the present invention that came into contact with the test sample in step (1) is measured. The fluorescence intensity can be measured using a commercially available fluorescence measuring device.

In step (3) of the detection method of the present invention, the presence or absence of a target molecule in the test sample is determined based on the change in fluorescence intensity measured in step (2). The change in fluorescence intensity can be obtained, for example, by calculating the difference between the fluorescence intensity (reference fluorescence intensity) measured after contact of a substance that does not bind to the peptide aptamer of the present invention with the detection agent of the present invention and the fluorescence intensity (measured fluorescence intensity) measured in step (2). If there is a difference between the reference fluorescence intensity and the measured fluorescence intensity, it can be determined that the test sample contains a target molecule that is detected by the detection agent of the present invention.

Alternatively, a standard curve relating the fluorescence intensity of the detection agent of the present invention to the amount of the target molecule can be created, and the presence or absence of the target molecule in the test sample can be determined from the measured fluorescence intensity based on the standard curve.

3. Screening method for peptide aptamers for target molecules Although the peptide aptamers of the present invention contain high concentrations of alcohol, the secondary structure of the peptide aptamers is unexpectedly not denatured. This demonstrates the existence of peptides whose secondary structure is not denatured by alcohol. Based on this discovery, the inventors came up with the idea of selecting a peptide library whose secondary structure is not denatured by alcohol from a random peptide library, and then more quickly screening candidate molecules for peptide aptamers that can bind to target molecules even in the presence of alcohol from the peptide library.
Therefore, the present invention also provides a method for screening peptide aptamers against a target molecule (the screening method of the present invention).

The screening method of the present invention comprises the following steps.
(1) A step of preparing a random peptide library consisting of peptides containing amino acids linked to fluorescent molecules (step (1) of the screening method of the present invention).
(2) A step of evaluating the stability of the secondary structure of each peptide contained in the library in the presence and absence of alcohol (step (2) of the screening method of the present invention).
(3) A step of selecting peptides whose secondary structure stability in the presence of alcohol is at least at the same level as that in the absence of alcohol (step (3) of the screening method of the present invention).
(4) contacting the selected peptide with a target molecule in the presence of alcohol (step (4) of the screening method of the present invention).
(5) Measuring the fluorescence intensity of the fluorescent molecule (step (5) of the screening method of the present invention).
(6) A step of selecting a peptide aptamer for the target molecule based on the change in fluorescence intensity (step (6) of the screening method of the present invention).

In step (1) of the screening method of the present invention, a random peptide library is prepared consisting of peptides containing amino acids linked to fluorescent molecules. The fluorescent molecules and the amino acids linked to the fluorescent molecules may be the same as those described in the detection agent of the present invention. The random peptides are not particularly limited, but may be in the form of a straight chain, a branched chain, or a dendrimer, and are preferably straight chain.

The length of the peptide is not particularly limited as long as it has binding activity to a predetermined target molecule, and may usually be about 50 amino acids or less, for example, about 30 amino acids or less, and preferably about 20 amino acids or less. If the total number of amino acids is small, chemical synthesis and mass production are easier, and there are also great benefits in terms of cost. In addition, chemical modification is also easier, and it is thought that the in vivo stability is high and the toxicity is low. There is no particular limit to the lower limit of the peptide length, but the length of the peptide may be, for example, about 5 amino acids or more, and preferably about 10 amino acids or more.

The peptide can be synthesized by a method known per se in the art. For example, the peptide can be produced by (a) chemical synthesis using a known peptide synthesis method using a peptide synthesizer or the like, (b) culturing a transformant containing DNA encoding the peptide, or (c) biochemical synthesis using a cell-free transcription/translation system with a nucleic acid encoding the peptide as a template.

In step (2) of the screening method of the present invention, the stability of the secondary structure of each peptide contained in the library in the presence and absence of alcohol is evaluated. The alcohol used in this step is not particularly limited, but examples include methanol, ethanol, propanol, isopropanol, etc.

The final concentration of the alcohol used in this step is not limited as long as it does not denature the peptide.
When the alcohol is methanol, the lower limit of the final concentration of methanol used in this step is usually 40% or more, preferably 45% or more, more preferably 50% or more, and the upper limit of the final concentration of methanol used in this step is usually 80% or less, preferably 60% or less, more preferably 55% or less.
When the alcohol is ethanol, the lower limit of the final concentration of ethanol used in this step is usually 40% or more, preferably 45% or more, more preferably 50% or more, and the upper limit of the final concentration of ethanol used in this step is usually 80% or less, preferably 60% or less, more preferably 55% or less.
When the alcohol is isopropanol, the lower limit of the final concentration of isopropanol used in this step is usually 40% or more, preferably 45% or more, more preferably 50% or more, and the upper limit of the final concentration of isopropanol used in this step is usually 80% or less, preferably 60% or less, more preferably 55% or less.

In this step, the stability of the secondary structure of each peptide contained in the library in the presence and absence of alcohol can be evaluated by a method known per se in the art. For example, the stability of the secondary structure of the peptide can be evaluated by a circular dichroism measurement method in the presence and absence of alcohol. Specifically, if the absolute values of the circular dichroism values (mdeg) of the peptide at wavelengths of 208 nm and 222 nm measured by the circular dichroism measurement method in the presence of alcohol are the same or higher than those in the presence of non-alcohol, the peptide can be evaluated as forming a more stable α-helix structure in the presence of alcohol compared to those in the presence of non-alcohol. Similarly, if the absolute values of the circular dichroism values (mdeg) of the peptide at wavelengths of 216 nm measured by the circular dichroism measurement method in the presence of alcohol are the same or higher than those in the presence of non-alcohol, the peptide can be evaluated as forming a more stable β-sheet structure in the presence of alcohol compared to those in the presence of non-alcohol.

In step (3) of the screening method of the present invention, peptides are selected that have the same or higher level of secondary structure stability in the presence of alcohol compared to the absence of alcohol. The peptides selected in this step have secondary structure stability in the presence of alcohol that is equal to or higher than that exhibited in the absence of alcohol, and therefore can be subjected to binding tests with target molecules while contained in an alcohol solvent.

In step (4) of the screening method of the present invention, the selected peptide is contacted with the target molecule in the presence of alcohol. The manner of contact between the selected peptide and the target molecule may vary depending on the state of the target molecule. When the target molecule exists as a liquid sample, the target molecule and the selected peptide can be mixed in the presence of alcohol to bring them into contact with each other. When the target molecule exists as a solid sample, the target molecule and the selected peptide may be directly contacted with each other, or the target molecule may be dissolved in a solvent and then mixed with the selected peptide to bring them into contact with each other. Examples of the solvent include water, buffer solutions (e.g., phosphate buffer, citrate buffer, acetate buffer, Tris buffer, etc.), and alcohol (e.g., methanol, ethanol, propanol, isopropanol, etc.).

The contact time and temperature between the selected peptide and the target molecule can be appropriately selected by those skilled in the art, but for example, contact at room temperature for one to several minutes may be sufficient.

In step (5) of the screening method of the present invention, the fluorescence intensity of the fluorescent molecule of the selected peptide that has come into contact with the target molecule in step (4) is measured. The fluorescence intensity can be measured using a commercially available fluorescence measuring device.

In step (6) of the screening method of the present invention, a peptide aptamer for the target molecule is selected based on the change in fluorescence intensity measured in step (5). The change in fluorescence intensity can be obtained, for example, by calculating the difference between the fluorescence intensity (reference fluorescence intensity) measured after contacting the selected peptide with a substance that does not bind to the selected peptide and the fluorescence intensity (measured fluorescence intensity) measured in step (5). If there is a difference between the reference fluorescence intensity and the measured fluorescence intensity, the selected peptide can be selected as a peptide aptamer for the target molecule.

Alternatively, a standard curve can be created relating the fluorescence intensity of the selected peptide to the amount of the target molecule, and the presence or absence of binding of the selected peptide to the target molecule can be determined based on the measured fluorescence intensity on the basis of the standard curve.

The present invention will be explained below using examples. However, the present invention is not limited to these examples.

(material and method)
＜DNA＞
DNA was purchased from Eurofin genomics.

<Preparation of NP-immobilized beads>
The N-terminal 110 residues of influenza virus nucleoprotein were expressed in COS7 cells as a fusion protein of red fluorescent protein (RFP) and flag tag as previously reported (K. Hagiwara et al. Biochemical and Biophysical Research Communications 394 (2010) 721-727). HeLa cell line was purchased from RIKEN Cell Bank (Tsukuba, Japan). The expressed protein was purified using anti-flag antibody magnetic beads, and sufficient purity was confirmed by SDS-PAGE and Western blotting. As a negative control, flag tagged RFP was also expressed and purified in the same manner. The purified fusion protein was separately immobilized on magnetic beads according to the manufacturer's protocol (Dynabeads MyOne Streptavidin C1, Invitrogen, CA, USA).

<Aptamer selection>
Two DNA libraries with random sequences [VVN: (VVN) ₅ TAG(VVVN) ₅ and NNK: (NNK) ₅ TAG(NNK) ₅ ] were prepared using a method previously reported [T. Maeno et al., Scientific Reports volume 8, Article number: 8271 (2018)]. The two templates were transcribed separately using the MEGAscript T7 transcription kit (Life Technologies) and then treated with DNase. After purification using the mRNA Clean & Concentrator kit (Zymo Research), the two mRNAs were mixed at a molar ratio (VVN:NNK = 9:1). This mRNA mixture was mixed with a reconstituted in vitro translation system (50 μL) (Shimizu Nat Biotechnol 19, 751-755 (2001)) lacking RF1 but containing 400 pmole of UAG suppressor tRNA carrying NBD-amPhe. The tRNA was synthesized as described in Chem Commun 50, 2962-2964 (2014). After 15 min of incubation at 37 °C for translation, the translation product was mixed with 200 μL of ice-cold WBT-RNasein buffer (WBT; 50 mM Tris/acetate, 150 mM NaCl, 50 mM magnesium acetate, 0.05% Tween 20, pH 7) and 5 μL of influenza virus NP-immobilized beads. The mixture was gently shaken at 4 °C for 30 min. To remove unbound complexes, the beads were washed eight times with ice-cold WBT-RNasin buffer. The beads were transferred to elution buffer (7 M urea, 50 mM Tris/acetate, pH 7.2; 150 mM sodium chloride, 50 mM magnesium acetate, 0.05% Tween-20) and the suspension was heated at 70 °C for 5 min. After purifying the mRNA using the mRNA Clean & Concentrator Kit, the mRNA was reverse transcribed into cDNA (PrimeScript ^TM Reverse Transcriptase, Takara Bio). The cDNA was amplified using PrimeSTAR GXL DNA polymerase and primers (Fwd_pCR-T7 150731 and RevtolA110617). After buffer exchange using NucAway (Invitrogen), the PCR product was used as a template for the next round of selection. To remove nonspecific binders, negative selection was performed from the third round to the seventh round using RFP-immobilized beads (Table 1). After the seventh round of selection, 20,289 base sequences were determined by massively parallel sequencing, and the top 11 peptide sequences were synthesized (Table 2).

Peptide synthesis
Eleven selected sequences were synthesized using solid-phase synthesis. The synthesized peptides were purified by reversed-phase HPLC using a C18 column (InertSustain C18 column, GL Sciences, Torrance, CA, USA or COSMOSILC18-AR-II, Nacalai Tesque, Japan) with a linear gradient of acetonitrile in water containing 0.1% trifluoroacetic acid (v/v). NPBP4 and NPBP5 were not well purified and were not used in further experiments. The masses of the nine purified peptides were verified by MALDI-TOF-MS (Microflex LT, Bruker Daltonics, USA).

To modify NPBP2 with polyethylene glycol or biotin, the cysteine residues of NPBP2 were modified with methyl-terminated polyethylene glycol compound (24 PEG units) maleimide (MM(PEG)24 Methyl-PEG-Maleimide, Thermo Fisher Scientific, USA) or maleimide-PEG-biotin (EZ-Link Maleimide-PEO2-Biotin, Thermo Fisher Scientific) according to the manufacturer's protocol. Each modified NPBP2 was purified by HPLC and confirmed by Maldi TOFMS.

Each peptide was dissolved in ultrapure water and the peptide concentration was measured using the extinction coefficient of NBD (475 nm, 25,000 M ^-1 cm ^-1 ) (Ladokhin, A. S., Isas, J. M., Haigler, H. T. & White, S. H. Biochemistry 41, 13617-13626 (2002).) or the extinction coefficient of FAM (450 nm, 75,000 M ^-1 cm ^-1 ).

<Purification of full-length NP>
The full-length influenza virus NP gene was cloned into the PET-28a expression vector as a fusion protein with a his-tag, and transformed into E. coli BL21(DE3). The transformed E. coli was cultured at 37°C, induced with IPTG (1 μM IPTG) at OD = 0.6, and further cultured at 37°C for 16 h. The cultured E. coli was suspended in a buffer (lysozyme 0.2 mg/mL, Triton 0.1%, RNaseA 10 μg/mL, DNaseI 5 μg/mL) and disrupted by ultrasonication (30 min). The full-length NP was captured on a Ni-NTA column (GE) and eluted with an elution buffer (50 mM HEPES (pH 7.6), 600 mM NH ₄ Cl, 40 mM KCl, 64 mM MgCl ₂ , 7.15 mM 2-mercaptoethanol, 200 mM imidazole). The purity of the full-length NP was confirmed by SDS-PAGE and Western blotting. The concentration of the full-length NP was determined using the SDS-PAGE band.

<Conventional dot blot method>
A commonly used dot blot method was used, except that NPBP was used instead of the primary and secondary antibodies. Specifically, 2 μl of the sample was spotted onto a PVDF membrane filter (Immobilon-FL PVDF Membrane, Merck Millipore, USA) that had been hydrophilized with methanol and then replaced with transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, 80% H ₂ O), and the membrane filter was dried on the bench. The membrane filter was soaked overnight at 4°C in methanol, transfer buffer, and 5% blocking reagent (ECL primeTM blocking agent, Cytiva) to block nonspecific sites on the membrane filter. The membrane filter was washed three times with TBST (Tris-buffered saline with Tween 20, 0.05 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween ^TM 20), and then incubated in TBST containing 1 μM NPBP at room temperature for 1 h. Finally, the membrane filter was immersed in TBST for 5 min, and this was repeated three times to wash the membrane filter.

First, stock solutions of full-length NP and the negative control bovine serum albumin (BSA) were prepared, and then serial dilutions of each stock solution were performed to prepare nine different total amounts of each protein (20, 15, 10, 7.5, 5, 2.5, 1, 0.5, and 0.25 μg).

<Simple dot blot method>
2 μl of sample was spotted onto a hydrophilized PVDF membrane filter, and the membrane filter was dried on the bench. The membrane filter was immersed in NPBP solution (50% TBST and 50% ethanol) at room temperature for 1 to 5 minutes. NPBP2 and NPBP8 containing NBD at 1 μM, and NPBP6 containing FAM at 0.25 μM were used.

<BLItz analysis>
The dissociation constants of NPBP and NP were determined using Biolayer Interferometry (BlItz, Pall ForteBio). All solutions used in BLItz contained 0.002% Tween20 and 0.01% BSA in the elution buffer. 0.2 μM of biotinylated NPBP was loaded onto a streptavidin-coated biosensor. Graded concentrations of full-length NP were used in the binding step: 15, 7.5, 3.8, and 1.8 μM. The dissociation constants between NPBP and full-length NP were determined using BLItz software.

(result)
Using the ribosome display method, fluorescent peptide aptamers against influenza virus NP were selected (Figure 1). Selection began with a random library sequence consisting of 33 nucleotides, with TAG in the center and the rest randomized. RF1 was removed from the in vitro transcription and translation system, and instead tRNA _CUA bound to aminophenylalanine (amPhe) modified with NBD was added to redefine the TAG stop codon as a sense codon. NBD was adopted as the source of the fluorescent properties of the peptide aptamer because the fluorescence intensity of NBD is environment-dependent, such that the fluorescence of NBD is effectively quenched in a hydrophilic environment and emitted in a hydrophobic environment. In general, viral proteins are prone to mutation, but the nuclear localization signal sequence region necessary for NP to function is thought to be relatively resistant to mutation, so in this example, the NP fragment was targeted. Specifically, the N-terminal 110 amino acids of NP, including the nuclear localization signal, were used. After seven rounds of selection using ribosome display, the enriched sequences were read and 11 peptide sequences were obtained as candidates for NP-binding peptides (NPBPs) (Table 2). These 11 peptides were synthesized by solid-phase synthesis. In addition, since the peptides that had lost the TAG sequence by mutation did not have aminophenylalanine modified with NBD, the N-terminal methionine of the peptide was modified with fluorescein for separate fluorescent labeling. NPBP4 and NPBP5 could not be purified, so the other nine NPBPs were used in the following experiments.

<Conventional dot blot method>
The binding ability of the nine synthesized NPBPs was examined using dot blot. In this example, NPBP was used instead of the primary and secondary antibodies for the dot blot method. Specifically, the membrane filter on which the protein was spotted was immersed in a solution containing each NPBP. Since each NPBP has NBD or fluorescein, the binding of NPBP to the protein spot can be visualized by irradiating it with blue light. Full-length NP was spotted as a positive control, and bovine serum albumin (BSA) was spotted as a negative control. As a result of dot blot, NPBP showed clear specific binding to full-length NP (NPBP2 is shown in Figure 2, and NPBP8 is shown in Figure 3). This result indicates that ribosome display using the selection of fragment-type NP works well.
We also confirmed the concentration dependency and reproducibility of NPBPs containing fluorescent NBD (NPBP2 and NPBP8) (Figures 4 to 6). The dissociation constants of NPBP2 and NPBP8 were 5.8 μM and 3.6 μM, respectively.

We also confirmed the binding of NPBP2 to full-length NP using biolayer interferometry (BLI). First, biotinylated NPBP2 was immobilized on a biosensor coated with streptavidin, and the process of binding and dissociation between the biosensor on which NPBP2 was immobilized and NP was observed (Figure 7). As a result, the dissociation constant of NPBP2 and NP was 3 μM, a value similar to the Kd calculated from the dot blot results. On the other hand, it was revealed that the binding to NP derived from human coronavirus was weaker than that to NP derived from influenza virus. A similar binding evaluation was also performed on NPBP11 and NPBP6, and it was revealed that, like NPBP2, they bound strongly to NP derived from influenza virus, but that their binding to NP other than influenza virus was weaker.

<Simple dot blot method>
Next, we investigated a system for instantly detecting NPs using the selected NPBPs. As mentioned above, the selected NPBPs can detect NPs in a dot blot assay using a PVDF membrane filter. Although nitrocellulose membrane filters are often used in the dot blot method, PVDF membrane filters have the advantage of having high protein binding capacity and higher sensitivity than nitrocellulose membrane filters. The protein binding capacity of PVDF membrane filters is due to its very hydrophobic surface, and proteins bind to PVDF through dipole-hydrophobic interactions. Since the surface of PVDF is very hydrophobic, in the conventional dot blot method, it is necessary to hydrophilize PVDF by immersing it in alcohol before binding water-soluble proteins to PVDF. On the other hand, alcohol irreversibly denatures proteins, so it is necessary to remove alcohol from the PVDF surface. Therefore, it is necessary to transfer the PVDF immersed in alcohol into an aqueous buffer and replace the alcohol on the PVDF surface with buffer. After this hydrophilization treatment using alcohol and buffer, proteins are spotted on PVDF and once dried, the proteins are immobilized on PVDF. Since the PVDF was dried once, in the conventional dot blot method, it was only possible to apply the PVDF to a liquid containing an antibody after re-hydrophilization. On the other hand, in the simple dot blot method, the inventors noticed that in the latter hydrophilization treatment, NPBP can be dissolved in alcohol, so that the hydrophilization treatment of PVDF and the binding of NPBP to proteins can be performed simultaneously. Since some alcohols can inactivate enveloped viruses, the inventors thought that it would be meaningful to use a liquid containing alcohol from the viewpoint of disinfection against infectious diseases. Therefore, the inventors investigated a system that instantly detects NP using NPBP dissolved in alcohol.

First, we investigated the hydrophilization ability of several alcohols that could inactivate viruses. Specifically, we investigated the optimal concentrations of methanol, ethanol, and isopropanol for hydrophilizing PVDF membrane filters. We observed the spread of spots of serially diluted alcohol solutions containing fluorescein on a dried PVDF membrane filter and determined the minimum percentage of each alcohol required to hydrophilize the PVDF membrane filter. As a result, we found that the minimum percentage of each alcohol required to hydrophilize the PVDF membrane filter was 70% for methanol, 50% for ethanol, and 30% for isopropanol (Figure 8).

Next, we investigated whether a simple dot blot method using NPBP2 could be performed.
After the membrane filter was hydrophilized with 50% ethanol, 2μg of influenza virus NP, 2μg of influenza virus NP (50% or 5% saliva solution), and 50% or 5% saliva alone were spotted on each membrane filter from the left. HEPES buffer (50mM HEPES pH7.6, 600mM ammonium chloride, 40mM KCl, 64mM _MgCl2 , 7.15mM mercaptoethanol, 200mM imidazole) was used as the dissolution buffer. The membrane filter was incubated for about 1 minute with NPBP2 (0.5μM) dissolved in 50%, 70% or 80% alcohol (upper row: ethanol, middle row: methanol, lower row: isopropanol), and the fluorescence was observed without washing.
As a result, when ethanol was used, NPBP2 showed reactivity not only to NP but also to NP dissolved in a saliva solution with a final concentration of 50%. Moreover, NPBP2 showed reactivity to NP not only when dissolved in ethanol with a final concentration of 50%, but also when dissolved in ethanol with a final concentration of 80%.
When methanol was used, NP was dissolved in a saliva solution with a final concentration of 5% because non-specific reactivity was observed against the saliva solution with a final concentration of 50%. As a result, NPBP2 showed reactivity not only with NP when dissolved in methanol with a final concentration of 80%, but also with NP dissolved in a saliva solution with a final concentration of 5%.
When isopropanol was used, NPBP2 showed reactivity to NP and to NP dissolved in a saliva solution with a final concentration of 50%. NPBP2 also showed reactivity to NP not only when dissolved in isopropanol with a final concentration of 50%, but also when dissolved in isopropanol with a final concentration of 80%.
Overall, ethanol was found to be more suitable than methanol or isopropanol for the sensitive detection of influenza virus NP by NPBP2 (Fig. 9), whereas ethanol or isopropanol was found to be more suitable for NPBP6 (Fig. 10).

Furthermore, it was confirmed that the above-mentioned simple dot blot method cannot be applied to antibodies because the antibodies are denatured when dissolved in alcohol (FIG. 11).
Specifically, after hydrophilization of a PVDF membrane filter with 50% ethanol, 4 μg of influenza virus NP, 2 μg of influenza virus NP, and 50% saliva solution were spotted on the membrane filter from the left. The membrane filter was incubated for 1 minute with an antibody against influenza virus NP diluted 200-fold with a buffer containing equal parts TBST and 100% ethanol, and observed without washing. As a result, the antibody could not detect influenza virus NP in 50% ethanol (Figure 11A).
In addition, the membrane filter spotted with influenza virus NP was completely dried, hydrophilized again with 50% ethanol, incubated with antibody dissolved in TBST for 1 minute, washed, and observed. This method differs from the usual dot blot method using antibodies in that there is no blocking treatment and the incubation time with antibodies is short. As a result, it was found that influenza virus NP could be barely detected using antibodies by immersing the membrane filter in a solution containing antibodies without alcohol and then washing the membrane filter (Figure 11B). This suggested that the dot blot method using antibodies, unlike the case using peptide aptamers, requires hydrophilization of the membrane filter, blocking treatment, and washing of the antibody for a sufficient time for antibody binding.
In other words, the simple dot blot method using the peptide aptamer of the present invention is a simple and rapid method since it does not require blocking treatment, antibody incubation time, etc.

Furthermore, we investigated whether the simple dot blot method could be performed on peptide aptamers other than NPBP2. As a result, it was found that the simple dot blot method could also be performed on NPBP6, one of the peptide aptamers for influenza virus NP (Figure 10). In addition, we also investigated the specificity for proteins other than influenza virus NP using the simple dot blot method. As a result, it was revealed that NPBP6 shows specificity (Figure 12). It was also found that the method can be applied to OvaBP6, which binds to the egg allergen ovomucoid, and Noro1, which binds to norovirus (Figures 13 and 14).

<A simple method for detecting targets in solutions containing ethanol>
Various concentrations of NP were added to NPBP6 at a final concentration of 0.1 μM, and the fluorescence intensity in the liquid state was observed. As a result, it was found that the fluorescence intensity increased in an NP concentration-dependent manner (Figure 15A). Even when 10% ethanol was present in the solution, the fluorescence intensity increased in an NP concentration-dependent manner, similar to the case without ethanol (Figure 15B).

<Identification of amino acids involved in the binding of NPBP6 to NP>
In order to investigate which amino acids are important for binding to NP among the amino acid sequence constituting NPBP6, mutant peptides were synthesized in which each of the constituent amino acids was replaced with alanine, and the binding ability of each peptide to NP was examined using the BLI method. The synthesized peptides are shown in Table 3.

Four micromolar amounts of each biotinylated peptide were loaded onto a streptavidin-coated biosensor, and 2.5 μM of NPs were used for the binding step.
W5A, L6A, and F8A were found to lose binding ability (Figure 16), suggesting that these five amino acids are important for binding to NP.

　By using the detection agent of the present invention, the presence or absence of a target molecule in a test sample can be detected simply by measuring the fluorescence intensity of a fluorescent molecule conjugated to a peptide aptamer. In addition, since the detection agent of the present invention contains a high concentration of alcohol, it is possible to reduce the risk of infection by simultaneously denaturing viruses and bacteria during detection. Therefore, the present invention can be useful as a practical detection agent for target molecules. This application is based on Patent Application No. 2023-058364 filed in Japan (filing date: March 31, 2023), the contents of which are incorporated in their entirety herein.

Claims (26)

A detection agent for a target molecule, comprising the following (1) and (2):
(1) An alcohol-tolerant peptide aptamer for a target molecule, comprising an amino acid linked to a fluorescent molecule;
(2) Alcohol. The fluorescent molecule has the following formula:
[In the formula, n is an integer from 0 to 6.]
or a compound represented by the following formula:
[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
The detection agent according to claim 1 , which is a compound represented by the formula: The detection agent according to claim 1 or 2, in which the target molecule is immobilized on a carrier. The detection agent according to claim 1 or 2, wherein the target molecule is contained in a liquid phase. The detection agent according to claim 1 or 2, wherein the target molecule is a protein that constitutes an influenza virus. The detection agent according to claim 5, wherein the protein constituting the influenza virus is a nucleoprotein. The alcohol-tolerant peptide aptamer for a target molecule, which comprises an amino acid linked to a fluorescent molecule, is represented by the following formula:
XM(Xaa1)(Xaa2)VWL(Xaa3)F(Xaa4-Xaa10) (SEQ ID NO: 1)
[wherein X is the following formula:
[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
where Xaa1-Xaa10 are any amino acids.
The detection agent according to claim 6, which is a compound represented by the formula: Xaa1 is a basic amino acid selected from the group consisting of R, H and K, or A;
Xaa2 is a basic amino acid selected from the group consisting of R, H and K, or A;
Xaa3 is a hydrophobic amino acid selected from the group consisting of G, P, A, I, L, M, V, F and W;
Xaa4 is a basic amino acid selected from the group consisting of R, H and K, or A;
Xaa5 is a basic amino acid selected from the group consisting of R, H and K, or A;
Xaa6 is a neutral amino acid selected from the group consisting of N, C, Q, S and T, or A;
Xaa7 is a neutral amino acid selected from the group consisting of N, C, Q, S and T, or A;
Xaa8 is a hydrophobic amino acid selected from the group consisting of G, P, A, I, L, M, V, F and W;
Xaa9 is a basic amino acid selected from the group consisting of R, H and K, or A; and
Xaa10 is a neutral amino acid selected from the group consisting of N, C, Q, S and T, or A;
The detection agent according to claim 7. The alcohol-tolerant peptide aptamer for a target molecule, which comprises an amino acid linked to a fluorescent molecule, is represented by the following formula:
XMKHVWLGFKRSSWRC (SEQ ID NO: 2)
[wherein X is the following formula:
[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
The detection agent according to claim 7, which is a compound represented by the formula: The detection agent according to claim 9, wherein m is 1 and n is 2. The alcohol-tolerant peptide aptamer for a target molecule, which comprises an amino acid linked to a fluorescent molecule, is represented by the following formula:
(1) XMKHVFLRRRRRWGFWC (SEQ ID NO: 3),
(2) MTTCRFSVYBMRLGFC (SEQ ID NO: 4),
(3) MTTCBGWSTBWASLP (SEQ ID NO: 5),
(4) MTTCGBSSIBVWGLNC (SEQ ID NO: 6),
(5) XMKHVLGFWRRRGWC (SEQ ID NO: 7),
(6) MTTCFRRGNBRBVFSC (SEQ ID NO: 8),
(7) XMTNVWWGWRRRWRLG (SEQ ID NO: 9),
(8) XMTTCRRRRGWRWLGWC (SEQ ID NO: 10), and
(9) XMKHVGWFGRLRRWC (SEQ ID NO: 11)
[In each formula, X represents the following formula:
[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
B is a compound represented by the following formula:
[In the formula, -COOH of adjacent amino acids and _-NH2 in the formula form a peptide bond, and -COOH in the formula and _-NH2 of another adjacent amino acid form a peptide bond.]
It is a modified phenylalanine represented by the formula:
The detection agent of claim 6, which is a compound selected from the group consisting of: The detection agent according to claim 11, wherein m is 1 and n is 2. The detection agent according to claim 1 or 2, wherein the target molecule is ovalbumin. The alcohol-tolerant peptide aptamer for a target molecule, which comprises an amino acid linked to a fluorescent molecule, is represented by the following formula:
XMTTCTRRRSRWNWICSWD (SEQ ID NO: 12)
[wherein X is the following formula:
[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
It is a compound represented by the formula:
The detection agent according to claim 13, which is a compound represented by the formula: The detection agent according to claim 14, wherein m is 0. The detection agent according to claim 1 or 2, wherein the target molecule is a nucleoprotein that constitutes norovirus. The alcohol-tolerant peptide aptamer for a target molecule, which comprises an amino acid linked to a fluorescent molecule, is represented by the following formula:
(1) XMKHVLFIFFRCGRSVLG (SEQ ID NO: 13), and
(2) XMTTCFYYRRSRTWVC (SEQ ID NO: 14)
[wherein X is the following formula:
[In the formula, m is 0 or 1, and n is an integer from 0 to 6.]
It is a compound represented by the formula:
17. The detection agent of claim 16, which is a compound selected from the group consisting of: The detection agent according to claim 17, wherein (1) m is 1 and n is 2, or (2) m is 0. The detection agent according to claim 1 or 2, wherein the peptide aptamer has a final concentration of 0.02 μM to 10 μM. The detection agent according to claim 1 or 2, wherein the alcohol is methanol, ethanol or isopropanol. The detection agent according to claim 1 or 2, wherein the final concentration of alcohol is 40% to 80%. A method for detecting a target molecule in a test sample, comprising the steps of:
(1) contacting the detection agent according to claim 1 or 2 with a test sample;
(2) measuring the fluorescence intensity of the fluorescent molecule; and (3) determining the presence or absence of the target molecule in the test sample based on the change in the fluorescence intensity. The detection method according to claim 22, wherein the test sample is immobilized on a carrier. The detection method according to claim 22, wherein the test sample is contained in a liquid phase. A method for screening a peptide aptamer against a target molecule, comprising the steps of:
(1) preparing a random peptide library consisting of peptides containing amino acids linked to fluorescent molecules;
(2) evaluating the stability of the secondary structure of each peptide contained in the library in the presence and absence of alcohol;
(3) selecting a peptide whose secondary structure stability in the presence of alcohol is equal to or greater than the same level as that in the absence of alcohol;
(4) contacting the selected peptide with a target molecule in the presence of alcohol;
(5) measuring the fluorescence intensity of the fluorescent molecule; and (6) selecting a peptide aptamer for the target molecule based on the change in fluorescence intensity. The screening method according to claim 25, wherein in step (2), the stability of the secondary structure of the peptide is evaluated by circular dichroism measurement.