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WO2024080776A1 - Sonde contenant du soufre pour la détection colorimétrique, et procédé d'accélération de l'activité enzymatique par introduction de soufre dans des acides nucléiques - Google Patents

Sonde contenant du soufre pour la détection colorimétrique, et procédé d'accélération de l'activité enzymatique par introduction de soufre dans des acides nucléiques Download PDF

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WO2024080776A1
WO2024080776A1 PCT/KR2023/015709 KR2023015709W WO2024080776A1 WO 2024080776 A1 WO2024080776 A1 WO 2024080776A1 KR 2023015709 W KR2023015709 W KR 2023015709W WO 2024080776 A1 WO2024080776 A1 WO 2024080776A1
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sulfur
colorimetric detection
colorimetric
probe
dna
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PCT/KR2023/015709
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English (en)
Korean (ko)
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정철희
신현
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고려대학교 산학협력단
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Priority claimed from KR1020230135804A external-priority patent/KR20240052677A/ko
Publication of WO2024080776A1 publication Critical patent/WO2024080776A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

  • the present invention relates to a sulfur-containing probe for colorimetric detection and a method for accelerating enzyme activity through sulfur introduction into nucleic acids.
  • the COVID-19 pandemic caused by the SARS-CoV-2 virus, continues to spread globally and is still not fully under control.
  • the representative method is fluorescence-based detection such as quantitative PCR using fluorescently labeled probes.
  • this method is very sensitive and specific, it has some limitations. Expensive equipment and skilled personnel are required, and the detection process is time-consuming. These limitations are a major constraint, especially in the current pandemic situation, where the importance of spreading and preventing infectious diseases is emphasized, so there is a need to develop new methods for point-of-care diagnosis that can be performed more quickly, user-friendly, and cost-effectively. .
  • the colorimetric diagnostic method has the advantage of being able to interpret detection results without expensive equipment or skilled personnel for detection, and being cost-effective for diagnosis, making it suitable for repetitive and large-scale mass screening compared to fluorescence analysis.
  • it is suitable for application to field-based nucleic acid diagnosis because it is easy to handle, and results can be confirmed simply and quickly.
  • the DNAzyme used in this colorimetric diagnosis is a type of DNA oligonucleotide that can perform a specific chemical reaction, and is known to have the ability to mimic the activity of peroxidase, especially when used in the G-quadruplex/hemin DNAzyme system.
  • This system is used in diagnostic methods that can produce colorimetric signals.
  • a G-quadruplex has a structure formed by four guanine-rich oligonucleotide strands that are joined together by Hoogsteen base pairs to form a stack of G-quadruplexes.
  • the present inventors have developed a new concept of DNAzyme that is not limited by sequence, so as to improve the shortcomings of conventional colorimetric diagnosis, by combining a sulfur atom that does not exist in nucleic acids but affects enzyme activity with a phosphorothioate bond (PS).
  • PS phosphorothioate bond
  • the object of the present invention is a sulfur-containing probe for colorimetric detection, wherein the probe has the form of a DNA oligonucleotide in which nucleobases are sequentially linked, and the sequential linkage of the nucleobases is adenine (A), guanine (G), At least one selected from cytosine (C) and thymine (T) is randomly linked, and the nucleotide phosphate backbone of the DNA oligonucleotide is modified with one or more phosphorothioates to form one or more sulfur.
  • A adenine
  • G guanine
  • T thymine
  • the nucleotide phosphate backbone of the DNA oligonucleotide is modified with one or more phosphorothioates to form one or more sulfur.
  • Another object of the present invention is to provide a composition for colorimetric detection, comprising the sulfur-containing probe for colorimetric detection of the present invention.
  • Another object of the present invention is to provide a kit for colorimetric detection containing the composition for colorimetric detection of the present invention.
  • Another object of the present invention is to provide a colorimetric detection method using the sulfur-containing probe for colorimetric detection of the present invention.
  • Another object of the present invention is to (1) select a target gene to be detected; (2) designing an oligonucleotide sequence complementary to the nucleic acid sequence of the target gene; and (3) modifying the nucleotide phosphate backbone in the complementary oligonucleotide with one or more phosphorothioates, wherein the complementary oligonucleotide has a length of 10 to 30 mer, with the phosphorothioate modifications on both sides.
  • A adenine
  • the present invention is a sulfur-containing probe for colorimetric detection, wherein the probe has the form of a DNA oligonucleotide in which nucleobases are sequentially linked, and the continuous linkage of the nucleobases is adenine (A), guanine (G), cytosine ( C) and thymine (T) are randomly linked, and the nucleotide phosphate backbone of the DNA oligonucleotide is modified with one or more phosphorothioates to form one or more sulfur atoms.
  • a sulfur-containing probe for colorimetric detection characterized in that it contains.
  • the sulfur-containing probe for colorimetric detection may be in the form of a DNA oligonucleotide in which 10 to 30 nucleobases are sequentially linked.
  • the sulfur-containing probe for colorimetric detection may enhance the enzyme activity of the colorimetric reaction mediator.
  • At least one nucleobase present on both sides of the modification may be adenine (A).
  • the DNA oligonucleotide containing one or more sulfur atoms may be a single-stranded DNA oligonucleotide.
  • the present invention also provides a composition for colorimetric detection, comprising the sulfur-containing probe for colorimetric detection of the present invention.
  • the composition may further include a colorimetric reagent.
  • the colorimetric reagent is ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), OPD (o-phenylenediamine dihydrochloride) 1 selected from the group consisting of DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbozole), TMB (3,3',5,5'-tetramethylbenzidine), AmplexRed, and Homovanilic acid. It may be more than one type; and one or more peroxides.
  • the composition may further include hemin.
  • the composition may further include hydrogen peroxide (H 2 O 2 ).
  • the present invention provides a kit for colorimetric detection containing the composition for colorimetric detection of the present invention.
  • the present invention provides a colorimetric detection method using the sulfur-containing probe for colorimetric detection of the present invention.
  • the present invention includes the steps of (1) selecting a target gene to be detected; (2) designing an oligonucleotide sequence complementary to the nucleic acid sequence of the target gene; and (3) modifying the nucleotide phosphate backbone in the complementary oligonucleotide with one or more phosphorothioates, wherein the complementary oligonucleotide has a length of 10 to 30 mer, with the phosphorothioate modifications on both sides.
  • a method for producing a sulfur-containing probe for colorimetric detection that has an effect of enhancing enzyme activity mediated by a colorimetric reaction, wherein at least one nucleobase present in the region is adenine (A).
  • the present invention solves the problem of colorimetric diagnosis with sequence limitations by introducing sulfur into nucleic acids.
  • the new concept of DNAzyme that exhibits peroxidase activity by introducing sulfur, an element that does not exist in nucleic acids according to the present invention can confirm positive and negative detection results depending on the DNA type, expanding the applicability of colorimetric diagnostic technology. It has a possible effect.
  • unlike existing DNAzymes that require a structure it has a specific structure for colorimetry and a highly versatile effect that can be widely applied to various sequences without additional modification, and is also a phosphorothioate that is easy to introduce sulfur into nucleic acids.
  • the advantage of using phosphorothioate modification is that it is inexpensive and easy to introduce.
  • sulfur modified single stranded DNA of sufficient length can interact with hemin to generate peroxidase-like activity and hybridize complementary sequences.
  • the reaction of the bases participating in the reaction with the substrate is limited by hydrogen bonding, and the effect of quenching the colorimetric color can be confirmed, enabling simple, rapid, and accurate colorimetric detection and diagnosis compared to conventional methods.
  • Figure 1 confirms the effect of introducing sulfur into nucleic acid.
  • A) shows the connection structure between PO bond (phosphodiester bond) and PS bond (phosphorothioate bond) nucleotides, respectively, and B) difference in enzyme activity depending on PS modification. This shows a schematic diagram, confirming the increase in enzyme activity when PS modification is introduced into ssDNA, C) Colorimetric analysis results in the presence of PO-DNA and PS-DNA were confirmed by measuring absorbance at 405 nm, D) This shows real-time absorbance data observed at 405 nm, and blank represents a group that does not contain nucleic acids.
  • Figure 2 confirms the optimal conditions for colorimetric analysis.
  • A) shows the absorbance spectrum at 405 nm after reaction at room temperature for 1 hour for each hemin concentration (leftmost), and shows the real-time absorbance curve for 1 hour (middle and Right)
  • B) This shows the results of colorimetric analysis by H 2 O 2 concentration, showing the absorbance spectrum at 405 nm after reaction at room temperature for 1 hour (far left), and the real-time absorbance curve for 1 hour (middle). , right).
  • Figure 3 verifies the effectiveness of the PS bond, A) the results of analyzing the absorbance according to the number of PS bonds introduced into the same randomly selected sequence, and B) consecutive nucleotides of the same length (A 20 , T 20 , C 20 , G 20 ), this is the absorbance analysis result according to the presence or absence of a single PS, and C) is the absorbance analysis result according to the number of PS inserted into the sequence in consecutive nucleotides (A 20 , T 20 , C 20 ) of the same length.
  • Figure 4 shows the absorbance analysis results for the experimental groups in Figure 3
  • A) shows the real-time absorbance analysis results for the experimental groups in Figure 3A
  • B) shows the real-time absorbance curve for the experimental groups in Figure 3B
  • C) to E) show the real-time absorbance curve corresponding to Figure 3C
  • F) to I) show the real-time absorbance curve corresponding to Figure 3.
  • Figure 5 shows the absorbance analysis results of PW17 G-quadruplex according to PS position.
  • Figure 6 compares the difference in enzyme activity in sequences with different lengths of consecutive bases composed of the same base depending on the presence or absence of PS.
  • A), B), C), and D) are A, T, C, and G bases, respectively. It shows the results of analysis on the type.
  • Figure 7 is a comparative analysis of enzyme activity according to the composition of the proximal nucleobase close to PS.
  • C) and D) confirm the enzyme activity of 3 different sequences generated based on the 0 PS sequence, and 2 PS_H is higher than 2 PS_L. It was confirmed that the enzyme activity efficiency was high.
  • Figure 9 shows the results of sequence-specific colorimetric detection using the H-OSD probe.
  • A) a schematic diagram showing the difference in enzyme activity according to DNA formation, and B) the absorbance measurement results according to the number of PS in dsDNA and ssDNA.
  • C) shows the mechanism of generating an absorbance signal from a scaffold-mediated strand displacement reaction, and D) shows the absorbance intensity results at 405 nm from a plate reacted at room temperature for 1 hour using an enzyme signal transduction system.
  • Figure 10 shows the optimal final MgSO 4 concentration in sequence-specific colorimetric diagnosis using an enzyme signal transduction system, which was analyzed using the samples in Figure 9D.
  • Figure 11 shows the real-time absorbance curve corresponding to A) Figure 9B.
  • the ssDNA mixture was prepared with 20 pmole DNA (PO and PS), and the dsDNA mixture was prepared with 20 pmole DNA (PO and PS) and 24 pmole complement DNA ( cPO-DNA) was prepared and used by annealing, and PO-dsDNA and PS-dsDNA were hybridized before absorbance analysis.
  • Figure 12 is an analysis of the specificity of sequence-specific colorimetric diagnosis using an enzyme signal transduction system.
  • the scaffold-mediated strand displacement reaction was performed in the same manner as Figure 9D, and 60 pmole of non-target DNA was used in the reaction.
  • A) Shows the absorbance results measured at 405 nm for a plate reacted at room temperature for 1 hour
  • B) Shows the real-time absorbance analysis results measured at 405 nm
  • Figure 13 analyzes the effect of the number of PS introduced into the H-OSD probe on enzyme activity.
  • the scaffold-mediated strand displacement reaction was performed in the same manner as Figure 9D, but this reaction was performed without including target DNA.
  • A) Shows the absorbance results measured at 405 nm for a plate reacted at room temperature for 1 hour
  • B) Shows the real-time absorbance analysis results measured at 405 nm
  • C) Shows a photograph of the well plate after detection of the enzyme signaling system. will be.
  • Figure 14 shows the results of sequence-specific colorimetric detection using an OSD probe.
  • A is a schematic diagram showing the difference in enzyme activity according to DNA formation
  • B is the absorbance using OSD probes containing different numbers of PS. This shows the measurement results.
  • Figure 15 shows the optimal final MgSO 4 concentration in sequence-specific colorimetric diagnosis using the enzyme signal transduction system for the OSD probe according to the present invention.
  • Figure 16 shows the real-time absorbance curve for the OSD probe of the present invention according to the presence or absence of the target sequence and the number of PS.
  • Figure 17 is an analysis of the specificity of sequence-specific colorimetric diagnosis using an enzyme signal transduction system for the OSD probe according to the present invention, in which a scaffold-mediated strand displacement reaction was performed and 60 pmole of non-target DNA was used in the reaction.
  • A) Shows the absorbance results measured at 405 nm for the plate reacted at room temperature for 1 hour
  • C) A photograph of the well plate after detection of the enzyme signaling system. It is shown.
  • the present invention relates to a sulfur-containing probe for nucleic acid-based colorimetric detection and a method for accelerating enzyme activity through the introduction of sulfur into nucleic acids.
  • the present invention is characterized by providing a new sulfur-containing probe for colorimetric detection that can improve the problems of conventional colorimetric analysis and accelerate enzyme activity to enable rapid and accurate colorimetric detection.
  • the sulfur-containing probe for colorimetric detection has the form of a DNA oligonucleotide in which nucleobases are sequentially linked, and the sequential linkage of the nucleobases includes adenine (A), guanine (G), cytosine (C), and At least one selected from thymine (T) is randomly linked, and the nucleotide phosphate backbone of the DNA oligonucleotide is modified with at least one phosphorothioate to contain at least one sulfur atom. It is characterized by having
  • the sulfur-containing probe for colorimetric detection of the present invention is a new concept DNAzyme that is not restricted by sequence, unlike the G-quadruplex used in conventional colorimetric diagnosis. It does not require a sequence forming a specific structure such as a G-quadruplex, and produces a colorimetric reaction. It has the characteristic of accelerating the enzyme activity of the medium.
  • the sulfur-containing probe for colorimetric detection of the present invention may be in the form of a DNA oligonucleotide in which 10 to 30 nucleobases are sequentially linked, preferably in the form of a DNA oligonucleotide in which 20 nucleobases are linked in series. .
  • the sulfur-containing probe for colorimetric detection of the present invention has the characteristic of introducing a sulfur element that does not exist in the original nucleic acid by modifying the phosphate backbone of the DNA oligonucleotide into phosphorothioate (PS: phosphorothioate modification).
  • PS phosphorothioate modification
  • PS At least one nucleobase on either side of may be adenine (A).
  • the length of the probe could affect enzyme activity, and as a result, no enzymatic activity was observed for the monomers dNTP and ⁇ -thio-dNTP regardless of the type of nucleotide constituting the probe. . Therefore, it was found that the length of the probe DNA can affect enzyme activity. This means that the degree of binding between DNA and hemin can vary depending on the length, and only when DNA and hemin can bind can PS and the nucleobase interact. It was found that a colorimetric reaction could be obtained.
  • the probe of the present invention for effective colorimetric detection may be a DNA oligonucleotide in which 10 to 30 nucleobases are sequentially linked, and preferably, it may be a DNA oligonucleotide in which 20 nucleobases are sequentially linked.
  • the sulfur-containing probe for colorimetric detection of the present invention has the characteristic of varying enzymatic activity depending on the type of nucleobase present at an adjacent position of PS.
  • adenine (A) and adenine (A) When PS is located between the bases, the enzyme activity efficiency was found to be the best compared to other base combinations. Next, when either nucleobase on both sides of PS is adenine (A), the enzyme activity is higher than that of other base combinations. It was found to be excellent. On the other hand, when PS was located between pyrimidine bases such as T or C, the enzyme activity rate was relatively low.
  • the position of PS can be preferentially selected in the following order: A*A, A*G, G*A, C*A, A*C, A*T, T*A, and most preferably A*A PS can be located in .
  • the * mark above indicates the location of PS.
  • the sulfur-containing probe for colorimetric detection of the present invention has the characteristic of having differences in enzyme activity depending on the form of DNA.
  • probes in the form of single-stranded DNA exhibit strong enzymatic activity, but probes in the form of double-stranded DNA have the characteristic of reduced enzymatic activity and no colorimetric reaction.
  • the sulfur-containing probe for colorimetric detection of the present invention has the form of single-stranded DNA.
  • the present invention also provides a composition for colorimetric detection, comprising the sulfur-containing probe for colorimetric detection of the present invention.
  • the sulfur-containing probe for colorimetric detection provided by the present invention can function as a new DNAzyme, and the term “DNAzyme” generally refers to a nucleic acid molecule having enzymatic activity, for example, it may have peroxidase activity. Additionally, the term may include deoxyribozymes, DNA enzymes, DNAzymes, catalytic DNA, DNA-based enzymes, etc., and may also include ribonuclease, RNA ligase, DNA phosphorylation, DNA adenylation, DNA deglycosylation, porphyrin metalation, thymine dimer photoreversion, and DNA cleavage. It may be that it makes chemical reactions possible.
  • the DNAzyme may include a sulfur-containing probe sequence for colorimetric detection of the present invention.
  • the composition for colorimetric detection of the present invention may include a colorimetric reagent, but the colorimetric reagent is not limited thereto, but is ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) , OPD (o-phenylenediamine dihydrochloride), DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbosol), TMB (3,3',5,5'-tetramethylbenzidine) , AmplexRed, and Homovanilic acid; and at least one peroxide.
  • the peroxide may be hydrogen peroxide (H 2 O 2 ).
  • composition may include hemin as a DNAzyme cofactor.
  • composition for colorimetric detection of the present invention may further include additional reagents and components used for colorimetric detection in the art.
  • the present invention can provide a kit for colorimetric detection containing the composition for colorimetric detection of the present invention, and can provide a colorimetric detection method using the sulfur-containing probe for colorimetric detection of the present invention.
  • the present invention can provide a method for producing a sulfur-containing probe for colorimetric detection that has the effect of enhancing enzyme activity mediated by a colorimetric reaction, which method includes the steps of (1) selecting a target gene to be detected; (2) designing an oligonucleotide sequence complementary to the nucleic acid sequence of the target gene; and (3) modifying the nucleotide phosphate backbone in the complementary oligonucleotide with one or more phosphorothioates, wherein the complementary oligonucleotide has a length of 10 to 30 mer, and the phosphorothioate modification is on both sides. It is characterized by being formed in a region where at least one nucleobase present is adenine (A).
  • A adenine
  • the sulfur-containing probe for colorimetric detection prepared by the method of the present invention accelerates the enzymatic activity of the colorimetric reaction mediator, enabling rapid and accurate detection of target nucleic acids through colorimetric reaction analysis.
  • the absorbance value increases by a colorimetric reaction and a colorimetric signal appears, whereas if the nucleic acid to be detected is not present in the sample, No colorimetric signal appears.
  • the probe according to the present invention may be a sulfur-containing probe forming a hairpin structure, and a single-stranded probe at the 5' or 3' end of the sulfur-containing probe.
  • the present invention provides a sulfur-containing probe for nucleic acid-based colorimetric detection and a nucleic acid. It relates to a method of accelerating enzyme activity through the introduction of sulfur.
  • the present invention is characterized by providing a new sulfur-containing probe for colorimetric detection that can improve the problems of conventional colorimetric analysis and accelerate enzyme activity to enable rapid and accurate colorimetric detection.
  • the sulfur-containing probe for colorimetric detection has the form of a DNA oligonucleotide in which nucleobases are sequentially linked, and the sequential linkage of the nucleobases includes adenine (A), guanine (G), cytosine (C), and At least one selected from thymine (T) is randomly linked, and the nucleotide phosphate backbone of the DNA oligonucleotide is modified with at least one phosphorothioate to contain at least one sulfur atom. It is characterized by having
  • the sulfur-containing probe for colorimetric detection of the present invention is a new concept DNAzyme that is not restricted by sequence, unlike the G-quadruplex used in conventional colorimetric diagnosis. It does not require a sequence forming a specific structure such as a G-quadruplex, and produces a colorimetric reaction. It has the characteristic of accelerating the enzyme activity of the medium.
  • the sulfur-containing probe for colorimetric detection of the present invention may be in the form of a DNA oligonucleotide in which 10 to 30 nucleobases are sequentially linked, preferably in the form of a DNA oligonucleotide in which 20 nucleobases are linked in series. .
  • the sulfur-containing probe for colorimetric detection of the present invention has the characteristic of introducing a sulfur element that does not exist in the original nucleic acid by modifying the phosphate backbone of the DNA oligonucleotide into phosphorothioate (PS: phosphorothioate modification).
  • PS phosphorothioate modification
  • PS At least one nucleobase on either side of may be adenine (A).
  • the length of the probe could affect enzyme activity, and as a result, no enzymatic activity was observed for the monomers dNTP and ⁇ -thio-dNTP regardless of the type of nucleotide constituting the probe. . Therefore, it was found that the length of the probe DNA can affect enzyme activity. This means that the degree of binding between DNA and hemin can vary depending on the length, and only when DNA and hemin can bind can PS and the nucleobase interact. It was found that a colorimetric reaction could be obtained.
  • the probe of the present invention for effective colorimetric detection may be a DNA oligonucleotide in which 10 to 30 nucleobases are sequentially linked, and preferably, it may be a DNA oligonucleotide in which 20 nucleobases are sequentially linked.
  • the sulfur-containing probe for colorimetric detection of the present invention has the characteristic of varying enzymatic activity depending on the type of nucleobase present at an adjacent position of PS.
  • adenine (A) and adenine (A) When PS is located between the bases, the enzyme activity efficiency was found to be the best compared to other base combinations. Next, when either nucleobase on both sides of PS is adenine (A), the enzyme activity is higher than that of other base combinations. It was found to be excellent. On the other hand, when PS was located between pyrimidine bases such as T or C, the enzyme activity rate was relatively low.
  • the position of PS can be preferentially selected in the following order: A*A, A*G, G*A, C*A, A*C, A*T, T*A, and most preferably A*A PS can be located in .
  • the * mark above indicates the location of PS.
  • the sulfur-containing probe for colorimetric detection of the present invention has the characteristic of having differences in enzyme activity depending on the form of DNA.
  • probes in the form of single-stranded DNA exhibit strong enzymatic activity, but probes in the form of double-stranded DNA have the characteristic of reduced enzymatic activity and no colorimetric reaction.
  • the sulfur-containing probe for colorimetric detection of the present invention has the form of single-stranded DNA.
  • the present invention also provides a composition for colorimetric detection, comprising the sulfur-containing probe for colorimetric detection of the present invention.
  • the sulfur-containing probe for colorimetric detection provided by the present invention can function as a new DNAzyme, and the term “DNAzyme” generally refers to a nucleic acid molecule having enzymatic activity, for example, it may have peroxidase activity. Additionally, the term may include deoxyribozymes, DNA enzymes, DNAzymes, catalytic DNA, DNA-based enzymes, etc., and may also include ribonuclease, RNA ligase, DNA phosphorylation, DNA adenylation, DNA deglycosylation, porphyrin metalation, thymine dimer photoreversion, and DNA cleavage. It may be that it makes chemical reactions possible.
  • the DNAzyme may include a sulfur-containing probe sequence for colorimetric detection of the present invention.
  • the composition for colorimetric detection of the present invention may include a colorimetric reagent, but the colorimetric reagent is not limited thereto, but is ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) , OPD (o-phenylenediamine dihydrochloride), DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbosol), TMB (3,3',5,5'-tetramethylbenzidine) , AmplexRed, and Homovanilic acid, and at least one peroxide may be, for example, hydrogen peroxide (H2O2).
  • ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid
  • OPD o-phenylenediamine dihydrochloride
  • DAB diaminobenzidine
  • AEC diamino-9-ethylcarbosol
  • composition may include hemin as a DNAzyme cofactor.
  • composition for colorimetric detection of the present invention may further include additional reagents and components used for colorimetric detection in the art.
  • the present invention can provide a kit for colorimetric detection containing the composition for colorimetric detection of the present invention, and can provide a colorimetric detection method using the sulfur-containing probe for colorimetric detection of the present invention.
  • the present invention can provide a method for producing a sulfur-containing probe for colorimetric detection that has the effect of enhancing enzyme activity mediated by a colorimetric reaction, which method includes the steps of (1) selecting a target gene to be detected; (2) designing an oligonucleotide sequence complementary to the nucleic acid sequence of the target gene; and (3) modifying the nucleotide phosphate backbone in the complementary oligonucleotide with one or more phosphorothioates, wherein the complementary oligonucleotide has a length of 10 to 30 mer, and the phosphorothioate modification is on both sides. It is characterized by being formed in a region where at least one nucleobase present is adenine (A).
  • A adenine
  • the sulfur-containing probe for colorimetric detection prepared by the method of the present invention accelerates the enzymatic activity of the colorimetric reaction mediator, enabling rapid and accurate detection of target nucleic acids through colorimetric reaction analysis.
  • the absorbance value increases by a colorimetric reaction and a colorimetric signal appears, whereas if the nucleic acid to be detected is not present in the sample, No colorimetric signal appears.
  • the probe according to the present invention may be a sulfur-containing probe that forms a hairpin structure, and the 5' or 3' end of the sulfur-containing probe has a single-stranded toehold sequence of 10 mer and a double-stranded stem structure of 20 bp behind it.
  • the complementary sequence is hybridized and can have a form designed as a loop structure composed of consecutive T 10 mers.
  • the probe of the present invention contains a sequence complementary to the target-binding site contained in the target nucleic acid, which is a single template strand, and the probe binds to the target nucleic acid from the toehold sequence, and the probe structure allows for branch migration. This occurs, resulting in a toehold-mediated single strand displacement reaction that pushes out the original hybridized complementary sequence.
  • a part of the sequence including the toehold binds complementary to the target nucleic acid, forming a new duplex structure through the hybridization process, and the existing sequence that was complementary to the probe is freely released in a single-stranded form.
  • the released single strand may contain PS, and when it contains PS, it was confirmed that the enzyme activity was strong, consistent with the previous experimental results of the present invention.
  • the probe of the present invention can not only be applied as an H-OSD that constitutes a probe through intramolecular hybridization, but also can be applied and utilized as an OSD that constitutes a probe through intermolecular hybridization.
  • OSD is composed of two complementary strands and can form a DNA duplex through intermolecular hybridization.
  • the DNA duplex is released by single strand displacement.
  • the substrate strand unlike the output strand, additionally contains a single strand of toehold sequence.
  • the scaffold sequence and the target nucleic acid are partially combined, causing the output strand to branch migrate, and then completely released.
  • the output strand converted to a single strand is PS. If it contains enzyme activity, It can be expressed strongly.
  • oligonucleotides used in this experiment were synthesized by Bioneer (Daejeon, Korea) and Integrated DNA Technologies, Inc. (Coralville, IA, USA). Additionally, the sequences of all DNA used in this experiment are shown in Table 1 below.
  • 27% (w/w) H 2 O 2 purchased from ThermoFisher Scientific, Inc.
  • All DNA mixtures were prepared with 1X isothermal amplification buffer (20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 , 0.1% Tween® 20, pH 8.8).
  • the ssDNA mixture was prepared with 40 pmole DNA, and the mononucleotide mixture was prepared with 40 pmole dNTP or ⁇ -thio-dNTP. Unless otherwise specified, all nucleic acids were used at a concentration of 40 pmole.
  • the DNA mixture was reacted at 95°C for 5 min and then slowly cooled to 25°C at a rate of 0.1°C/s, after which the DNA mixture was maintained at a temperature of 25°C for at least 5 min before use.
  • G-quadruplex analytes were prepared using 1X isothermal amplification buffer (20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 , 0.1% Tween® 20, pH 8.8).
  • the PW17 mixture was prepared by reacting with 40 pmole DNA (PO or 1 PS), and the analyte was reacted at 95°C for 5 min, then slowly cooled to 25°C at a rate of 0.1°C/s and incubated at room temperature for 5 min. Reacted for minutes.
  • the total volume of the analysis sample was set to 122.5 ⁇ L. 1.5 ⁇ L of 75 ⁇ M hemin, 100 ⁇ L of 100 mM citrate buffer containing 0.025% (w/v) ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) at 25°C. C, pH 4.0) and 1 ⁇ L of 3% H 2 O 2 were added to 20 ⁇ L of the DNA mixture. Afterwards, 120 ⁇ L of the final sample solution was transferred to a 96-well transparent plate and incubated at 24-25°C for 1 hour.
  • ABTS 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
  • the absorption intensity at 405 nm for the reaction mixture was then recorded every minute for a total of 60 cycles using a Tecan Spark® M10 multimode microplate reader (Mannedorf, Switzerland). Additionally, images were acquired by loading the analysis samples into a 96-well transparent plate (Corning cat. No. 9017) and then taking pictures using EOS-7D (Canon, Japan).
  • the probe solution was 25 ⁇ M hairpin probe strand in 1 was prepared by annealing. Afterwards, the probe solution was reacted at 95°C for 5 minutes, cooled slowly to 25°C at a rate of 0.1°C/s, and then reacted at room temperature for 1 hour, and the probe was stored at 4°C before use in the experiment. did.
  • the probe solution was 25 ⁇ M release strand in 1 and 30 ⁇ M scaffold strands were prepared by annealing. Afterwards, the probe solution was reacted at 95°C for 5 minutes, cooled slowly to 25°C at a rate of 0.1°C/s, and then reacted at room temperature for 1 hour, and the probe was stored at 4°C before use in the experiment. did.
  • 1X isothermal amplification buffer (20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 , 0.1% Tween® 20, pH 8.8), 6mM MgSO 4 (final 8mM), 60 pmole of target DNA and 1.6
  • the toehold-mediated strand displacement reaction was started by adding ⁇ L of the probe solution, and the reaction was performed at 25°C for 1 hour.
  • sulfur atoms which do not originally exist in nucleic acids, are introduced into nucleic acids through PS (phosphorothioate bond) modification, we analyzed whether the sulfur atoms affect the colorimetric reaction ( Figure 1A). At this time, sulfur atoms may be included in various types of nucleic acids.
  • the present inventors found that when a sulfur atom is introduced into a single-stranded DNA backbone to induce modification, enzyme activity can be activated simply and conveniently even if there is no sequence forming a specific structure such as a G-quadruplex.
  • a 20 refers to a nucleotide consisting of 20 adenine (A) bases in a row.
  • the absorbance value increased for the nucleotides of A 20 and G 20 , while the absorbance value did not change significantly for the nucleotides of C 20 and T 20 .
  • the enzyme activity was found to be increased compared to the blank both with and without PS ( Figures 3B and 4B), which means that the G-quadruplex structure can be partially formed even without PS. This appears to be due to a relative increase in enzyme activity, and the fact that enzyme activity tended to decrease when PS was introduced appears to have decreased enzyme activity by increasing structural instability during the formation of intermolecular G-quadruplexes. .
  • the present inventors confirmed whether increasing the number of PS would increase enzyme activity, but the G 20 sequence was not used in this analysis due to its self-assembly characteristics (e.g., intermolecular G-quadruplex).
  • the enzyme activity did not increase even though the number of PS was increased to 8, whereas in the case of A 20 , the enzyme activity appeared to increase as the number of PS increased ( Figure 3C, Figure 4C ⁇ 4E).
  • dNTP and ⁇ -thio-dNTP were used as monomeric nucleotides.
  • the general form of dNTP is the original form containing an oxygen atom in ⁇ -phosphate, while ⁇ -thio-dNTP refers to a sulfur atom introduced into ⁇ -phosphate instead of a non-crosslinked oxygen atom.
  • a repeating sequence formed by continuously repeating each base to become 10 mer, 20 mer, and 30 mer was used for A/C/T, and for G, a repeating sequence of 10 mer and 20 mer was used.
  • PS was introduced into the middle of each DNA sequence, and a single oligonucleotide containing PS was prepared and used.
  • the T-based nucleotide sequence was designed to contain a repeated combination of the 4th to 5th nucleobases and the 14th to 15th nucleobases, and between the 4th and 5th nucleobases and the 14th and 15th nucleobases.
  • the present inventors selected and used a random DNA sequence with 50% GC content that does not form secondary structures instead of the sequence used in Figure 1.
  • the random sequence showed significant enzymatic activity (Figure 3A).
  • two PSs were introduced to distinguish sufficient efficiency. PS was introduced by selecting the combination showing the highest efficiency according to the base combination ranking in Table 2, and PS was introduced within the same sequence showing low efficiency. PS was introduced by selecting the position of the combination, and the two sequences designated as low efficiency (L) and high efficiency (H) in Table 3 below were used in the experiment.
  • the present inventors formed dsDNA by combining complementary sequences to ssDNA.
  • the present inventors prepared a sequence complementary to the sequence used in Table 2 above and evaluated the enzyme activity after passing through appropriate DNA hybridization conditions.
  • the probe for the enzyme-generated signal transduction system was designed in a hairpin format called H-OSD (intramolecular DNA hybridization).
  • H-OSD intramolecular DNA hybridization
  • a hairpin stem of appropriate length was selected and used to ensure the stability of the duplex domain and sufficient enzyme activity even when multiple PS molecules are introduced.
  • the H-OSD probe consists of a 21bp hairpin stem with a 10nt toehold at the 5' end, which is complementary to the target sequence up to the loop sequence.
  • the toehold region hybridizes with the target sequence, opening the PS-containing strand and activating the enzyme activity. Conversely, in the absence of the target sequence, the enzyme activity remains inactive.
  • Example 4 H-OSD in the form of a hairpin through intramolecular hybridization was used for sequence-specific colorimetric detection.
  • Example 5 a sulfur atom using PS modification was added to the probe constructed through intermolecular DNA hybridization.
  • GC clamping was added to the end of the part forming the DNA duplex to reduce the instability of overall intermolecular hybridization due to the influence of DNA end breathing and PS mentioned above. Stability was improved.
  • OSD internal molecular DNA hybridization
  • a stem structure of appropriate length was included to ensure stability even as the number of PS increases.
  • the OSD may contain a toehold region of 10 nt toward the 5' or 3' end.
  • a 20bp substrate strand and a 20nt output strand that acts on enzyme activity were used after going through appropriate DNA hybridization conditions.
  • the probe operates in the same manner as in Example 4, and when the target sequence is present, the output strand containing PS is released through a toehold-mediated strand displacement reaction and enzyme activity occurs ( Figures 14A, 14B, Figure 16). On the other hand, in the absence of the target sequence, the enzyme activity remains inactive. Reaction conditions were further optimized to reduce the possibility of signal leakage due to increased DNA instability of the probe (FIG. 15). Additionally, in the presence of a non-target sequence, the probe did not react to release the output strand containing PS, the structure remained stable, and only background signals were detected (FIG. 17). Through these results, it was confirmed that the OSD composed of intermolecular hybridization according to the present invention has excellent usability as a probe considering factors that increase DNA structure stability such as GC clamping.
  • the new probe in which the sulfur atom for colorimetric detection is introduced into the nucleic acid developed in the present invention can increase enzyme activity without sequence-dependent restrictions, so the enzyme generation signal transduction system utilizing it can be used for sequence-specific colorimetric detection using H-OSD. It can be useful.

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Abstract

La présente invention concerne une sonde contenant du soufre pour la détection colorimétrique, et un procédé pour accélérer l'activité enzymatique par l'introduction de soufre dans des acides nucléiques. Plus particulièrement, la présente invention concerne : une sonde contenant du soufre pour la détection colorimétrique, la sonde se présentant sous la forme d'un oligonucléotide d'ADN dans lequel les nucléobases sont connectées de manière continue, la connexion continue des nucléobases est caractérisée en ce qu'une ou plusieurs choisies parmi l'adénine (A), la guanine (G), la cytosine (C) et la thymine (T) sont connectées de manière aléatoire et la chaîne principale de phosphate du nucléotide de l'oligonucléotide d'ADN est modifiée par un ou plusieurs phosphorothioates de manière à inclure un ou plusieurs atomes de soufre ; une composition contenant la sonde pour la détection colorimétrique ; un kit pour la détection colorimétrique ; un procédé de détection colorimétrique ; et un procédé de préparation de la sonde contenant du soufre pour la détection colorimétrique, présentant l'effet d'améliorer l'activité enzymatique médiée par la détection colorimétrique.
PCT/KR2023/015709 2022-10-14 2023-10-12 Sonde contenant du soufre pour la détection colorimétrique, et procédé d'accélération de l'activité enzymatique par introduction de soufre dans des acides nucléiques WO2024080776A1 (fr)

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KR10-2022-0132471 2022-10-14
KR20220132471 2022-10-14
KR1020230135804A KR20240052677A (ko) 2022-10-14 2023-10-12 비색 검출을 위한 황 함유 프로브 및 핵산 내 황 도입을 통한 효소 활성 가속화 방법
KR10-2023-0135804 2023-10-12

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002153284A (ja) * 2000-11-24 2002-05-28 Canon Inc 末端標識プローブアレイの製造方法、並びにそれを用いた標的物質量の評価方法
KR20140064735A (ko) * 2011-05-17 2014-05-28 디엑스테리티 다이아그노스틱스 인코포레이티드 표적 핵산 검출을 위한 방법 및 조성물
KR20200068738A (ko) * 2017-11-29 2020-06-15 주식회사 파나진 표적핵산 증폭방법 및 표적핵산 증폭용 조성물
KR20210038541A (ko) * 2018-07-25 2021-04-07 센스 바이오디텍션 리미티드 핵산 검출 방법
KR20220018266A (ko) * 2020-08-06 2022-02-15 한국과학기술원 포스포로티오에이트 dna로 수식된 헤어핀 프로브 기반의 등온 핵산증폭기술을 이용한 표적핵산 검출방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002153284A (ja) * 2000-11-24 2002-05-28 Canon Inc 末端標識プローブアレイの製造方法、並びにそれを用いた標的物質量の評価方法
KR20140064735A (ko) * 2011-05-17 2014-05-28 디엑스테리티 다이아그노스틱스 인코포레이티드 표적 핵산 검출을 위한 방법 및 조성물
KR20200068738A (ko) * 2017-11-29 2020-06-15 주식회사 파나진 표적핵산 증폭방법 및 표적핵산 증폭용 조성물
KR20210038541A (ko) * 2018-07-25 2021-04-07 센스 바이오디텍션 리미티드 핵산 검출 방법
KR20220018266A (ko) * 2020-08-06 2022-02-15 한국과학기술원 포스포로티오에이트 dna로 수식된 헤어핀 프로브 기반의 등온 핵산증폭기술을 이용한 표적핵산 검출방법

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