WO2024080776A1 - Sulfur-containing probe for colorimetric detection, and method for accelerating enzymatic activity through sulfur introduction into nucleic acids - Google Patents
Sulfur-containing probe for colorimetric detection, and method for accelerating enzymatic activity through sulfur introduction into nucleic acids Download PDFInfo
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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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Classifications
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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
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- 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.
Abstract
The present invention relates to a sulfur-containing probe for colorimetric detection, and a method for accelerating enzymatic activity through sulfur introduction into nucleic acids. Particularly, the present invention relates to: a sulfur-containing probe for colorimetric detection, wherein the probe has the form of a DNA oligonucleotide in which nucleobases are continuously connected, the continuous connection of nucleobases is characterized in that any one or more selected from adenine (A), guanine (G), cytosine (C) and thymine (T) are randomly connected and the nucleotide phosphate backbone of the DNA oligonucleotide is modified with one or more phosphorothioates so as to include one or more sulfur atoms; a composition containing the probe for colorimetric detection; a kit for colorimetric detection; a colorimetric detection method; and a method for preparing the sulfur-containing probe for colorimetric detection, having the effect of enhancing enzymatic activity mediated by colorimetric detection.
Description
본 발명은 비색 검출을 위한 황 함유 프로브 및 핵산 내 황 도입을 통한 효소 활성 가속화 방법에 관한 것이다.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.
SARS-CoV-2 바이러스로 인해 COVID-19 전염병은 전 세계적으로 계속 확산되고 있으며, 아직도 완전히 통제되지 않고 있다. 팬데믹에 대응하여 바이러스를 탐지하는 다양한 방법 중에서 대표적인 방법이 형광 표지 프로브를 사용한 정량적 PCR과 같은 형광 기반 검출이다. 이 방법은 매우 민감하고 구체적이지만 몇 가지 제한 사항이 있다. 값비싼 장비와 숙련된 인력이 필요하며 탐지 프로세스에 시간이 많이 걸린다. 이러한 한계는 특히 감염성 질병의 확산 및 예방의 중요성이 강조되는 현재의 팬데믹 상황에서 큰 제약이 되고 있어 현장 진단을 위한 보다 신속하고 사용자 친화적이며 비용 효율적으로 수행할 수 있는 새로운 방법의 개발이 필요하다.The COVID-19 pandemic, caused by the SARS-CoV-2 virus, continues to spread globally and is still not fully under control. Among various methods to detect viruses in response to a pandemic, the representative method is fluorescence-based detection such as quantitative PCR using fluorescently labeled probes. Although 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. .
반면, 비색 진단 방법은 고가의 장비나 검출을 위한 숙련된 인원 없이 검출 결과를 해석할 수 있고, 진단 비용 효율적으로 형광 분석에 비해 반복적이고 대량의 집단 스크리닝에 적합한 장점이 있다. 뿐만 아니라 다루기 쉽고 간단하고 신속하게 결과를 확인할 수 있어 현장기반핵산진단에 응용하기가 적합하다.On the other hand, 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. In addition, 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.
그러나 이러한 비색 진단에 사용되는 DNAzyme은 특정 화학반응을 수행할 수 있는 DNA 올리고뉴클레오티드의 일종으로, 특히 G-quadruplex/hemin DNAzyme 시스템에 사용될 때 퍼옥시다제의 활성을 모방하는 능력이 있는 것으로 알려져 있다. 이 시스템은 비색 신호를 생성할 수 있는 진단 방법에 사용된다. G-quadruplex는 Hoogsteen 염기쌍에 의해 서로 결합되어 G-4중체의 스택을 형성하는 구아닌이 풍부한 4개의 올리고뉴클레오티드 가닥으로 형성된 구조를 갖는다. 이 구조에 hemin이 첨가되면 과산화수소 매개의 산화가 일어날 수 있으며, 이는 ABTS, 루미놀, OPD, TMB 등과 같은 기질의 산화로 이어져 비색진단이 가능하다. 그러나 이 방법은 비색 진단을 위해 G-quadruplex 구조가 필요하다는 한계가 있으며, G-quadruplex 구조를 이용한 서열 특이적 프로브의 설계가 어려울 뿐만 아니라 비특이적 증폭으로부터 위양성(false positive)의 구별이 불가능하다는 문제점이 있다.However, 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. When hemin is added to this structure, hydrogen peroxide-mediated oxidation can occur, which leads to oxidation of substrates such as ABTS, luminol, OPD, and TMB, enabling colorimetric diagnosis. However, this method has the limitation that a G-quadruplex structure is required for colorimetric diagnosis, and not only is it difficult to design a sequence-specific probe using the G-quadruplex structure, but it is also impossible to distinguish false positives from non-specific amplification. there is.
따라서 이러한 문제점을 개선할 수 있으며 보다 편리하고 효과적인 새로운 비색 진단 방법의 개발이 필요하다.Therefore, it is necessary to develop a new colorimetric diagnostic method that can improve these problems and is more convenient and effective.
이에 본 발명자들은 서열의 제한을 받지 않는 새로운 개념의 DNAzyme으로 종래 비색 진단이 갖는 단점을 개선할 수 있도록, 핵산에 존재하지 않지만 효소 활성에 영향을 주는 황 원자를 포스포로티오에이트 결합(PS)을 통해 핵산에 도입한 새로운 비색 검출용 프로브를 고안하였고, 이를 이용할 경우, 효소 활성을 가속화시켜 보다 신속하고 정확하게 비색 진단이 가능하다는 것을 확인함으로써 본 발명을 완성하였다.Accordingly, 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). We designed a new colorimetric detection probe introduced into nucleic acids, and completed the present invention by confirming that using this probe accelerates enzyme activity and enables more rapid and accurate colorimetric diagnosis.
그러므로 본 발명의 목적은, 비색 검출용 황 함유 프로브로서, 상기 프로브는 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태를 가지며, 상기 핵염기의 연속적인 연결은 아데닌(A), 구아닌(G), 시토신(C) 및 티민(T) 중에서 선택되는 어느 하나 이상이 무작위적으로 연결되어 있고, 상기 DNA 올리고뉴클레오티드 중 뉴클레오티드 포스페이트 백본이 하나 이상의 포스포로티오에이트로 변형되어(phosphorothioate modification) 하나 이상의 황(sulfur) 원자를 포함하고 있는 것을 특징으로 하는, 비색 검출용 황 함유 프로브를 제공하는 것이다.Therefore, 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. ) To provide a sulfur-containing probe for colorimetric detection, characterized in that it contains an atom.
본 발명의 다른 목적은 본 발명의 비색 검출용 황 함유 프로브를 포함하는, 비색 검출용 조성물을 제공하는 것이다.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.
본 발명의 또 다른 목적은 본 발명의 비색 검출용 조성물을 포함하는 비색 검출용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for colorimetric detection containing the composition for colorimetric detection of the present invention.
본 발명의 또 다른 목적은 본 발명의 비색 검출용 황 함유 프로브를 이용한 비색 검출 방법을 제공하는 것이다.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.
본 발명의 또 다른 목적은, (1) 검출 대상의 타겟 유전자를 선정하는 단계; (2) 상기 타겟 유전자의 핵산서열에 대한 상보적인 올리고뉴클레오티드 서열을 디자인하는 단계; 및 (3) 상기 상보적인 올리고뉴클레오티드에서 뉴클레오티드 포스페이트 백본을 하나 이상의 포스포로티오에이트로 변형시키는 단계를 포함하며, 상기 상보적인 올리고뉴클레오티드는 10 내지 30mer의 길이를 가지며, 포스포로티오에이트 변형은 양쪽 옆에 존재하는 핵염기가 적어도 하나는 아데닌(A)인 영역에서 이루어지는 것을 특징으로 하는, 비색 반응 매개의 효소 활성 증진 효과를 갖는 비색 검출용 황 함유 프로브의 제조방법을 제공하는 것이다.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. To provide a method for producing a sulfur-containing probe for colorimetric detection that has an effect of enhancing enzyme activity mediated by a colorimetric reaction, characterized in that at least one nucleobase present in the region is adenine (A).
따라서 본 발명은, 비색 검출용 황 함유 프로브로서, 상기 프로브는 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태를 가지며, 상기 핵염기의 연속적인 연결은 아데닌(A), 구아닌(G), 시토신(C) 및 티민(T) 중에서 선택되는 어느 하나 이상이 무작위적으로 연결되어 있고, 상기 DNA 올리고뉴클레오티드 중 뉴클레오티드 포스페이트 백본이 하나 이상의 포스포로티오에이트로 변형되어(phosphorothioate modification) 하나 이상의 황(sulfur) 원자를 포함하고 있는 것을 특징으로 하는, 비색 검출용 황 함유 프로브를 제공한다.Therefore, 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. Provided is a sulfur-containing probe for colorimetric detection, characterized in that it contains.
본 발명의 일실시예에 있어서, 상기 비색 검출용 황 함유 프로브는 10개 내지 30개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태를 갖는 것일 수 있다.In one embodiment of the present invention, 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.
본 발명의 일실시예에 있어서, 상기 비색 검출용 황 함유 프로브는 비색 반응 매개의 효소 활성을 증진시키는 것일 수 있다.In one embodiment of the present invention, the sulfur-containing probe for colorimetric detection may enhance the enzyme activity of the colorimetric reaction mediator.
본 발명의 일실시예에 있어서, DNA 올리고뉴클레오티드의 포스페이트 백본을 포스포로티오에이트로 변형시킬 경우, 상기 변형의 양쪽 옆에 존재하는 핵염기는 적어도 하나가 아데닌(A)인 것일 수 있다.In one embodiment of the present invention, when the phosphate backbone of a DNA oligonucleotide is modified with phosphorothioate, at least one nucleobase present on both sides of the modification may be adenine (A).
본 발명의 일실시예에 있어서, 상기 하나 이상의 황(sulfur) 원자를 포함하는 DNA 올리고뉴클레오티드는 단일 가닥 DNA 올리고뉴클레오티드일 수 있다.In one embodiment of the present invention, the DNA oligonucleotide containing one or more sulfur atoms may be a single-stranded DNA oligonucleotide.
또한 본 발명은 본 발명의 비색 검출용 황 함유 프로브를 포함하는, 비색 검출용 조성물을 제공한다.The present invention also provides a composition for colorimetric detection, comprising the sulfur-containing probe for colorimetric detection of the present invention.
본 발명의 일실시예에 있어서, 상기 조성물은 비색계 시약을 더 포함하는 것일 수 있다.In one embodiment of the present invention, the composition may further include a colorimetric reagent.
본 발명의 일실시예에 있어서, 상기 비색계 시약은 ABTS(2,2'-아지노-bis(3-에틸벤조싸이아졸린-6-설폰산), OPD(o-페닐렌다이아민 다이하이드로클로라이드), DAB(다이아미노벤지딘), AEC(3-아미노-9-에틸카보졸), TMB(3,3',5,5'-테트라메틸벤지딘), AmplexRed, 및 Homovanilic acid로 이루어진 군으로부터 선택되는 1종 이상; 및 1종 이상의 과산화물일 수 있다.In one embodiment of the present invention, 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.
본 발명의 일실시예에 있어서, 상기 조성물에는 헤민(hemin)을 더 포함할 수 있다.In one embodiment of the present invention, the composition may further include hemin.
또한 본 발명의 일실시예에 있어서, 상기 조성물에는 과산화수소(H2O2)를 더 포함할 수 있다.Additionally, in one embodiment of the present invention, the composition may further include hydrogen peroxide (H 2 O 2 ).
또한 본 발명은 본 발명의 비색 검출용 조성물을 포함하는 비색 검출용 키트를 제공한다.Additionally, the present invention provides a kit for colorimetric detection containing the composition for colorimetric detection of the present invention.
또한 본 발명은 본 발명의 비색 검출용 황 함유 프로브를 이용한 비색 검출 방법을 제공한다.Additionally, the present invention provides a colorimetric detection method using the sulfur-containing probe for colorimetric detection of the present invention.
나아가 본 발명은, (1) 검출 대상의 타겟 유전자를 선정하는 단계; (2) 상기 타겟 유전자의 핵산서열에 대한 상보적인 올리고뉴클레오티드 서열을 디자인하는 단계; 및 (3) 상기 상보적인 올리고뉴클레오티드에서 뉴클레오티드 포스페이트 백본을 하나 이상의 포스포로티오에이트로 변형시키는 단계를 포함하며, 상기 상보적인 올리고뉴클레오티드는 10 내지 30mer의 길이를 가지며, 포스포로티오에이트 변형은 양쪽 옆에 존재하는 핵염기가 적어도 하나는 아데닌(A)인 영역에서 이루어지는 것을 특징으로 하는, 비색 반응 매개의 효소 활성 증진 효과를 갖는 비색 검출용 황 함유 프로브의 제조방법을 제공한다.Furthermore, 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. Provided is 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).
본 발명은 황을 핵산에 도입하여 서열의 제한을 갖는 비색 진단의 문제점을 해결할 수 있도록 하였다. 즉, 본 발명에 따른 핵산에 존재하지 않는 원소인 황을 도입하여 퍼옥시다제 활성을 보이는 새로운 개념의 DNAzyme은 DNA형태에 따라 검출 양성 및 음성의 결과를 확인할 수 있어 비색 진단 기술 응용성을 확장할 수 있는 효과를 갖는다. 또한, 구조가 필수적으로 필요한 기존 DNAzyme과 다르게 비색을 위한 특이적인 구조 및 추가적인 변형 없이도 다양한 시퀀스에 폭넓게 적용할 수 있는 범용성이 높은 효과를 가지며, 또한 핵산 내로 황(sulfur) 도입이 쉬운 포스포로티오에이트 변형(phosphorothioate modification)을 이용함으로써 가격이 저렴하고 도입이 쉬운 장점이 있다. 또한, 모노머 형태에 포함된 황은 비색 활성이 없는 반면 충분한 길이의 황 변형 단일 가닥의 DNA(sulfur modified single stranded DNA)는 hemin과 상호작용하여 과산화효소 유사 활성을 발생시킬 수 있으며, 상보적인 서열을 혼성화시키는 경우 반응에 참여하는 염기들이 수소결합으로 기질과의 반응이 제한되어 비색이 소광되는 효과를 확인할 수 있으므로, 종래 방법에 비해 간편하고 신속하며 정확하게 비색 검출 및 진단이 가능한 효과를 갖는다.The present invention solves the problem of colorimetric diagnosis with sequence limitations by introducing sulfur into nucleic acids. In other words, 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. In addition, 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. In addition, while sulfur contained in monomeric form has no colorimetric activity, sulfur modified single stranded DNA of sufficient length can interact with hemin to generate peroxidase-like activity and hybridize complementary sequences. In this case, 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.
도 1은 핵산에 황을 도입한 효과를 확인한 것으로, A) PO 결합(phosphodiester bond)과 PS 결합(phosphorothioate bond)된 뉴클레오티드 간의 연결 구조를 각각 나타낸 것이고, B) PS 변형 여부에 따른 효소 활성의 차이에 대한 모식도를 나타낸 것으로, PS 변형이 ssDNA에 도입될 경우, 효소 활성의 증가를 확인한 것이며, C) PO-DNA 및 PS-DNA 존재 하에서 비색 분석결과를 405 nm에서흡광도 측정으로 확인한 것이며, D) 405 nm에서 관찰된 실시간 흡광도 데이터를 나타낸 것으로, blank는 핵산이 포함되지 않은 군을 나타낸 것이다.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.
도 2는 비색 분석의 최적 조건을 확인한 것으로, A) hemin 농도별 1시간 상온에서 반응 후, 405nm에서의 흡광도 스펙트럼을 나타낸 것이고(가장 왼쪽), 1시간 동안의 실시간 흡광도 곡선을 나타낸 것이며(가운데 및 오른쪽), B) H2O2 농도별 비색 분석 결과를 나타낸 것으로, 1시간 상온에서 반응 후, 405nm에서의 흡광도 스펙트럼을 나타낸 것이고(가장 왼쪽), 1시간 동안의 실시간 흡광도 곡선을 나타낸 것이다(가운데, 오른쪽).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).
도 3은 PS 결합의 유효성을 검증한 것으로, A) 무작위 선택된 동일 서열에 도입된 PS 결합 수에 따른 흡광도를 분석한 결과이고, B) 동일 길이의 연속적인 뉴클레오티드(A20, T20, C20, G20)에서 단일 PS 유무에 따른 흡광도 분석결과이며, C) 동일 길이의 연속적인 뉴클레오티드(A20, T20, C20)에서 서열에 삽입된 PS의 개수에 따른 흡광도 분석결과를 나타낸 것이다.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.
도 4는 도 3의 실험군들에 대한 흡광도 분석 결과를 나타낸 것으로, A)는 도 3A 실험군들에 대한 실시간 흡광도 분석 결과를 나타낸 것이고, B)는 도 3B 실험군들에 대한 실시간 흡광도 곡선을 나타낸 것이며, C)~E)는 도 3C에 해당하는 실시간 흡광도 곡선을 나타낸 것이고, F)~I) 도 3에 해당하는 실시간 흡광도 곡선을 나타낸 것이다.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, and 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, and F) to I) show the real-time absorbance curve corresponding to Figure 3.
도 5는 PS 위치에 따른 PW17 G-quadruplex의 흡광도 분석결과를 나타낸 것으로, A) PW17 G-quadruplex 서열에서 PS 위치가 서로 다른 16개의 서로 다른 서열에 대한 흡광도 측정 결과이고, B) 흡광도 검출 후 웰 플레이트 사진을 나타낸 것이며, C)~D) 도 5A에 해당하는 실시간 흡광도 곡선을 나타낸 것이다.Figure 5 shows the absorbance analysis results of PW17 G-quadruplex according to PS position. A) The absorbance measurement results for 16 different sequences with different PS positions in the PW17 G-quadruplex sequence, B) Well after absorbance detection It shows a photograph of the plate, and C) to D) show the real-time absorbance curve corresponding to Figure 5A.
도 6은 PS의 유무에 따라 동일 염기로 구성된 연속염기의 길이가 상이한 서열에서의 효소 활성 차이를 비교한 것으로, A), B), C) 및 D)는 각각 A, T, C, G 염기 종류에 대한 분석 결과를 나타낸 것이다.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.
도 7은 PS에 가까운 근위 핵염기 조성에 따른 효소 활성을 비교 분석한 것으로, A) 근위 핵염기와 PS와의 상호작용을 통한 효소 활성 조절에 대한 평가로서, TTTN*NTTTTTTTTN*NTTTTT 로부터 파생되고 무작위로 나열된 20개의 염기로 구성된 총 16개의 시퀀스 라이브러리를 생성하였고, "N"은 염기 A, T, G 및 C를 나타낸 것이며, 혼합물은 80 pmole DNA로 준비하였다. B) 총 16개 시퀀스 라이브러리를 효소 활성 결과에 따라 순위로 나타낸 것이며, C) 및 D)는 0 PS 서열을 기반으로 생성한 3개의 다른 서열에 대한 효소 활성을 확인한 것으로, 2 PS_H는 2 PS_L보다 효소 활성 효율이 높은 것을 확인한 것이다.Figure 7 is a comparative analysis of enzyme activity according to the composition of the proximal nucleobase close to PS. A) Evaluation of the regulation of enzyme activity through the interaction between the proximal nucleobase and PS, derived from TTTN*NTTTTTTTTN*NTTTTT and randomly A total of 16 sequence libraries consisting of the 20 bases listed were generated, “N” indicates bases A, T, G, and C, and the mixture was prepared with 80 pmole DNA. B) A total of 16 sequence libraries are ranked according to enzyme activity results, 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.
도 8에서 A) 및 B)는 도 7A에 해당하는 실시간 흡광도 곡선과 웰 플레이트 사진을 나타낸 것이고, C) 도 7C에 해당하는 실시간 흡광도 곡선을 나타낸 것이다.In Figure 8, A) and B) show the real-time absorbance curve and well plate photo corresponding to Figure 7A, and C) show the real-time absorbance curve corresponding to Figure 7C.
도 9는 H-OSD 프로브를 이용한 서열 특이적 비색 검출 결과를 나타낸 것으로, A) DNA 형성에 따른 효소 활성의 차이를 모식도로 나타낸 것이고, B) dsDNA 및 ssDNA에서 PS 수에 따른 흡광도 측정 결과를 나타낸 것이며, C) 발판 매개 가닥 변위 반응으로부터 흡광도 신호의 생성 메커니즘을 나타낸 것이고, D) 효소 신호 전달 시스템을 사용하여 1시간 상온에서 반응시킨 플레이트로부터 405 nm에서의 흡광도 강도 결과를 나타낸 것이다.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.
도 10은 효소 신호 전달 시스템을 이용한 서열 특이적 비색 진단에 있어서, 최적의 최종 MgSO4 농도를 확인한 것으로, 도 9D의 시료들을 사용하여 분석하였으며, A) 1시간 실온에서 반응 후, 405nm에서 측정한 흡광도 스펙트럼을 나타낸 것이고, B) 검출 후 웰 플레이트의 사진을 나타낸 것이며, C) 1시간 동안 실시간 흡광도 곡선을 나타낸 것이다.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. A) Measured at 405 nm after reaction at room temperature for 1 hour. This shows the absorbance spectrum, B) shows a photograph of the well plate after detection, and C) shows the real-time absorbance curve for 1 hour.
도 11은 A) 도 9B에 해당하는 실시간 흡광도 곡선을 나타낸 것으로 ssDNA 혼합물은 20 pmole DNA(PO 및 PS)로 제조하여 사용하였고, dsDNA 혼합물은 20 pmole DNA(PO 및 PS)를 24 pmole 보체 DNA(ⓒPO-DNA)와 어닐링하여 제조하여 사용하였으며, PO-dsDNA와 PS-dsDNA는 흡광도 분석 이전에 혼성화하여 수행하였다. B) 도 9D에 해당하는 실시간 흡광도 곡선을 나타낸 것이다.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 ( ⓒPO-DNA) was prepared and used by annealing, and PO-dsDNA and PS-dsDNA were hybridized before absorbance analysis. B) Shows the real-time absorbance curve corresponding to Figure 9D.
도 12는 효소 신호 전달 시스템을 이용한 서열 특이적 비색 진단의 특이성을 분석한 것으로, 발판 매개 가닥 치환 반응은 도 9D와 동일한 방법으로 수행하였고, 60 pmole의 비표적 DNA를 반응에 사용하였다. A) 1시간 실온에서 반응시킨 플레이트를 405 nm에서의 측정한 흡광도 결과를 나타낸 것이고, B) 405 nm에서 측정된 실시간 흡광도 분석결과이며, C) 효소 신호 전달 시스템 검출 후, 웰 플레이트의 사진을 나타낸 것이다.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, and C) Shows a photograph of the well plate after detection of the enzyme signaling system. will be.
도 13은 H-OSD 프로브에 도입된 PS 수가 효소 활성이 미치는 영향을 분석한 것으로, 발판 매개 가닥 치환 반응은 도 9D와 동일한 방법으로 진행하였으나, 이 반응에는 표적 DNA를 포함하지 않고 수행하였다. A) 1시간 실온에서 반응시킨 플레이트를 405 nm에서의 측정한 흡광도 결과를 나타낸 것이고, B) 405 nm에서 측정된 실시간 흡광도 분석결과이며, C) 효소 신호 전달 시스템 검출 후, 웰 플레이트의 사진을 나타낸 것이다.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, and C) Shows a photograph of the well plate after detection of the enzyme signaling system. will be.
도 14는 OSD 프로브를 이용한 서열 특이적 비색 검출 결과를 나타낸 것으로, 도 14에서 A는 DNA 형성에 따른 효소 활성의 차이를 모식도로 나타낸 것이고, B는 서로 다른 PS 개수를 포함하는 OSD 프로브를 이용한 흡광도 측정 결과를 나타낸 것이다.Figure 14 shows the results of sequence-specific colorimetric detection using an OSD probe. In Figure 14, A is a schematic diagram showing the difference in enzyme activity according to DNA formation, and B is the absorbance using OSD probes containing different numbers of PS. This shows the measurement results.
도 15는 본 발명에 따른 OSD 프로브에 대한 효소 신호 전달 시스템을 이용한 서열 특이적 비색 진단에 있어서, 최적의 최종 MgSO4 농도를 확인한 것으로, A) 1시간 실온에서 반응 후, 405nm에서 측정한 흡광도 스펙트럼을 나타낸 것이고, B) 검출 후 웰 플레이트의 사진을 나타낸 것이며, C) 1시간 동안 실시간 흡광도 곡선을 나타낸 것이다.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. A) Absorbance spectrum measured at 405 nm after reaction at room temperature for 1 hour. , B) a photograph of the well plate after detection, and C) a real-time absorbance curve for 1 hour.
도 16은 타겟 서열 유무 및 PS 개수에 따른 본 발명의 OSD 프로브에 대한 실시간 흡광도 곡선을 나타낸 것이다.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.
도 17은 본 발명에 따른 OSD 프로브에 대한 효소 신호 전달 시스템을 이용한 서열 특이적 비색 진단의 특이성을 분석한 것으로, 발판 매개 가닥 치환 반응을 수행하였고, 60 pmole의 비표적 DNA를 반응에 사용한 결과로서, A) 1시간 실온에서 반응시킨 플레이트를 405 nm에서의 측정한 흡광도 결과를 나타낸 것이고, B) 405 nm에서 측정된 실시간 흡광도 분석결과이며, C) 효소 신호 전달 시스템 검출 후, 웰 플레이트의 사진을 나타낸 것이다.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, B) Shows the real-time absorbance analysis results measured at 405 nm, C) A photograph of the well plate after detection of the enzyme signaling system. It is shown.
본 발명은 핵산 기반 비색 검출을 위한 황 함유 프로브 및 핵산 내 황 도입을 통한 효소 활성 가속화 방법에 관한 것이다.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.
본 발명은 종래 비색 분석의 문제점을 개선하고 효소 활성을 가속화시켜 신속하고 정확하게 비색 검출을 가능하게 할 수 있는 새로운 비색 검출용 황 함유 프로브를 제공한다는 점에 특징이 있다.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.
구체적으로 본 발명에 따른 비색 검출용 황 함유 프로브는 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태를 가지며, 상기 핵염기의 연속적인 연결은 아데닌(A), 구아닌(G), 시토신(C) 및 티민(T) 중에서 선택되는 어느 하나 이상이 무작위적으로 연결되어 있고, 상기 DNA 올리고뉴클레오티드 중 뉴클레오티드 포스페이트 백본이 하나 이상의 포스포로티오에이트로 변형되어(phosphorothioate modification) 하나 이상의 황(sulfur) 원자를 포함하고 있는 것을 특징으로 한다.Specifically, the sulfur-containing probe for colorimetric detection according to the present invention 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
본 발명의 비색 검출용 황 함유 프로브는 종래 비색 진단에 사용되는 G-quadruplex과 달리 서열의 제한을 받지 않는 새로운 개념의 DNAzyme으로서, G-quadruplex와 같은 특정 구조를 이루는 서열이 필요하지 않고, 비색 반응 매개의 효소 활성을 가속화시킬 수 있는 특징을 갖는다.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.
본 발명의 상기 비색 검출용 황 함유 프로브는 10개 내지 30개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태일 수 있으며, 바람직하게는 20개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태일 수 있다.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. .
또한, 본 발명의 비색 검출용 황 함유 프로브는 DNA 올리고뉴클레오티드의 포스페이트 백본을 포스포로티오에이트로 변형시켜(PS: phosphorothioate modification) 원래 핵산에는 존재하지 않는 황 원소가 도입되어 있는 특징을 가지며, 이때 PS의 양쪽 옆에 존재하는 핵염기는 적어도 하나가 아데닌(A)일 수 있다.In addition, 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). In this case, PS At least one nucleobase on either side of may be adenine (A).
본 발명의 일실시예에서는, 본 발명의 비색 검출용 황 함유 프로브에 대하여, 황 원자가 비색 반응에 영향을 미치는지 확인하였는데, 황 원자가 도입된 프로브에서 흡광도의 신호 반응이 뚜렷하게 관찰되는 것으로 나타났고, 반면 황 원자가 도입되지 않은 프로브에서는 흡광도의 신호 반응이 관찰되지 않았다.In one embodiment of the present invention, it was confirmed whether sulfur atoms affect the colorimetric reaction of the sulfur-containing probe for colorimetric detection of the present invention, and it was found that a signal response of absorbance was clearly observed in the probe into which sulfur atoms were introduced. No signal response in absorbance was observed in the probe without sulfur atoms introduced.
또한 본 발명의 다른 일실시예에서는, 포스페이트 백본이 포스포로티오에이트로 변형된 PS의 수가 비색 반응의 효소 활성에 영향을 주는지를 분석하였다. 그 결과, PS의 수가 증가할수록 효소 활성이 증가하는 것으로 나타났으며 PS의 수가 효소 활성에 영향을 준다는 것을 확인하였다.Additionally, in another example of the present invention, it was analyzed whether the number of PSs in which the phosphate backbone was modified to phosphorothioate affected the enzyme activity of the colorimetric reaction. As a result, it was found that enzyme activity increased as the number of PS increased, and it was confirmed that the number of PS affects enzyme activity.
다른 일실시예에서는 프로브의 길이가 효소 활성에 영향을 줄 수 있는지를 분석하였으며, 그 결과, 단량체인 dNTP와 α-thio-dNTP는 프로브를 구성하는 뉴클레오티드의 종류에 관계없이 효소 활성이 관찰되지 않았다. 따라서 프로브 DNA의 길이가 효소 활성에 영향을 줄 수 있음을 알 수 있었는데, 이는 DNA와 hemin의 결합 정도는 길이에 따라 달라질 수 있고, DNA와 hemin이 결합할 수 있어야만 PS와 핵염기가 상호작용하여 비색 반응을 나타낼 수 있음을 알 수 있었다.In another example, it was analyzed whether the length of the probe could affect enzyme activity, and as a result, no enzymatic activity was observed for the monomers dNTP and α-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.
따라서 효과적인 비색 검출을 위한 본 발명의 프로브는 10개 내지 30개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드일 수 있고, 바람직하게는 20개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드일 수 있다.Therefore, 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.
이 외에도 본 발명의 비색 검출용 황 함유 프로브는 PS의 인접 위치에 존재하는 핵염기의 종류에 따라 효소 활성이 달라지는 특징을 갖는데, 본 발명의 실시예의 실험결과, 아데닌(A)과 아데닌(A) 염기 사이에 PS가 위치하는 경우, 효소 활성 효율이 다른 염기의 조합군에 비해 가장 우수한 것으로 나타났고, 다음으로 PS가 위치된 양쪽의 핵염기 중 어느 하나가 아데닌(A)인 경우, 효소 활성이 우수한 것으로 나타났다. 한편, T 또는 C와 같은 피리미딘 염기들 사이에 PS가 위치하게 되면 효소 활성 비율이 상대적으로 낮게 나타났다.In addition, 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. As a result of an experiment in an example of the present invention, 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.
그러므로 우수한 비색 검출 효과를 도출하기 위해서는, DNA 올리고뉴클레오티드의 포스페이트 백본을 포스포로티오에이트로 변형시킬 경우, 상기 변형의 양쪽 옆에 존재하는 핵염기는 적어도 하나가 아데닌(A)이 되도록 하는 것이 중요하다. 바람직하게, PS의 위치는 A*A, A*G, G*A, C*A, A*C, A*T, T*A의 순서로 우선하여 선택할 수 있으며, 가장 바람직하게는 A*A에 PS가 위치되도록 할 수 있다. 상기 *표시는 PS의 위치를 나타낸 것이다.Therefore, in order to obtain an excellent colorimetric detection effect, when the phosphate backbone of a DNA oligonucleotide is modified with phosphorothioate, it is important to ensure that at least one nucleobase on both sides of the modification is adenine (A). . Preferably, 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.
또한 본 발명의 비색 검출용 황 함유 프로브는 DNA 형태에 따라 효소 활성의 차이를 갖는 특징이 있다. 즉, 단일가닥 DNA의 형태를 갖는 프로브의 경우, 효소 활성이 강하게 나타나지만 이중가닥 DNA의 형태를 갖는 프로브는 효소 활성이 감소하여 비색 반응이 나타나지 않는 특징이 있다.In addition, 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. In other words, 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.
그러므로 본 발명의 비색 검출용 황 함유 프로브는 단일가닥 DNA의 형태를 갖는다.Therefore, the sulfur-containing probe for colorimetric detection of the present invention has the form of single-stranded DNA.
또한 본 발명은 상기 본 발명의 비색 검출용 황 함유 프로브를 포함하는, 비색 검출용 조성물을 제공한다.The present invention also provides a composition for colorimetric detection, comprising the sulfur-containing probe for colorimetric detection of the present invention.
본 발명에서 제공하는 상기 비색 검출용 황 함유 프로브는 새로운 DNAzyme로 작용할 수 있으며, 용어 “DNAzyme” 통상적으로 효소활성을 가지는 핵산분자를 의미하며, 예를 들어 과산화효소 활성을 갖는 것일 수 있다. 또한, Deoxyribozymes, DNA enzymes, DNAzymes, catalytic DNA, DNA 기반 효소 등을 포함하는 용어일 수 있고, Ribonuclease, RNA ligase일 수도 있고, DNA phosphorylation, DNA adenylation, DNA deglycosylation, porphyrin metalation, thymine dimer photoreversion, DNA cleavage 등의 화학반응을 가능하게 하는 것일 수도 있다.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.
여기서 DNAzyme은 본 발명의 비색 검출용 황 함유 프로브 서열을 포함할 수 있다.Here, the DNAzyme may include a sulfur-containing probe sequence for colorimetric detection of the present invention.
상기 본 발명의 비색 검출용 조성물은 비색계 시약을 포함할 수 있는데, 비색계 시약은 이에 제한되지는 않으나, ABTS(2,2'-아지노-bis(3-에틸벤조싸이아졸린-6-설폰산), OPD(o-페닐렌다이아민 다이하이드로클로라이드), DAB(다이아미노벤지딘), AEC(3-아미노-9-에틸카보졸), TMB(3,3',5,5'-테트라메틸벤지딘), AmplexRed, 및 Homovanilic acid로 이루어진 군으로부터 선택되는 1종 이상; 및 1종 이상의 과산화물을 포함할 수 있다. 상기 과산화물로는 예를 들어, 과산화수소(H2O2)일 수 있다.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. For example, the peroxide may be hydrogen peroxide (H 2 O 2 ).
또한 상기 조성물에는 DNAzyme 코펙터(cofactor)로서 헤민(hemin)을 포함할 수 있다.Additionally, the composition may include hemin as a DNAzyme cofactor.
이 외에도 본 발명의 비색 검출용 조성물은 당업계에서 비색 검출에 사용되는 부가적인 시약 및 성분들을 추가로 더 포함할 수 있다.In addition, the composition for colorimetric detection of the present invention may further include additional reagents and components used for colorimetric detection in the art.
또한, 본 발명은 본 발명의 비색 검출용 조성물을 포함하는 비색 검출용 키트를 제공할 수 있으며, 본 발명의 비색 검출용 황 함유 프로브를 이용한 비색 검출 방법을 제공할 수 있다.In addition, 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.
나아가 본 발명은, 비색 반응 매개의 효소 활성 증진 효과를 갖는 비색 검출용 황 함유 프로브의 제조방법을 제공할 수 있는데, 상기 방법은 (1) 검출 대상의 타겟 유전자를 선정하는 단계; (2) 상기 타겟 유전자의 핵산서열에 대한 상보적인 올리고뉴클레오티드 서열을 디자인하는 단계; 및(3) 상기 상보적인 올리고뉴클레오티드에서 뉴클레오티드 포스페이트 백본을 하나 이상의 포스포로티오에이트로 변형시키는 단계를 포함하며, 상기 상보적인 올리고뉴클레오티드는 10 내지 30mer의 길이를 가지며, 포스포로티오에이트 변형은 양쪽 옆에 존재하는 핵염기가 적어도 하나는 아데닌(A)인 영역에서 이루어지는 것을 특징으로 한다.Furthermore, 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).
본 발명의 방법으로 제조된 비색 검출용 황 함유 프로브는 비색 반응 매개의 효소 활성을 가속화시켜 신속하고 정확하게 비색 반응 분석을 통해 타겟 핵산을 검출할 수 있다. 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.
예를 들어, 본 발명의 프로브를 사용할 경우, 검출 대상의 핵산이 시료에 존재하는 경우, 비색 반응에 의해 흡광도 값이 증가하여 비색 신호가 나타나는 반면, 검출 대상의 핵산이 시료에 존재하지 않는 경우, 비색 신호가 나타나지 않는다.For example, when using the probe of the present invention, if the nucleic acid to be detected is present in the sample, 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.
또한, 본 발명에 따른 상기 프로브는 헤어핀 구조를 형성한 황 함유 프로브일 수 있으며, 상기 황 함유 프로브의 5’ 또는 3’ 말단에 단일 가닥 toeh본 발명은 핵산 기반 비색 검출을 위한 황 함유 프로브 및 핵산 내 황 도입을 통한 효소 활성 가속화 방법에 관한 것이다.In addition, 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.
본 발명은 종래 비색 분석의 문제점을 개선하고 효소 활성을 가속화시켜 신속하고 정확하게 비색 검출을 가능하게 할 수 있는 새로운 비색 검출용 황 함유 프로브를 제공한다는 점에 특징이 있다.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.
구체적으로 본 발명에 따른 비색 검출용 황 함유 프로브는 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태를 가지며, 상기 핵염기의 연속적인 연결은 아데닌(A), 구아닌(G), 시토신(C) 및 티민(T) 중에서 선택되는 어느 하나 이상이 무작위적으로 연결되어 있고, 상기 DNA 올리고뉴클레오티드 중 뉴클레오티드 포스페이트 백본이 하나 이상의 포스포로티오에이트로 변형되어(phosphorothioate modification) 하나 이상의 황(sulfur) 원자를 포함하고 있는 것을 특징으로 한다.Specifically, the sulfur-containing probe for colorimetric detection according to the present invention 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
본 발명의 비색 검출용 황 함유 프로브는 종래 비색 진단에 사용되는 G-quadruplex과 달리 서열의 제한을 받지 않는 새로운 개념의 DNAzyme으로서, G-quadruplex와 같은 특정 구조를 이루는 서열이 필요하지 않고, 비색 반응 매개의 효소 활성을 가속화시킬 수 있는 특징을 갖는다.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.
본 발명의 상기 비색 검출용 황 함유 프로브는 10개 내지 30개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태일 수 있으며, 바람직하게는 20개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태일 수 있다.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. .
또한, 본 발명의 비색 검출용 황 함유 프로브는 DNA 올리고뉴클레오티드의 포스페이트 백본을 포스포로티오에이트로 변형시켜(PS: phosphorothioate modification) 원래 핵산에는 존재하지 않는 황 원소가 도입되어 있는 특징을 가지며, 이때 PS의 양쪽 옆에 존재하는 핵염기는 적어도 하나가 아데닌(A)일 수 있다.In addition, 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). In this case, PS At least one nucleobase on either side of may be adenine (A).
본 발명의 일실시예에서는, 본 발명의 비색 검출용 황 함유 프로브에 대하여, 황 원자가 비색 반응에 영향을 미치는지 확인하였는데, 황 원자가 도입된 프로브에서 흡광도의 신호 반응이 뚜렷하게 관찰되는 것으로 나타났고, 반면 황 원자가 도입되지 않은 프로브에서는 흡광도의 신호 반응이 관찰되지 않았다.In one embodiment of the present invention, it was confirmed whether sulfur atoms affect the colorimetric reaction of the sulfur-containing probe for colorimetric detection of the present invention, and it was found that a signal response of absorbance was clearly observed in the probe into which sulfur atoms were introduced. No signal response in absorbance was observed in the probe without sulfur atoms introduced.
또한 본 발명의 다른 일실시예에서는, 포스페이트 백본이 포스포로티오에이트로 변형된 PS의 수가 비색 반응의 효소 활성에 영향을 주는지를 분석하였다. 그 결과, PS의 수가 증가할수록 효소 활성이 증가하는 것으로 나타났으며 PS의 수가 효소 활성에 영향을 준다는 것을 확인하였다.Additionally, in another example of the present invention, it was analyzed whether the number of PSs in which the phosphate backbone was modified to phosphorothioate affected the enzyme activity of the colorimetric reaction. As a result, it was found that enzyme activity increased as the number of PS increased, and it was confirmed that the number of PS affects enzyme activity.
다른 일실시예에서는 프로브의 길이가 효소 활성에 영향을 줄 수 있는지를 분석하였으며, 그 결과, 단량체인 dNTP와 α-thio-dNTP는 프로브를 구성하는 뉴클레오티드의 종류에 관계없이 효소 활성이 관찰되지 않았다. 따라서 프로브 DNA의 길이가 효소 활성에 영향을 줄 수 있음을 알 수 있었는데, 이는 DNA와 hemin의 결합 정도는 길이에 따라 달라질 수 있고, DNA와 hemin이 결합할 수 있어야만 PS와 핵염기가 상호작용하여 비색 반응을 나타낼 수 있음을 알 수 있었다.In another example, it was analyzed whether the length of the probe could affect enzyme activity, and as a result, no enzymatic activity was observed for the monomers dNTP and α-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.
따라서 효과적인 비색 검출을 위한 본 발명의 프로브는 10개 내지 30개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드일 수 있고, 바람직하게는 20개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드일 수 있다.Therefore, 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.
이 외에도 본 발명의 비색 검출용 황 함유 프로브는 PS의 인접 위치에 존재하는 핵염기의 종류에 따라 효소 활성이 달라지는 특징을 갖는데, 본 발명의 실시예의 실험결과, 아데닌(A)과 아데닌(A) 염기 사이에 PS가 위치하는 경우, 효소 활성 효율이 다른 염기의 조합군에 비해 가장 우수한 것으로 나타났고, 다음으로 PS가 위치된 양쪽의 핵염기 중 어느 하나가 아데닌(A)인 경우, 효소 활성이 우수한 것으로 나타났다. 한편, T 또는 C와 같은 피리미딘 염기들 사이에 PS가 위치하게 되면 효소 활성 비율이 상대적으로 낮게 나타났다.In addition, 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. As a result of an experiment in an example of the present invention, 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.
그러므로 우수한 비색 검출 효과를 도출하기 위해서는, DNA 올리고뉴클레오티드의 포스페이트 백본을 포스포로티오에이트로 변형시킬 경우, 상기 변형의 양쪽 옆에 존재하는 핵염기는 적어도 하나가 아데닌(A)이 되도록 하는 것이 중요하다. 바람직하게, PS의 위치는 A*A, A*G, G*A, C*A, A*C, A*T, T*A의 순서로 우선하여 선택할 수 있으며, 가장 바람직하게는 A*A에 PS가 위치되도록 할 수 있다. 상기 *표시는 PS의 위치를 나타낸 것이다.Therefore, in order to obtain an excellent colorimetric detection effect, when the phosphate backbone of a DNA oligonucleotide is modified with phosphorothioate, it is important to ensure that at least one nucleobase on both sides of the modification is adenine (A). . Preferably, 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.
또한 본 발명의 비색 검출용 황 함유 프로브는 DNA 형태에 따라 효소 활성의 차이를 갖는 특징이 있다. 즉, 단일가닥 DNA의 형태를 갖는 프로브의 경우, 효소 활성이 강하게 나타나지만 이중가닥 DNA의 형태를 갖는 프로브는 효소 활성이 감소하여 비색 반응이 나타나지 않는 특징이 있다.In addition, 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. In other words, 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.
그러므로 본 발명의 비색 검출용 황 함유 프로브는 단일가닥 DNA의 형태를 갖는다.Therefore, the sulfur-containing probe for colorimetric detection of the present invention has the form of single-stranded DNA.
또한 본 발명은 상기 본 발명의 비색 검출용 황 함유 프로브를 포함하는, 비색 검출용 조성물을 제공한다.The present invention also provides a composition for colorimetric detection, comprising the sulfur-containing probe for colorimetric detection of the present invention.
본 발명에서 제공하는 상기 비색 검출용 황 함유 프로브는 새로운 DNAzyme로 작용할 수 있으며, 용어 “DNAzyme” 통상적으로 효소활성을 가지는 핵산분자를 의미하며, 예를 들어 과산화효소 활성을 갖는 것일 수 있다. 또한, Deoxyribozymes, DNA enzymes, DNAzymes, catalytic DNA, DNA 기반 효소 등을 포함하는 용어일 수 있고, Ribonuclease, RNA ligase일 수도 있고, DNA phosphorylation, DNA adenylation, DNA deglycosylation, porphyrin metalation, thymine dimer photoreversion, DNA cleavage 등의 화학반응을 가능하게 하는 것일 수도 있다.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.
여기서 DNAzyme은 본 발명의 비색 검출용 황 함유 프로브 서열을 포함할 수 있다.Here, the DNAzyme may include a sulfur-containing probe sequence for colorimetric detection of the present invention.
상기 본 발명의 비색 검출용 조성물은 비색계 시약을 포함할 수 있는데, 비색계 시약은 이에 제한되지는 않으나, ABTS(2,2'-아지노-bis(3-에틸벤조싸이아졸린-6-설폰산), OPD(o-페닐렌다이아민 다이하이드로클로라이드), DAB(다이아미노벤지딘), AEC(3-아미노-9-에틸카보졸), TMB(3,3',5,5'-테트라메틸벤지딘), AmplexRed, 및 Homovanilic acid로 이루어진 군으로부터 선택되는 1종 이상; 및 1종 이상의 과산화물을 포함할 수 있다. 상기 과산화물로는 예를 들어, 과산화수소(H2O2)일 수 있다.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).
또한 상기 조성물에는 DNAzyme 코펙터(cofactor)로서 헤민(hemin)을 포함할 수 있다.Additionally, the composition may include hemin as a DNAzyme cofactor.
이 외에도 본 발명의 비색 검출용 조성물은 당업계에서 비색 검출에 사용되는 부가적인 시약 및 성분들을 추가로 더 포함할 수 있다.In addition, the composition for colorimetric detection of the present invention may further include additional reagents and components used for colorimetric detection in the art.
또한, 본 발명은 본 발명의 비색 검출용 조성물을 포함하는 비색 검출용 키트를 제공할 수 있으며, 본 발명의 비색 검출용 황 함유 프로브를 이용한 비색 검출 방법을 제공할 수 있다.In addition, 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.
나아가 본 발명은, 비색 반응 매개의 효소 활성 증진 효과를 갖는 비색 검출용 황 함유 프로브의 제조방법을 제공할 수 있는데, 상기 방법은 (1) 검출 대상의 타겟 유전자를 선정하는 단계; (2) 상기 타겟 유전자의 핵산서열에 대한 상보적인 올리고뉴클레오티드 서열을 디자인하는 단계; 및(3) 상기 상보적인 올리고뉴클레오티드에서 뉴클레오티드 포스페이트 백본을 하나 이상의 포스포로티오에이트로 변형시키는 단계를 포함하며, 상기 상보적인 올리고뉴클레오티드는 10 내지 30mer의 길이를 가지며, 포스포로티오에이트 변형은 양쪽 옆에 존재하는 핵염기가 적어도 하나는 아데닌(A)인 영역에서 이루어지는 것을 특징으로 한다.Furthermore, 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).
본 발명의 방법으로 제조된 비색 검출용 황 함유 프로브는 비색 반응 매개의 효소 활성을 가속화시켜 신속하고 정확하게 비색 반응 분석을 통해 타겟 핵산을 검출할 수 있다. 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.
예를 들어, 본 발명의 프로브를 사용할 경우, 검출 대상의 핵산이 시료에 존재하는 경우, 비색 반응에 의해 흡광도 값이 증가하여 비색 신호가 나타나는 반면, 검출 대상의 핵산이 시료에 존재하지 않는 경우, 비색 신호가 나타나지 않는다.For example, when using the probe of the present invention, if the nucleic acid to be detected is present in the sample, 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.
또한, 본 발명에 따른 상기 프로브는 헤어핀 구조를 형성한 황 함유 프로브일 수 있으며, 상기 황 함유 프로브의 5’ 또는 3’ 말단에 단일 가닥 toehold 서열 10 mer와 그 뒤로 이중 가닥 stem 구조 20 bp로 서로 상보적인 서열이 혼성화되어 있고，연속된 T 10 mer로 구성된 loop 구조로 설계된 형태를 가질 수 있다.In addition, 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.
본 발명의 프로브는 단일 주형 가닥인 표적 핵산에 포함된 타겟 결합 사이트(target-binding site)에 상보적인 서열을 포함하고 있으며, 프로브는 발판(toehold) 서열부터 표적 핵산에 결합하며 프로브 구조가 branch migration이 일어나 결과적으로 원래의 혼성화되어 있던 상보적인 서열을 밀어내는 발판 매개 단일가닥 치환 반응(toehold-mediated strand displacement reaction)이 발생된다.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.
이 경우, toehold를 포함한 서열 일부가 표적 핵산에 상보적으로 결합하며 혼성화 과정을 통해 새로운 이중체 구조를 형성하게 되며, 또한，프로브의 상보적으로 결합하고 있던 기존 서열은 단일 가닥 형태로 자유롭게 방출된다. 방출된 단일 가닥은 PS를 포함할 수 있으며, PS를 포함하는 경우 앞선 본 발명의 실험 결과와 동일하게 효소 활성이 강하게 나타나는 것을 확인하였다.In this case, 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.
그러므로 본 발명의 프로브는 분자 내 혼성화(intramolecular hybridization)를 통해 프로브를 구성하는 H-OSD로 적용할 수 있을 뿐만 아니라, 분자 간 혼성화를 통해 프로브를 구성하는 OSD의 적용 및 활용도 가능하다.Therefore, 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.
OSD는 두 개의 서로 상보적인 가닥으로 구성되어 분자 간 혼성화(intermolecular hybridization)를 통해 DNA 이중체를 형성할 수 있다．DNA 이중체는 단일가닥 치환(strand displacement)에 의해 방출되는 output 가닥과 substrate 가닥으로 나누어진다．substrate 가닥은 output 가닥과 다르게 추가적으로 단일 가닥의 발판서열(toehold sequence)를 포함하고 있다.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. With output strand and substrate strand The substrate strand, unlike the output strand, additionally contains a single strand of toehold sequence.
상보적인 표적 핵산이 공급되는 경우 발판 서열과 표적 핵산이 부분적으로 결합하면서 output 가닥을 branch migration 시키며 이후엔 완전히 방출시킨다．이때, 단일 가닥으로 상태가 변환된 output 가닥이 PS를 포함하고 있는 경우 효소 활성을 강하게 나타낼 수 있다.When a complementary target nucleic acid is supplied, the scaffold sequence and the target nucleic acid are partially combined, causing the output strand to branch migrate, and then completely released. At this time, the output strand converted to a single strand is PS. If it contains enzyme activity, It can be expressed strongly.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.
<재료준비 및 실험방법><Material preparation and experiment method>
재료 및 시약Materials and Reagents
본 실험에 사용한 모든 올리고뉴클레오티드는 Bioneer(대한민국, 대전) 및 Integrated DNA Technologies, Inc.(Coralville, IA, 미국)에서 합성한 것을 사용하였다. 또한, 본 실험에 사용한 모든 DNA의 서열은 하기 표 1에 나타내었다. 구연산, 구연산나트륨 삼염기 이수화물(sodium citrate tribasic dihydrate), ABTS(2,2'-Azino-bis-(3-에틸벤조티아졸린-6-술폰산) 이암모늄염), 헤민 및 DMSO(디메틸 설폭사이드)는 시그마 사에서 구입한 것을 사용하였다. 또한 27%(w/w) H2O2는 ThermoFisher Scientific, Inc.(Waltham, MA, USA)에서 구입한 것으로 사용하였고, 10X 등온 증폭 완충액과 디옥시 뉴클레오티드(dNTP) 용액 세트(100mM)는 New England Biolabs, Inc.(Beverly, MA, USA)에서 구입하여 사용하였다. 96웰 투명 플레이트는 Corning(Corning, NY, U.S.A.)에서 준비한 것을 사용하였으며, α-thio-dATP, α-thio-dGTP, α-thio-dCTP는 TriLink BioTechnologies(San Diego, CA, USA)에서 구입한 것을 사용하였고, dTTPαS(α-thio-dTTP)는 Jena Bioscience GmbH(독일 예나)에서 구입하여 사용하였다. NaOH 및 HCl은 iNtRON Biotechnology, Inc.(대한민국 성남)에서 구입한 것을 사용하였고, 2mM Hemin 스톡 용액은 DMSO를 이용하여 만들었으며, -20°C에서 보관하였다. 하기 표에서 * 표기는 phosphorothioate bond(PS) 변형을 나타낸 것이다.All 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. Citric acid, sodium citrate tribasic dihydrate, 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), hemin, and dimethyl sulfoxide (DMSO). was used purchased from Sigma. In addition, 27% (w/w) H 2 O 2 purchased from ThermoFisher Scientific, Inc. (Waltham, MA, USA) was used, and 10X isothermal amplification buffer and deoxynucleotide (dNTP) solution set (100mM) were purchased from New. It was purchased and used from England Biolabs, Inc. (Beverly, MA, USA). The 96-well transparent plate was prepared by Corning (Corning, NY, USA), and α-thio-dATP, α-thio-dGTP, and α-thio-dCTP were purchased from TriLink BioTechnologies (San Diego, CA, USA). dTTPαS (α-thio-dTTP) was purchased from Jena Bioscience GmbH (Jena, Germany). NaOH and HCl were purchased from iNtRON Biotechnology, Inc. (Seongnam, Korea), and a 2mM Hemin stock solution was prepared using DMSO and stored at -20°C. In the table below, * indicates phosphorothioate bond (PS) modification.
모노뉴클레오티드 및 DNA 준비Mononucleotide and DNA preparation
모든 DNA 혼합물은 1X 등온증폭 완충액(20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 2mM MgSO4, 0.1% Tween® 20, pH 8.8)으로 준비하였다. ssDNA 혼합물은 40 pmole DNA로 제조하였고, 모노뉴클레오티드 혼합물은 40 pmole의 dNTP 또는 α-thio-dNTP로 제조하였다. 달리 명시하지 않는 한 모든 핵산은 40 pmole의 농도로 사용하였다. DNA 혼합물을 95°C에서 5분 동안 반응 후, 0.1°C/s의 속도로 25°C까지 천천히 냉각시켰고, 이후 DNA 혼합물은 사용하기 최소 5분 전에 25°C의 온도가 되도록 유지하였다.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 α-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.
PW17 G-quadruplex 분석PW17 G-quadruplex analysis
G-quadruplex 분석물질은 1X 등온증폭 완충액(20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 2mM MgSO4, 0.1% Tween® 20, pH 8.8)을 이용하여 준비하였다. PW17 혼합물은 40 pmole DNA(PO 또는 1 PS)과 반응시켜 준비하였고, 분석물은 95°C에서 5분 동안 반응시킨 후, 0.1°C/s의 속도로 25°C까지 천천히 냉각하고 실온에서 5분 동안 반응시켰다.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.
흡광도 측정 및 영상 획득Absorbance measurement and image acquisition
분석 시료의 총 부피는 122.5 μL가 되도록 하였다. 75μM hemin 1.5μL, 0.025%(w/v) ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-술폰산)디암모늄 염)을 함유한 100mM 구연산염 버퍼 완충액 100μL(25°C, pH 4.0) 및 1 μL의 3% H2O2를 20 μL의 DNA 혼합물에 첨가하였다. 이후 120 μL의 최종 샘플 용액을 96-웰 투명 플레이트로 옮기고 24~25°C에서 1시간 동안 반응시켰다. 그런 뒤, 반응 혼합물에 대한 405 nm에서의 흡수 강도를 Tecan Spark® M10 다중 모드 마이크로플레이트 판독기(스위스 Mannedorf)를 사용하여 총 60회 동안 1분마다 기록하였다. 또한 영상은 분석 샘플을 96-well 투명 플레이트(Corning cat. No. 9017)에 로딩한 후, EOS-7D(Canon, Japan)를 이용하여 촬영함으로써 획득하였다.The total volume of the analysis sample was set to 122.5 μL. 1.5 μL of 75 μM hemin, 100 μL 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 μL of 3% H 2 O 2 were added to 20 μL of the DNA mixture. Afterwards, 120 μL of the final sample solution was transferred to a 96-well transparent plate and incubated at 24-25°C for 1 hour. 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).
효소성 프로브 준비Enzymatic probe preparation
프로브 용액은 1X 등온증폭 완충액(20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 2mM MgSO4, 0.1% Tween® 20, pH 8.8) 및 6mM MgSO4(최종 8mM)에서 25μM 헤어핀 프로브 가닥을 어닐링하여 준비하였다. 이후 프로브 용액을 95°C에서 5분간 반응시키고 0.1°C/s의 속도로 25°C까지 천천히 냉각시킨 후, 실온에서 1시간 동안 반응시켰으며, 상기 프로브는 실험에 사용 전 4°C에 보관하였다.The probe solution was 25 μM 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.
효소성 프로브 준비 - OSDEnzymatic Probe Preparation - OSD
프로브 용액은 1X 등온증폭 완충액(20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 2mM MgSO4, 0.1% Tween® 20, pH 8.8) 및 6mM MgSO4(최종 8mM)에서 25μM 방출 가닥과 ３0μM 발판 가닥을 어닐링하여 준비하였다. 이후 프로브 용액을 95°C에서 5분간 반응시키고 0.1°C/s의 속도로 25°C까지 천천히 냉각시킨 후, 실온에서 1시간 동안 반응시켰으며, 상기 프로브는 실험에 사용 전 4°C에 보관하였다.The probe solution was 25 μM release strand in 1 and 30μM 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.
H-OSD 및 OSD 프로브를 이용한 서열 특이적 비색 분석Sequence-specific colorimetric analysis using H-OSD and OSD probes
1X 등온증폭 완충액(20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 2mM MgSO4, 0.1% Tween® 20, pH 8.8), 6mM MgSO4(최종 8mM), 60pmole 의 타겟 DNA 및 1.6 μL의 프로브 용액을 첨가하여 발판 매개 가닥 치환 반응(toehold-mediated strand displacement reaction)을 시작하였으며, 반응은 25℃에서 1시간 동안 수행하였다. 이후 1.5μL의 75μM hemin, 0.025%(w/v) ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-술폰산)디암모늄염)을 함유하는 100μL의 100mM 구연산염 완충액(pH 4.0) 및 1 μL의 3% H2O2를 20 μL의 반응 생성물에 첨가하고 혼합하였다. 120 μL의 최종 샘플 용액을 96웰 투명 플레이트에 옮기고 1시간 동안 24°C에서 유지시킨 후, 반응 혼합물에 대한 405 nm에서의 흡수 강도를 Tecan Spark® M10 다중 모드 마이크로플레이트 판독기(스위스 M_nnedorf)를 사용하여 총 60회 동안 1분마다 기록하였고, 96-well 투명 플레이트(Corning cat. No. 9017)에 로딩된 분석 샘플들은 EOS-7D(Canon, Japan)를 이용하여 영상 촬영하였다.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 μL of the probe solution, and the reaction was performed at 25°C for 1 hour. This was followed by 100 μL of 100 mM citrate buffer (pH) containing 1.5 μL of 75 μM hemin, 0.025% (w/v) ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt). 4.0) and 1 μL of 3% H2O2 were added to 20 μL of reaction product and mixed. 120 μL of the final sample solution was transferred to a 96-well transparent plate and kept at 24 °C for 1 h, after which the absorption intensity at 405 nm for the reaction mixture was measured using a Tecan Spark® M10 multimode microplate reader (M_nnedorf, Switzerland). Recording was recorded every minute for a total of 60 times, and the analysis samples loaded into a 96-well transparent plate (Corning cat. No. 9017) were imaged using EOS-7D (Canon, Japan).
<실시예 1><Example 1>
단일 가닥 DNA(ssDNA)로 황 원자의 도입Introduction of sulfur atoms into single-stranded DNA (ssDNA)
핵산에는 원래 존재하지 않는 황 원자를 PS(phosphorothioate bond) 변형을 통해 핵산에 도입할 경우, 황 원자가 비색 반응에 영향을 미치는지를 분석하였다(도 1A). 이때 황 원자는 다양한 형태의 핵산에 포함될 수 있다.When 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.
그 결과, PS가 ssDNA에 도입한 경우, 효소 활성이 확인되었으며 효소 활성이 활성화되어 높은 흡광도(O.D.)의 신호 반응이 관찰되었다. 반면, PO(phosphodiester bond)가 도입된 ssDNA에서는 흡광도 신호 반응이 거의 관찰되지 않았다(도 1B).As a result, when PS was introduced into ssDNA, enzyme activity was confirmed, and a signal reaction with high absorbance (O.D.) was observed as the enzyme activity was activated. On the other hand, almost no absorbance signal response was observed in ssDNA into which a phosphodiester bond (PO) was introduced (Figure 1B).
따라서 본 발명자들은 단일 가닥의 DNA 백본에 황 원자의 도입시켜 변형을 유도할 경우, G-quadruplex와 같은 특정 구조를 이루는 서열이 없더라도 간단하고 편리하게 효소 활성을 활성화시킬 수 있음을 알 수 있었다.Therefore, 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.
또한, 황을 핵산 내로 도입하는 인공적인 결합 변형인 PS 결합(phosphorothioate bond)을 특정 서열에 도입하여 효소 활성의 효과를 관찰하였다. 이때 특정 서열로는 SARS-CoV-2 바이러스의 N 유전자 내의 20mer 올리고뉴클레오티드(PO-DNA: 5'-ATACAAAACATTCCCACCAA-3')를 선택하였고, 또한, 동일 특정 서열에 대해 PS 결합 변형을 유도한 DNA(PS-DNA: 5'-ATAC*AAAA*CATT*CCCA*CCAA-3')를 준비하였으며, 이때 PS가 서열 내에서 고르게 분포되도록 설계하였고, 별표(*)는 황 도입부위를 나타낸 것이다. 또한, PS-DNA 존재 시 색상 변화를 최대화하기 위해 hemin과 H2O2의 농도는 최적화시킨 농도를 사용하였고(도 2), 구연산염 완충액과 ABTS 농도는 설정된 대로 사용하였다. 각 PO-DNA와 PS-DNA는 헤민, H2O2, ABTS가 포함된 반응 완충액과 혼합하였고, 이후 흡광도 세기를 측정하여 색상 변화를 실시간으로 모니터링하였다.In addition, the effect of enzyme activity was observed by introducing a PS bond (phosphorothioate bond), an artificial bond modification that introduces sulfur into a nucleic acid, into a specific sequence. At this time, a 20mer oligonucleotide (PO-DNA: 5'-ATACAAAACATTCCCACCAA-3') within the N gene of the SARS-CoV-2 virus was selected as the specific sequence, and DNA that induced PS bond modification for the same specific sequence ( PS-DNA: 5'-ATAC*AAAA*CATT*CCCA*CCAA-3') was prepared, and in this case, PS was designed to be evenly distributed within the sequence, and the asterisk (*) indicates the sulfur introduction site. In addition, to maximize the color change in the presence of PS-DNA, optimized concentrations of hemin and H2O2 were used (Figure 2), and citrate buffer and ABTS concentrations were used as set. Each PO-DNA and PS-DNA were mixed with a reaction buffer containing hemin, H2O2, and ABTS, and then the color change was monitored in real time by measuring the absorbance intensity.
그 결과, 몇 분 후에 PO-DNA와 PS-DNA 사이에 서로 다른 흡광 신호 반응 결과가 나타나기 시작했으며, 흡광도는 PS-DNA가 있는 경우 약 2.0까지 크게 증가하는 것으로 나타난 반면, PO-DNA 처리 군에서는 음성 대조군(Blank)과 유사한 흡광 값을 나타내었다(도 1C, 1D).As a result, after a few minutes, different absorbance signal responses began to appear between PO-DNA and PS-DNA, and the absorbance was found to increase significantly up to about 2.0 in the presence of PS-DNA, whereas in the PO-DNA treatment group, It showed similar absorbance values as the negative control (Blank) (Figures 1C, 1D).
이러한 비색 분석결과를 통해 본 발명자들은 황 원자를 PS로 변형시킨 DNA를 비색 검출을 위한 프로브로 사용할 경우, 효소 활성을 가속화시켜 보다 신속하고 정확하게 비색 진단 결과를 도출할 수 있음을 알 수 있었다.Through these colorimetric analysis results, the present inventors found that when DNA with sulfur atoms modified with PS is used as a probe for colorimetric detection, enzyme activity can be accelerated and colorimetric diagnostic results can be derived more quickly and accurately.
<실시예 2><Example 2>
PS 변형(PS modification)이 효소활성에 미치는 영향분석Analysis of the effect of PS modification on enzyme activity
본 발명자들은 PS의 수에 따라 효소 활성이 어떻게 변화하는지 확인하기 위해, 다양한 PS 수(0, 1, 2, 4, 8)를 핵산 내에 도입시켜 실험을 수행하였다.To determine how enzyme activity changes depending on the number of PS, the present inventors performed an experiment by introducing various numbers of PS (0, 1, 2, 4, 8) into nucleic acid.
그 결과, PS의 수가 증가함에 따라 효소 활성이 증가하는 것으로 나타났다(도 3A 및 도 4A). 이는 핵산에 포함된 PS의 수가 증가할수록 효소 활성이 증가한다는 것을 의미하며, 이러한 결과는 PS가 효소 활성 발생에 긍정적으로 기여하며, PS의 수가 효소 활성에 중요한 역할을 한다는 것을 알 수 있었다.As a result, enzyme activity appeared to increase as the number of PS increased (Figures 3A and 4A). This means that enzyme activity increases as the number of PS contained in the nucleic acid increases. These results show that PS contributes positively to the generation of enzyme activity, and that the number of PS plays an important role in enzyme activity.
다음으로, 본 발명자들은 PS와 nucleobases 간의 상관관계를 분석하였다. 이를 위해 각각의 A, C, G, T 뉴클레오티드에 대해 20개의 연속된 뉴클레오티드를 갖는 서열을 준비하고 PS를 도입한 경우와 도입하지 않은 경우에 대해 실험을 수행하였다. 참고로, A20은 20개의 아데닌(A) 염기가 연속적으로 이루어진 뉴클레오티드를 말한다.Next, the present inventors analyzed the correlation between PS and nucleobases. For this purpose, sequences with 20 consecutive nucleotides for each A, C, G, and T nucleotide were prepared, and experiments were performed with and without PS introduced. For reference, A 20 refers to a nucleotide consisting of 20 adenine (A) bases in a row.
그 결과, PS가 존재하는 경우, A20과 G20의 뉴클레오티드에서는 흡광도 값이 증가하는 것으로 나타난 반면, C20과 T20의 뉴클레오티드에서는 흡광도 값이 크게 변하지 않는 것으로 나타났다. 또한, 다른 종류의 뉴클레오티드와 달리 G20의 경우, PS가 있거나 없는 경우 모두 블랭크에 비해 효소 활성이 증가한 것으로 나타났는데(도 3B 및 도 4B), 이는 G-quadruplex 구조가 PS 없이도 부분적으로 형성될 수 있어 상대적으로 효소 활성이 증가하였기 때문인 것으로 보이며, PS가 도입되었을 때 효소 활성이 PO에 비해 감소하는 경향을 나타낸 것은 PS 첨가가 분자간 G-quadruplex 형성 시 구조적 불안정성을 증가시켜 효소 활성을 감소시킨 것으로 보인다. 또한 표준 및 잘 알려진 G-quadruplex 구조에서 PS의 효과를 추가로 확인했지만 유의미한 효과는 확인되지 않았다(도 5). 반면에, A20의 효과는 극적이지는 않지만 흡광도 값이 약간 증가하는 것으로 관찰되었는데, A20은 G20과 달리 특정 구조를 형성하지 않아 ssDNA만으로 효소 활성이 나타났다. 이를 통해 퓨린 염기가 피리미딘 염기보다 효소 활성에 더 효율적으로 관여한다는 것을 알 수 있었다.As a result, in the presence of PS, 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 . In addition, unlike other types of nucleotides, in the case of G 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. . Additionally, the effect of PS was further confirmed in standard and well-known G-quadruplex structures, but no significant effect was identified (Figure 5). On the other hand, the effect of A 20 was not dramatic, but a slight increase in absorbance value was observed. Unlike G 20 , A 20 did not form a specific structure, showing enzyme activity only with ssDNA. This showed that purine bases participate in enzyme activity more efficiently than pyrimidine bases.
또한, 본 발명자들은 PS의 수를 증가시킬 경우, 효소 활성이 증가하는지를 확인하였는데, 이때 G20 서열은 자기 조립 특성(예: 분자간 G-quadruplex)으로 인해 이 분석에서는 사용하지 않았다. C20과 T20의 경우, PS 수를 8까지 증가시켰음에도 불구하고 효소 활성이 증가하지 않았으며, 반면, A20의 경우 PS 수가 증가할수록 효소 활성이 증가하는 것으로 나타났다(도 3C, 도 4C~4E). 이러한 결과를 통해 PS가 핵염기 중에서 특히 아데닌에 보다 효과적인 영향을 미친다는 것을 알 수 있으며, PS의 양도 중요한 역할을 하는 것을 알 수 있었다.Additionally, 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). In the case of C 20 and T 20 , 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). These results show that PS has a more effective effect on nucleobases, especially adenine, and that the amount of PS also plays an important role.
또한, 본 발명자들은 4개의 DNA 뉴클레오티드 A, C, G 및 T 각각에 대해 뉴클레오티드의 길이가 효소 활성에 영향을 미치는지 조사하기 위해, 단량체 뉴클레오티드와 단일가닥 올리고뉴클레오티드를 사용하였다. 단량체 뉴클레오티드로는 dNTP와 α-thio-dNTP를 사용하였다. dNTP의 일반적인 형태는 α-인산염에 산소 원자를 포함하는 원래 형태인 반면, α-thio-dNTP는 α-인산염에 비가교 산소 원자 대신 황 원자를 도입시킨 것을 말한다. 단일 올리고뉴클레오티드는 A/C/T의 경우 10 mer, 20 mer, 30 mer가 되도록 각 염기가 연속적으로 반복되어 형성된 반복 서열을 사용하였고, G의 경우 10 mer, 20 mer의 반복 서열을 사용하였다. 또한 각 DNA 서열의 중간에 PS를 도입하여 PS를 포함하는 단일 올리고뉴클레오티드를 준비하여 사용하였다.Additionally, the present inventors used monomeric nucleotides and single-stranded oligonucleotides to investigate whether nucleotide length affects enzyme activity for each of the four DNA nucleotides A, C, G, and T. dNTP and α-thio-dNTP were used as monomeric nucleotides. The general form of dNTP is the original form containing an oxygen atom in α-phosphate, while α-thio-dNTP refers to a sulfur atom introduced into α-phosphate instead of a non-crosslinked oxygen atom. For the single oligonucleotide, 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. In addition, PS was introduced into the middle of each DNA sequence, and a single oligonucleotide containing PS was prepared and used.
그 결과, 단량체인 dNTP와 α-thio-dNTP의 경우, 뉴클레오티드의 종류에 관계없이 효소 활성이 관찰되지 않았다. 또한, A(아데닌)의 길이가 증가함에 따라 효소 활성은 증가하는 것으로 나타났는데, 이는 DNA 길이가 DNA와 hemin의 결합에 긍정적인 영향을 미치는 것으로 보여진다. 이러한 결과를 통해 DNA와 hemin의 결합 정도는 길이에 따라 달라지며, DNA와 hemin이 결합할 수 있어야만 PS와 핵염기가 상호작용할 수 있다는 것을 알 수 있었다. 즉, 황 원자가 도입된 단량체의 경우 황 원자가 도입이 되었더라도 단량체는 DNA와 hemin이 결합할 수 없게 되므로 효소 활성을 나타낼 수 있다. 또한, ssDNA는 π-π 스태킹 상호작용을 통해 hemin의 포르피린 고리와 결합할 수 있으며, 길이가 길어질수록 DNA와 hemin이 보다 안정적으로 결합할 수 있어 높은 효소 활성을 유도할 수 있음을 알 수 있었다(도 6 및 도 4F~4I).As a result, in the case of monomers dNTP and α-thio-dNTP, enzyme activity was not observed regardless of the type of nucleotide. In addition, enzyme activity was found to increase as the length of A (adenine) increased, which suggests that DNA length has a positive effect on the binding of DNA and hemin. These results showed that the degree of binding between DNA and hemin varies depending on the length, and that PS and nucleobases can only interact if DNA and hemin can bind. In other words, in the case of a monomer into which a sulfur atom is introduced, even if a sulfur atom is introduced, the monomer cannot bind DNA and hemin, so it can exhibit enzymatic activity. In addition, it was found that ssDNA can bind to the porphyrin ring of hemin through π-π stacking interaction, and as the length increases, DNA and hemin can bind more stably, leading to higher enzyme activity ( Figure 6 and Figures 4F-4I).
<실시예 3><Example 3>
PS의 인접 위치에서 근접 뉴클레오티드 조합이 효소 활성에 미치는 영향 분석Analysis of the effect of close nucleotide combinations at adjacent positions of PS on enzyme activity
상기 실시예들의 실험을 통해 DNA와 hemin이 π-π 스태킹 상호작용을 형성하고, PS의 영향으로 효소 활성이 촉진된다는 것을 확인할 수 있었다. 또한, DNA의 길이와 DNA를 구성하는 핵염기의 타입에 따라 효소 활성이 달라지는 것을 확인할 수 있었다. 이에 본 발명자들은 다양한 핵염기 조합에 대한 효소 활성을 평가하기 위해 T(티민)를 뉴클레오티드 백본으로 선택하였다. 전자 밀도가 낮은 연속적인 T 서열을 기반으로 한 분석은 PS에 인접한 핵염기의 다양한 조합에 대한 효소 활성을 보다 명확하게 확인할 수 있기 때문이다. T는 주로 G-quadruplex 구조에서 스페이서 역할을 하며 실제 효율 향상에는 크게 기여하지 않는다. 그러나 T 비율이 높은 서열에서는 전체 효소 활성이 약할 수 있다. 따라서 T 기반의 뉴클레오티드 서열에서 4번째~5번째의 핵염기와 14번째~15번째의 핵염기 조합을 반복하여 포함하도록 하였고, 4번째 및 5번째의 핵염기 사이와 14번째 및 15번째의 핵염기 사이에 하나의 PS를 각각 포함하여 길이가 20nt인 16개의 서로 다른 T 기반 서열을 준비하여 사용하였다(하기 표 2 참조).Through the experiments of the above examples, it was confirmed that DNA and hemin form a π-π stacking interaction and that enzyme activity is promoted under the influence of PS. In addition, it was confirmed that enzyme activity varies depending on the length of DNA and the type of nucleobases that make up DNA. Accordingly, the present inventors selected T (thymine) as the nucleotide backbone to evaluate the enzyme activity for various nucleobase combinations. This is because analysis based on continuous T sequences with low electron density can more clearly confirm enzyme activity for various combinations of nucleobases adjacent to PS. T mainly functions as a spacer in the G-quadruplex structure and does not significantly contribute to improving actual efficiency. However, in sequences with high T ratios, overall enzyme activity may be weak. Therefore, 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. Sixteen different T-based sequences each with a length of 20 nt, including one PS in between, were prepared and used (see Table 2 below).
그 결과, PS에 인접한 염기 조합의 유형과 PS 양쪽에 있는 염기의 순서에 따라 효소 활성이 달라지는 것으로 나타났으며, 효소 활성 효율에 따른 서열 순위는 상기 표 2 및 도 7A에 나타내었다. 구체적으로 16개 서열 중에서 AA가 가장 높은 효율을 나타내었고, AA를 구성하는 A 염기는 G-quadruplex의 효소 활성을 향상시키는 핵심 염기임을 알 수 있었다. 이를 통해 본 발명자들은 핵염기 중에서 A가 PS가 삽입되어 효소 활성을 나타내기 위한 위치에서 우선적인 염기로 사용할 수 있음을 알 수 있었다. 또한, 순위 목록에서는 AN 또는 NA가 높은 순위를 차지하고 있어 A를 포함하는 헤테로 조합도 AA 다음으로 사용할 수 있는 우선 순위의 염기임을 알 수 있었다. 이와는 대조적으로, GG는 A와 같은 퓨린 염기임에도 불구하고 상대적으로 낮은 순위를 보였고, AG 및 GA와 같은 조합을 제외하면 GX(X = T, C, G) 또는 X*G는 순위가 더 낮은 것으로 나타났다. 이는 G가 A보다 환원 잠재력이 낮기 때문인 것으로 보인다. 퓨린 염기는 효소 활성에 참여할 수 있는 전자의 수가 더 많고, 퓨린과 피리미딘 염기의 조합도 상대적으로 좋은 효율을 나타낼 수 있음을 확인하였다. 피리미딘만으로 구성된 서열은 순위 목록의 맨 아래에서 확인되어 효소 활성이 낮은 것으로 나타났는데, T 또는 C와 같은 피리미딘 염기는 일반적으로 퓨린에 비해 전자 밀도가 낮기 때문에 효소 활성이 더 낮은 것을 알 수 있었다(도 7A 및 도 8A~8B).As a result, it was found that enzyme activity varies depending on the type of base combination adjacent to PS and the order of bases on both sides of PS, and the sequence ranking according to enzyme activity efficiency is shown in Table 2 and Figure 7A. Specifically, among the 16 sequences, AA showed the highest efficiency, and the A base constituting AA was found to be a key base that improves the enzymatic activity of G-quadruplex. Through this, the present inventors were able to find that among the nucleobases, A can be used as a preferential base at the position where PS is inserted to exhibit enzyme activity. In addition, in the ranking list, AN or NA were ranked high, showing that hetero combinations containing A are also the bases of priority that can be used after AA. In contrast, GG was ranked relatively low despite being the same purine base as A, and excluding combinations such as AG and GA, GX (X = T, C, G) or appear. This appears to be because G has a lower reduction potential than A. It was confirmed that purine bases have a larger number of electrons that can participate in enzyme activity, and that the combination of purine and pyrimidine bases can also show relatively good efficiency. Sequences consisting of only pyrimidines were found at the bottom of the ranking list and were shown to have low enzymatic activity. Pyrimidine bases such as T or C generally have lower electron density than purines, so they were found to have lower enzymatic activity. (Figure 7A and Figures 8A-8B).
나아가 본 발명자들은 도 1에 사용된 서열 대신 2차 구조를 형성하지 않는 50% GC 함량을 갖는 무작위 DNA 서열을 선택하여 사용하였다. 앞서 실시예에서 단 하나의 PS가 도입된 경우에도 무작위 서열은 상당한 효소 활성을 보였다(도 3A). 본 실험에서는 충분한 효율성을 구별하기 위해 2개의 PS를 도입한 것을 사용하였는데, 상기 표 2의 염기 조합 순위에 따라 가장 높은 효율을 나타내는 조합을 선택하여 PS를 도입하였고, 동일한 서열 내에서 낮은 효율을 나타내는 조합의 위치를 선택하여 PS를 도입하여, 하기 표 3의 저효율(L)과 고효율(H)로 지정된 두 개의 서열을 실험에 사용하였다.Furthermore, 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. In the previous example, even when only one PS was introduced, the random sequence showed significant enzymatic activity (Figure 3A). In this experiment, 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.
분석 결과, 도 7A 및 7B의 결과와 유사하게 PS 양쪽에 있는 염기가 AA 조합인 경우, 효소 활성이 우수한 것으로 나타났다(도 7C~7D 및 도 8C). 이를 통해 효과적인 비색 검출을 위해서는, PS의 위치를 PS 양쪽에 있는 염기가 적어도 하나 이상의 아데닌(A)이 있는 부위가 되도록 도입시키는 것이 유리하다는 것을 알 수 있었다.As a result of the analysis, similar to the results in Figures 7A and 7B, when the bases on both sides of PS were a combination of AA, the enzyme activity was found to be excellent (Figures 7C-7D and Figure 8C). Through this, it was found that for effective colorimetric detection, it is advantageous to introduce the position of PS so that the base on both sides of PS is a site where at least one adenine (A) is located.
<실시예 4><Example 4>
효소 신호전달 시스템(enzymogenic signaling system)을 이용한 서열 특이적 비색 분석Sequence-specific colorimetric analysis using an enzyme signaling system
ssDNA에 PS를 도입시키면 효소 활성이 강력하게 향상됨을 상기 실시예의 결과를 통해 확인할 수 있었다. 이러한 과정은 다음과 같은 단계에 의해 진행되는데, 첫째, hemin이 적절한 길이의 ssDNA에 결합하고, 둘째, PS는 hemin 결합 염기 근처의 전자 밀도를 증가시키며, 셋째, 염기의 전자 밀도가 높아져 hemin과 상호작용하여 효소 활성이 가속화된다. 여기서 hemin의 효소 활성을 촉진하는데 직접적인 역할을 하는 것이 바로 염기이다.It was confirmed through the results of the above example that introducing PS into ssDNA strongly improved enzyme activity. This process proceeds by the following steps: first, hemin binds to ssDNA of an appropriate length, second, PS increases the electron density near the hemin-binding base, and third, the electron density of the base increases and interacts with hemin. It acts to accelerate enzyme activity. Here, it is the base that plays a direct role in promoting the enzymatic activity of hemin.
이에 본 발명자들은 ssDNA와 dsDNA의 핵산 형태의 차이에 대한 효소 활성을 비교하기 위해, ssDNA에 상보적인 서열을 결합시켜 dsDNA를 형성하여 사용하였다.Accordingly, in order to compare the enzyme activity of ssDNA and dsDNA due to differences in nucleic acid form, the present inventors formed dsDNA by combining complementary sequences to ssDNA.
그 결과, PS-ssDNA 형태일 때는 효소 활성이 강하지만, 상보적인 ssDNA가 결합하여 dsDNA를 형성하면 효소 활성이 감소되는 것으로 나타났고, 또한 특정 표적 DNA 또는 RNA가 dsDNA 내에서 상호작용하여 PS-ssDNA 부분을 방출하면 효소 활성이 다시 향상되는 것으로 나타났다(도 9). 이러한 메커니즘을 활용하면 표적 DNA나 RNA에 의해 활성화될 수 있는 효소 생성 시스템을 구축할 수 있다.As a result, the enzyme activity was strong when in the form of PS-ssDNA, but the enzyme activity was reduced when complementary ssDNA combined to form dsDNA. In addition, specific target DNA or RNA interacted within dsDNA to form PS-ssDNA. Release of the portion showed that enzyme activity improved again (Figure 9). Using this mechanism, it is possible to build an enzyme production system that can be activated by target DNA or RNA.
또한 본 발명자들은 이러한 내용의 검증을 위해, 상기 표 2에서 사용한 서열에 대한 상보적인 서열을 준비하고 적절한 DNA 혼성화 조건을 거친 후 효소 활성을 평가하였다.Additionally, to verify this, 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.
그 결과, 흡광도 분석에서 PS를 함유한 dsDNA에서는 효소 활성이 감소하는 것으로 나타났다. 한편, PS 수가 증가함에 따라 dsDNA의 효소 활성 억제 효율이 감소하는 것으로 나타났는데(도 9A, 9B), 이러한 원인은 dsDNA 내의 분자간 DNA 혼성화에서 DNA end breathing과 PS의 영향으로 인해 전체 DNA 혼성화의 불안정성이 증가하여 잠재적으로 두 ssDNA 가닥 모두에서 염기가 노출되기 때문일 수 있다. 이렇게 증가된 불안정성은 hemin, H2O2 및 ABTS가 노출된 염기와 상호 작용할 수 있기 때문에 원하지 않는 효소 활성의 가능성을 높였을 수 있다.As a result, absorbance analysis showed that enzyme activity decreased in dsDNA containing PS. Meanwhile, as the number of PS increased, the efficiency of dsDNA enzyme activity inhibition was found to decrease (Figures 9A, 9B). This was due to the instability of overall DNA hybridization due to the influence of DNA end breathing and PS on intermolecular DNA hybridization within dsDNA. This may be due to the increase, potentially exposing bases on both ssDNA strands. This increased instability may have increased the likelihood of undesired enzyme activity because hemin, H2O2, and ABTS may interact with exposed bases.
나아가 분자간 DNA 혼성화로 인한 신호 누출 가능성을 완화하기 위해 효소 발생 신호 전달 시스템용 프로브는 H-OSD(분자내 DNA 혼성화)라고 불리는 헤어핀 형식으로 설계하였다. 이 구성에서는 다중 PS 분자가 도입되는 경우에도 이중 영역의 안정성과 충분한 효소 활성을 보장하기 위해 적절한 길이의 헤어핀 줄기를 선택하여 사용하였다. H-OSD 프로브는 5'말단에 10nt 발판(toehold)이 있는 21bp 헤어핀 줄기로 구성되며 이는 루프 서열까지 표적 서열에 상보적이다. 발판(toehold) 영역은 표적 서열과 혼성화되어 PS를 함유하는 가닥이 열리고 효소 활성이 활성화된다. 반대로 표적 서열이 없으면 효소 활성은 비활성 상태로 유지된다. 이 상태에서는 헤어핀 구조가 유지되며 루프서열은 노출 시에도 배경 증가를 방지하기 위해 T로만 구성된다(도 9C, 9D). 최적의 흡광도 구배를 달성하고 표적 존재 또는 부재에 대한 DNA 불안정성으로 인한 신호 누출 위험을 방지하기 위해 추가 조건을 최적화하였다(도 10). 또한, 비표적 서열이 존재하는 경우에도 헤어핀은 닫힌 채로 유지되었으며, 도입된 비표적의 신호만 검출되었다(도 11 및 도 12). 또한 H-OSD 프로브의 PS 분자 수를 최대 8개까지 증가시켜 백그라운드 제어 기능을 평가하였는데, 분자 내 DNA 혼성화는 더 안정적이며, PS 첨가로 인한 증가된 dsDNA 불안정성에 영향을 덜 받는 것으로 나타났다(도 13).Furthermore, to mitigate the possibility of signal leakage due to intermolecular DNA hybridization, the probe for the enzyme-generated signal transduction system was designed in a hairpin format called H-OSD (intramolecular DNA hybridization). In this configuration, 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. In this state, the hairpin structure is maintained and the loop sequence consists only of Ts to prevent background increase even upon exposure (Figures 9C, 9D). Additional conditions were optimized to achieve optimal absorbance gradients and avoid the risk of signal leakage due to DNA instability in the presence or absence of target (Figure 10). Additionally, even in the presence of off-target sequences, the hairpin remained closed, and only the signal of the introduced off-target was detected (Figures 11 and 12). Additionally, the background control function was evaluated by increasing the number of PS molecules in the H-OSD probe up to eight, and intramolecular DNA hybridization was found to be more stable and less affected by the increased dsDNA instability caused by PS addition (Figure 13 ).
<실시예 ５><Example 5>
효소 신호전달 시스템(enzymogenic signaling system)을 이용한 서열 특이적 비색 분석Sequence-specific colorimetric analysis using an enzyme signaling system
나아가 본 발명자들은 실시예 4와 또 다른 방식으로 조성한 프로브를 준비하였다. 실시예 4에서는 분자내 혼성화를 통한 헤어핀 형태의 H-OSD를 활용하여 서열 특이적 비색 검출에 활용하였는데, 이와는 또 달리, 실시예 5에서는 분자간 DNA 혼성화를 통해 구성한 프로브에 PS 변형을 이용한 황 원자를 도입하는 경우 PS를 포함한 단일 가닥의 방출로 인한 효소 활성의 활성화 향상효과를 분석하였다.Furthermore, the present inventors prepared a probe formulated in a method different from Example 4. In Example 4, H-OSD in the form of a hairpin through intramolecular hybridization was used for sequence-specific colorimetric detection. In contrast, in Example 5, a sulfur atom using PS modification was added to the probe constructed through intermolecular DNA hybridization. When introduced, the effect of improving activation of enzyme activity due to the release of a single strand containing PS was analyzed.
분자간 DNA 혼성화를 통한 신호 누출 가능성을 완화하는 목적으로 앞서 언급된 DNA end breathing과 PS의 영향으로 인한 전체 분자간 혼성화의 불안정성을 낮추기 위하여 DNA 이중체를 형성하는 부분의 말단에 GC clamping을 추가하여 DNA 구조 안정성을 높이도록 하였다. 이로써 효소 발생 신호 전달 시스템용 프로브인 OSD(분자간 DNA 혼성화)라 불리는 hemi-duplex 형식의 프로브를 설계하였다. GC clamping region외에 PS의 개수의 증가에도 안정성을 보장하기 위해 적절한 길이의 stem 구조를 포함시켰다. OSD는 5’말단 또는 3’말단 방향으로 10nt의 발판(toehold) 영역을 포함시킬 수 있다． 20bp의 substrate 가닥과 효소활성에 작용하는 20nt의 output 가닥을 적절한 DNA 혼성화 조건을 거친 후 활용하였다. 프로브는 실시예 4와 동일한 방식으로 작동하며 표적 서열이 있는 경우 toehold-mediated strand displacement 반응을 통해 PS를 포함한 output 가닥이 방출되며 효소 활성이 발생한다(도 14A, 14B, 도 16). 반면, 표적 서열이 없는 경우 효소 활성은 비활성 상태가 유지된다. 프로브의 DNA 불안정성의 증가로 인한 신호 누출의 가능성을 낮추기 위하여 반응 조건을 추가로 최적화하였다(도 15). 또한, 비표적 서열이 존재하는 경우 프로브는 PS를 포함한 output 가닥을 방출시키는 반응을 하지 않으며 구조가 안정적으로 유지되며 백그라운드 신호만 검출되었다(도 17). 이러한 결과를 통해, 본 발명에 따른 분자 간 혼성화로 구성되는 OSD의 경우 GC clamping 등 DNA 구조 안정성을 높이는 요소를 고려하여 프로브로서 활용 가능성이 우수하다는 것을 확인하였다.In order to alleviate the possibility of signal leakage 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. As a result, a hemi-duplex type probe called OSD (intermolecular DNA hybridization), which is a probe for an enzyme-generated signal transduction system, was designed. In addition to the GC clamping region, 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.
이상의 결과를 통해 본 발명자들은 포스포로티오에이트 결합(PS) 변형을 이용하여 황 원자를 핵산에 도입할 경우, 효소 활성을 크게 향상시킬 수 있음을 알 수 있었고, 이는 PS에 의해 변형된 핵염기 내의 전자 밀도가 hemin과의 상호작용을 통해 효소 활성의 가속화를 촉진한다는 것을 알 수 있었다.Through the above results, the present inventors were able to see that introducing a sulfur atom into a nucleic acid using phosphorothioate bond (PS) modification can greatly improve enzyme activity, which is achieved by increasing the enzyme activity within the nucleobase modified by PS. It was found that electron density promotes acceleration of enzyme activity through interaction with hemin.
그러므로 본 발명에서 개발한 비색 검출용 황 원자가 핵산 내로 도입된 새로운 프로브는 서열 의존적 제한 없이 효소 활성을 증가시킬 수 있으므로, 이를 활용한 효소 발생 신호 전달 시스템은 H-OSD를 사용하여 서열별 비색 검출에 유용하게 활용할 수 있다.Therefore, 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.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
Claims (12)
- 비색 검출용 황 함유 프로브로서,A sulfur-containing probe for colorimetric detection, comprising:상기 프로브는 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태를 가지며,The probe has the form of a DNA oligonucleotide in which nucleobases are sequentially linked,상기 핵염기의 연속적인 연결은 아데닌(A), 구아닌(G), 시토신(C) 및 티민(T) 중에서 선택되는 어느 하나 이상이 무작위적으로 연결되어 있고,The continuous linkage of the nucleobases is randomly linked to one or more selected from adenine (A), guanine (G), cytosine (C), and thymine (T),상기 DNA 올리고뉴클레오티드 중 뉴클레오티드 포스페이트 백본이 하나 이상의 포스포로티오에이트로 변형되어(phosphorothioate modification) 하나 이상의 황(sulfur) 원자를 포함하고 있는 것을 특징으로 하는, 비색 검출용 황 함유 프로브.A sulfur-containing probe for colorimetric detection, wherein the nucleotide phosphate backbone of the DNA oligonucleotide is modified with one or more phosphorothioates to contain one or more sulfur atoms.
- 제1항에 있어서,According to paragraph 1,상기 비색 검출용 황 함유 프로브는 10개 내지 30개의 핵염기가 연속적으로 연결된 DNA 올리고뉴클레오티드의 형태를 갖는 것을 특징으로 하는, 비색 검출용 황 함유 프로브.The sulfur-containing probe for colorimetric detection is characterized in that it has the form of a DNA oligonucleotide in which 10 to 30 nucleobases are sequentially linked.
- 제1항에 있어서,According to paragraph 1,상기 비색 검출용 황 함유 프로브는 비색 반응 매개의 효소 활성을 증진시키는 것을 특징으로 하는, 비색 검출용 황 함유 프로브.The sulfur-containing probe for colorimetric detection is a sulfur-containing probe for colorimetric detection, characterized in that it enhances the enzyme activity of the colorimetric reaction mediator.
- 제1항에 있어서,According to paragraph 1,DNA 올리고뉴클레오티드의 포스페이트 백본을 포스포로티오에이트로 변형시킬 경우, 상기 변형의 양쪽 옆에 존재하는 핵염기는 적어도 하나가 아데닌(A)인 것을 특징으로 하는, 비색 검출용 황 함유 프로브.A sulfur-containing probe for colorimetric detection, wherein when the phosphate backbone of a DNA oligonucleotide is modified with phosphorothioate, at least one nucleobase present on both sides of the modification is adenine (A).
- 제1항에 있어서,According to paragraph 1,상기 하나 이상의 황(sulfur) 원자를 포함하는 DNA 올리고뉴클레오티드는 단일 가닥 DNA 올리고뉴클레오티드인 것을 특징으로 하는, 비색 검출용 황 함유 프로브.A sulfur-containing probe for colorimetric detection, wherein the DNA oligonucleotide containing at least one sulfur atom is a single-stranded DNA oligonucleotide.
- 제1항 내지 제5항 중 어느 한 항의 비색 검출용 황 함유 프로브를 포함하는, 비색 검출용 조성물.A composition for colorimetric detection, comprising the sulfur-containing probe for colorimetric detection of any one of claims 1 to 5.
- 제6항에 있어서,According to clause 6,상기 조성물은 비색계 시약을 더 포함하는 것을 특징으로 하는, 비색 검출용 조성물.A composition for colorimetric detection, characterized in that the composition further comprises a colorimetric reagent.
- 제7항에 있어서,In clause 7,상기 비색계 시약은 ABTS(2,2'-아지노-bis(3-에틸벤조싸이아졸린-6-설폰산), OPD(o-페닐렌다이아민 다이하이드로클로라이드), DAB(다이아미노벤지딘), AEC(3-아미노-9-에틸카보졸), TMB(3,3',5,5'-테트라메틸벤지딘), AmplexRed, 및 Homovanilic acid로 이루어진 군으로부터 선택되는 1종 이상; 및 1종 이상의 과산화물인 것을 특징으로 하는, 비색 검출용 조성물.The colorimetric reagents include ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), OPD (o-phenylenediamine dihydrochloride), DAB (diaminobenzidine), and AEC. (3-amino-9-ethylcarbozole), TMB (3,3',5,5'-tetramethylbenzidine), AmplexRed, and one or more peroxides selected from the group consisting of Homovanilic acid; A composition for colorimetric detection, characterized in that.
- 제6항에 있어서,According to clause 6,상기 조성물에는 헤민(hemin)을 더 포함하는 것을 특징으로 하는, 비색 검출용 조성물.A composition for colorimetric detection, characterized in that the composition further contains hemin.
- 제6항의 비색 검출용 조성물을 포함하는 비색 검출용 키트.A colorimetric detection kit comprising the colorimetric detection composition of claim 6.
- 제1항의 비색 검출용 황 함유 프로브를 이용한 비색 검출 방법.A colorimetric detection method using the sulfur-containing probe for colorimetric detection of claim 1.
- (1) 검출 대상의 타겟 유전자를 선정하는 단계;(1) selecting a target gene for detection;(2) 상기 타겟 유전자의 핵산서열에 대한 상보적인 올리고뉴클레오티드 서열을 디자인하는 단계; 및(2) designing an oligonucleotide sequence complementary to the nucleic acid sequence of the target gene; and(3) 상기 상보적인 올리고뉴클레오티드에서 뉴클레오티드 포스페이트 백본을 하나 이상의 포스포로티오에이트로 변형시키는 단계를 포함하며,(3) modifying the nucleotide phosphate backbone in the complementary oligonucleotide with one or more phosphorothioates,상기 상보적인 올리고뉴클레오티드는 10 내지 30mer의 길이를 가지며, 포스포로티오에이트 변형은 양쪽 옆에 존재하는 핵염기가 적어도 하나는 아데닌(A)인 영역에서 이루어지는 것을 특징으로 하는,The complementary oligonucleotide has a length of 10 to 30 mer, and the phosphorothioate modification is characterized in that at least one nucleobase on both sides is adenine (A).비색 반응 매개의 효소 활성 증진 효과를 갖는 비색 검출용 황 함유 프로브의 제조방법.Method for producing a sulfur-containing probe for colorimetric detection that has the effect of enhancing enzyme activity through colorimetric reaction.
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