WO2014207515A1

WO2014207515A1 - Fluorescence method for detecting nuclease activity

Info

Publication number: WO2014207515A1
Application number: PCT/IB2013/059297
Authority: WO
Inventors: Erkan MOZIOGLU; Muslum AKGOZ; Tanil Kocagoz; Candan TAMERLER
Original assignee: Tubitak (Turkiye Bilimsel Ve Teknolojik Arastirma Kurumu)
Priority date: 2013-06-25
Filing date: 2013-10-11
Publication date: 2014-12-31

Abstract

The present invention provides a method of detecting a nuclease activity of DNA or RNA oligomers containing a fluorophor at one terminus (5' or 3'). When the fluorophore is attached to an oligomer covalently, its fluorescence intensity is quenched by the oligomer. After nuclease digestion, the fluorophore is released and the flourescence signal of the fluorophor increases. The differences of these fluorescence intensities make it easy to characterize the DNA or RNA nuclease activity. The invention is a fluorescence method for detecting nucleases and inhibitors thereof by using the oligomer attached to only a single fluorescent molecule (a fluorophore). It does not need any other substances such as second fluorescent molecule, non-fluorescent molecule (a quencher molecule), graphene oxide, or cationic polymers to produce signals after nuclease digestion.

Description

Fluorescence method for detecting nuclease activity

Since nucleases have widespread use in molecular biology, it is important to determine their functions as simple and fast as possible. In addition to conventional biochemical methods, the activity of nuclease enzymes can also be determined using fluorescence based methods. Molecular beacons are one of these structures, made up of a fluorophore and quencher molecule that are bound to each end of ssDNA linker (Li et al.2000). Molecular beacon structure is designed to form a hairpin structure to keep both of these molecules in close proximity. When excited, quencher molecule prevents the fluorophore molecule being detected. After digestion of the ssDNA linker with an enzyme, the fluorophore and quencher are separated from each other, thus the corresponding fluorescence emission signal can be detected (Fig. 1A).

I n one study, it was postulated that, a single fluorescent molecule, attached to a ssDNA linker was conjugated to polymer surface and FRET occurs between florescent molecule and the cationic polymer (Feng et al.2007). After enzyme digestion of the ssDNA, an increase in the signal was attributed to the release of the fluorescent molecule (Feng et al.2007).

Here, we suggest a new single fluorescent sensor composed of only one fluorescent molecule attached to DNA or RNA oligomer. I n this system, since the intensity of the fluorescent molecule is quenched by binding to this DNa or RNA oligomer, upon digestion of the oligomer, fluorescence signal increases dramatically (Fig. IB). This molecular mechanism can be used for the determination of activity of DNA or RNA nucleases (DNase or RNase).

Summary of the Invention

The present invention provides a method which uses a DNA oligomer or an RNA oligomer which is labeled with a fluorophore at the 5'-terminus or 3'-terminus. When The DNA or RNA oligomer is labeled with a fluorophor covalently, fluorescence is quenched. This is called "quenching state". In the presence of nuclease enzymes such as SI nuclease, the DNA or RNA oligomer is cut, and then the fluorophore is separated from the DNA or RNA oligomer. Fluorescence of the fluorophore is recovered and the fluorescence signal inreases. This is called "fluorescence state". Therefore, by detecting change of the fluorescence intensity, nucleases and inhibitors thereof can be detected. The method of the invention is simple, rapid, and sensitive for nuclease detection. Brief Description of Drawings

Fig. 1. Single molecule fluorescein sensor model. The structure of molecular beacons are made up of a fluorophore and a non-fluorescent quencher molecule in close proximity. After digestion, the fluorophore and quencher are separated from each other, thus the corresponding fluorescence emission signal can be detected (A). The structure of single fluorescent sensor composed of only one fluorescent molecule attached to ssDNA molecule. Because the intensity of the fluorescent molecule is quenched by binding to ssDNA, upon digestion of the ssDNA, fluorescence signal can be detected (B).

Fig. 2. Single molecule fluorescein detection of enzyme activity. Fluorescein-ssDNA concentration was kept fixed and SI nuclease concentration changed (A). SI nuclease concentration was kept fixed and Fluorescein-ssDNA concentration changed (B). The insert shows smaller time frame. Red arrow at 5^th minute indicates the addition of enzyme.

Fig. 3. Digestion of Fluorescein-ssDNA. Fluorescein-ssDNA was incubated with SI nuclease for 2.5 hr and it was run on the 12% nondenaturing polyacrylamide gel with the original molecule.

Detailed Description of the Invention

The invention is a fluorescence method which is used for measurement of DNA nucleases and RNA nucleases activities.

Nucleases are a kind of enzymes and they can hydrolyze the phosphodiester bonds in a nucleic acid backbone. They are classified according to their substrate: DNA nucleases and RNA nucleases. The nucleases are very important for biological processes. For example, DNA replication, repair, recombination. These nucleases are also used for biotechnological processes. Because of their important roles in biological processes, they are attractive for development of many therapeutics. For these, several methods have been used. These are gel electrophoresis, radioactive labeling method, HPLC techniques, and enzyme-linked immunosorbent assays (Fude Feng et al., Adv Mater 2007, 19, 3490-3495). These techniques have some difficulties such as use of radioactive substances, or expensive apparatus, being of labor intensive and time-consuming. Because of these limitations, the researchers are interested in new methods based on fluorescence. For measurement of nucleases activity based on fluorescence, fluorescence-quenching techniques have been used. These are based on "molecular beacon" techniques. According to this technique, DNA molecules labeled with fluorescent molecules like fluorescein at one end and quencher molecules like DABCYL at the other end have been used. When these molecules are in a solution, these fluorescent molecules and quencher molecules are in close proximity to each other, because of their hair-pin structure. This structure causes the fluorescence of the fluorescent molecule to be quenched by FRET (Fluorescence Resonance Energy Transfer). After cleaving these structures by nucleases, these fluorescent and quencher molecules are separated from each other. As a result, fluorescence intensity increases. Some researchers developed a method to detect nuclease activity by using this principle (Jefrey J. et al., Nucleic Acid Research, 28 (11), 2000). After then, another method was developed for this purpose. According to this, DNA oligomer labeled with only one fluorescent molecule together with some cationic polymers were used (Fude Feng et al., Adv Mater 2007, 19, 3490-3495). Their DNA complexes and cationic conjugated polymer were used as a probe for detection of nuclease enzyme activity. These cationic polymers were needed to produce fluorescence signal after digestion in this technique.

Fluorescent molecules like fluorescein have been commonly used both nucleic acids (DNA and RNA) and other molecules like lipids and carbohydrates. They can be used as covalently bonded , or free in solution. Using these approaches, proteinase and lipase activities can be determined (Drake C.R et al., Curr.Org.Synth., 8 (4), 2011; Guiltbaut G.G et al., Analytical Chemistry, 36 (2), 1964). Some enzymes like phosphatase and peptidase were fulfilled patents (EP0217371 (A2); MXPA05006448 (A); DE19843873 (Al)). Using molecular beacon techniques, in measurement of nucleases activities, new methods were developed and fulfilled patents (CN102321759 (A); MXPA05006448 (A)). Furthermore, using DNA oligomer or RNA oligomer labeled with fluorescent molecules and quencher molecules at 5' and 3' end, two different patents were fulfilled for measurement of both DNA nuclease (CN102321759 (A)) and RNA nuclease (MXPA05006448 (A)) activities.

The invention is a fluorescence method which does not need fluorescent molecules together with quencher molecules, but only single fluorescent molecule is enough to measure nuclease activity. So, it is different from other patents and techniques published. 90 The invention is a fluorescence method which does not use any cationic polymers, or any substances like graphene oxide to produce fluorescence signal after nuclease digestions.

The invention is a fluorescence method which is simple, inexpensive, rapid, and sensitive in measurement of DNA or RNA nuclease activities.

• CN 102321759 A - Fluorescence method for detecting SI nuclease and inhibitor 95 thereof

The invention is a fluorescence method for detecting SI nuclease and inhibitors thereof by using oligonucleotide and graphene oxide. The method uses graphene oxide as a solid carrier, allows a dye-labelled oligonucleotide probe to adhere to the surface of graphene oxide by the interaction between graphene oxide and nucleic acid bases, and performs 100 fluorescence quenching.

• US 4719097 A -Phosphates of resorufin derivatives and compositions thereof for the determination of the activity of phosphatases

The present invention provides phosphates of resorufin derivatives and also provides processes for the preparation of these compounds and diagnostic agents containing them 105 for the detection of phosphatases.

• DE 19843873 Al - Assay for cell-surface protease activity useful for diagnostics, uses a fluorogenic substrate comprising rhodamine 110 and fluorescence or laser scanning microscopy

The invention relates to a method for the determination of protease activity on cell 110 surfaces. The method for determining cell-surface protease activity, is new and comprises protease-catalyzed hydrolysis of a fluorogenic substrate comprising rhodamine 110 (R110) having an amino acid or peptide residue attached to one amino group and an anchor group attached to the other amino group, and detection using fluorescence microscopy, laser scanning microscopy or flow cytometry.

115 · WO 2004059012 Al - Assay for RNAse H activity

The present invention provides a method of detecting a nuclease-mediated cleavage of a target nucleic acid through hybridizing a target nucleic acid to a fluorescently labeled oligonucleotide probe complementary to the target nucleic acid and containing a flourophor at one terminus and a quenching group at the other terminus.

120

Example 1

Materials and Methods

The single fluorescent sensor could detect enzymes at lower concentrations. Fixed concentration of fluorescein molecule attached to the 15-mer ssDNA (5'-Fluorescein-

125 GCGGAGCGTGGCAGG) (Nutiu et al.2005) was mixed with different concentrations of SI nuclease (EN0321, Thermo Scientific, USA) and florescence signal was recorded over time (Fig. 2A). For the purpose, 40 nM Fluorescein-ssDNAs was prepared in reaction buffer (200 mM sodium acetate; 1,5 M NaCI, 10 mM ZnS04, pH 4,5) and 200 μΙ was transferred to the wells of 96 well plates. The plates were excited with 485nm filters and emission intensities

130 were recorded from 535nm filters, during the first 5 min. SI nucleases also were diluted in reaction buffer as 100 U, 50 U, 10 U, 5 U, 1 U, 0,5 U ve 0 U. Then, 5 μΙ of these diluted enzymes were transferred to the wells, and their emission intensities were measured. All experiments were conducted at 37 °C with Beckman Plate Reader DTX880. As seen in Fig. 2A, the response of Fluorescein-ssDNA sensor is concentration dependent and it could

135 respond to concentrations of as low as 0.0488 U/ul of SI nuclease enzyme in one minute.

After 20 minutes of incubation, 10 fold less enzyme concentration could be also detected with this sensor.

The relationship between substrate concentration and initial rate was also investigated. Increasing concentrations of the Fluorescein-ssDNA was incubated with fixed concentration

140 of SI nuclease enzyme and fluorescence was recorded over time (Fig. 2B). For the purpose, Fluorescein-ssDNA were diluted in reaction buffer as 160 nM, 80 nM, 40 nM, 20 nM, 10 nM, 5 nM, and 0 nM. 200 μΙ of were transferred to wells in 96-well plate as. At 485nm/535nm excitation and emission wave length, their emission intensities were measured during 5 min. Then, 5 μΙ of 1U SI nuclease was added; 5 μΙ of reaction buffer was added as a negative

145 control. Emission intensities were measured during 2 h. All experiments were conducted at 37 °C. Since the initial fluorescent signal of different concentrations of Fluorescein-ssDNA molecules are different (Fig. 2B legends) time zero fluorescent signal was subtracted from all the data points for each concentration to compare the increase. As seen in Fig. 2B, increasing concentrations of the substrate leads to an increase at the initial rate of SI 150 nuclease enzyme.

The degradation of Fluorescein-ssDNA was confirmed by running Fluorescein-ssDNA and enzyme mixture on 12,5% non-denaturing PAGE (Fig. 3). This data supports that the increase of fluorescence intensity obtained this study is the result of free fluorescein molecules.

In conclusion, we propose a new, low cost, rapid and sensitive fluorescence method to 155 measure activities of nucleases.

Claims

WHAT IS CLAIMED IS?

1. A method for detecting a nuclease-mediated cleavage of a target nucleic acid, which method comprises:

(a) the target nucleic acid is a fluorescently labeled oligonucleotide containing a fluorophor at one terminus, wherein (i) the fluorescently labeled oligonucleotide is DNA oligomer or RNA oligomer and (ii) the fluorophore is a fluorescent molecule (iii) the fluorescently labeled oligonucleotide has a fluorescence signal because of its fluorescent molecule, but the fluorescence intensity of the fluorescent molecule is quenched by the DNA oligomer or the RNA oligomer which is attached to the fluorescent molecule;

(b) contacting the fluorescently labeled oligonucleotide with an agent having nuclease activity in an amount sufficient to selectively cleave the target nucleic acid and thereby release the fluorescent molecule; and

(c) detecting the release of the fluorescent molecule by measuring a increase in the fluorescent signal of the fluorophor as compared to the signal of the fluorescently labeled oligonucleotide before cleavage.

2. The method of claim 1, wherein the agent is an enzyme having an nuclease activity.

3. The method of claim 1, wherein the nuclease reaction is performed in the presence of a compound, wherein a difference in the rate of the increase in the fluorescent signal of the fluorophor during the nuclease reaction, as compared to the increase observed when the same reaction is conducted in the absence of the compound, is indicative of the ability of the compound to either inhibit or enhance the nuclease activity of the agent.

4. The method of claim 1, which further comprises monitoring the fluorescence signal of the fluorophor during the nuclease reaction.

5. A method for measuring a nuclease activity of an agent, which method comprises:

(a) fluorescently labeled oligonucleotide containing a flourophor at one terminus, wherein (i) the fluorescently labeled oligonucleotide is DNA oligomer or RNA oligomer and (ii) the fluorophore is a fluorescent molecule (iii) the fluorescently labeled oligonucleotide has a fluorescence signal because of its fluorescent molecule, but the fluorescence intensity of the fluorescent molecule is quenched by the DNA oligomer or the RNA oligomer which is attached to the fluorescent molecule;

(b) contacting the fluorescently labeled oligonucleotide with the agent in an amount sufficient to selectively cleave the target nucleic acid and thereby release the fluorescent molecule; and

(c) measuring a increase in the flourescence signal of the fluorophor as compared to the signal of the fluorescently labeled oligonucleotide before.

6. The method of claim 5, wherein the agent is an sample having an nuclease activity.

7. The method of claim 5, wherein the agent is obtained from the natural sources such as plants, animals, microorganisms, or other living organisms or by synthesizing chemically like drugs.

8. The method of claim 5, wherein the nuclease reaction is performed in the presence of a compound, wherein a difference in the rate of the increase in the fluorescence signal of the flourophor during the nuclease reaction, as compared to the increase observed when the same reaction is conducted in the absence of the compound, is indicative of the ability of the compound to either inhibit or enhance the nuclease activity of the agent.

9. The method of claim 5, which further comprises monitoring the fluorescence signal of the fluorophor during the nuclease reaction.

10. A method for screening a modulator of the nuclease activity of an agent, which method comprises:

(a) a fluorescently labeled oligonucleotide containing a flourophor at one terminus, wherein (i) the fluorescently labeled oligonucleotide is DNA oligomer or RNA oligomer and (ii) the fluorophore is a fluorescent molecule (iii) the fluorescently labeled oligonucleotide has a fluorescence signal because of its fluorescent molecule, but the fluorescence intensity of the fluorescent molecule is quenched by the DNA oligomer or the RNA oligomer which is attached to the fluorescent molecule. (b) contacting the fluorescently labeled oligonucleotide of a first sample with the agent in an amount sufficient to selectively cleave the target nucleic acid and thereby release the fluorescent molecule;

(c) contacting the fluorescently labeled oligonucleotide of a second sample with the agent in an amount sufficient to selectively cleave the target nucleic acid and thereby release the fluorescent molecule in the presence of a candidate compound, which is being tested for its ability to modulate the nuclease activity of the agent;

(d) detecting the release of the fluorescent molecule in each sample by measuring a increase in the fluorescence signal of the fluorophor as compared to the signal of the fluorescently labeled oligonucleotide before; and

(e) comparing the rate of the increase in the fluorescence signal of the fluorophor in the two samples, wherein a difference in the rate of the increase in the fluorescence signal of the fluorophor during the nuclease reaction in the two samples is indicative of the ability of the compound to either inhibit or enhance the nuclease activity of the agent.

11. The method of claim 10, wherein a greater extent or relative rate of increase of the 80 fluorescence signal of the fluorophor in the second sample compared to the first sample indicates that the candidate compound is an agent agonist.

12. The method of claim 10, wherein a lesser extent or relative rate of increase of the fluorescence signal of the fluorophor in the second sample compared to the first sample indicates that the candidate compound is an agent antagonist.

85 13. A kit for measuring a nuclease activity of an agent, comprising a fluorescently labeled oligonucleotide containing a fluorophor at one terminus, wherein (i) the fluorescently labeled oligonucleotide is DNA oligomer or RNA oligomer and (ii) the fluorophore is a fluorescent molecule (iii) the fluorescently labeled oligonucleotide has a fluorescence signal because of its fluorescent molecule, but the fluorescence intensity of the

90 fluorescent molecule is quenched by the DNA oligomer or the RNA oligomer which is attached to the fluorescent molecule.

14. The kit of claim 13, further comprising the agent.

15. The kit of claim 13, wherein the agent is the enzyme consisting of RNase or DNase activity.

16. An assay mixture for measuring a nuclease activity of an agent, comprising a fluorescently labeled oligonucleotide containing a fluorophor at one terminus, wherein (i) the fluorescently labeled oligonucleotide is DNA oligomer or RNA oligomer and (ii) the fluorophore is a fluorescent molecule (iii) the fluorescently labeled oligonucleotide has a fluorescence signal because of its fluorescent molecule, but the fluorescence intensity of the fluorescent molecule is quenched by the DNA oligomer or the RNA oligomer which is attached to the fluorescent molecule.

17. The assay mixture of claim 16, wherein the agent is the enzyme consisting of RNase or DNase activity.