ES2947987A1 - METHOD AND KIT FOR THE QUANTITATIVE ASSESSMENT OF TELOMERASE ACTIVITY (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Method and kit for the quantitative assessment of telomerase activity. The present invention relates to a method for the quantitative assessment of telomerase activity without the use of DNA amplification techniques, such as PCR. The method uses the primers defined by SEQ ID NO: 1 and 2 together with DNA polymerase, d-NTPs, a double-stranded DNA-specific intercalating agent and the appropriate conditions for the telomerase of the sample to synthesize the G strand of the telomere and the DNA polymerase synthesizes the C chain, its activity being measured by the specific intercalating agent of double-stranded DNA. Since DNA amplification is not carried out, the DNA polymerase used may be thermolabile. The invention also refers to a kit to carry out this method and the uses of both.

Description

DESCRIPTION

METHOD AND KIT FOR THE QUANTITATIVE VALUATION OF THE ACTIVITY OF

TELOMERASE

TECHNIQUE SECTOR

The invention falls within the sector of the detection and analysis of enzymatic activity, specifically the quantitative determination of telomerase activity without using the PCR technique.

BACKGROUND OF THE INVENTION

Telomerase is an enzyme that is of great importance in medicine since the loss of its activity is associated with cellular senescence, thereby affecting the replicative capacity, and the shortening of telomeres. Furthermore, it is used to detect different types of cancer, hence the interest in finding a quantitative assessment method that allows its enzymatic activity to be easily detected.

The function of telomerase consists of elongating the ends of the single-stranded chain of telomeres by adding the TTAGGG nucleotide sequence.

The structure of telomeric DNA (tDNA) consists of a double-stranded portion formed by the G and C strands and a single-stranded structure formed by the G strand, in addition to a set of proteins with which they form the specialized multiprotein complex called shelterin/telosome.

Telomerase activity is a marker of the proliferation capacity of cells that manifests itself in reproductive organs, embryonic tissues and tumor cells.

There are several methods to assess this enzyme, most of them based on the polymerase chain reaction (PCR) with different modifications.

The most common methods are TRAP (repeat amplification protocols). telomeric) described by Kim (Kim, NW. et al. (1994). Specific association of human telomerase activity with immortal cells and cancer, Science 266(5193): 2011-2015) and Piatyszek (Piatyszek, MA. et al. ( 1995). Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods in Cell Science 17: 1-15) based on the PCR technique. The TRAP method allows detecting the presence of telomerase activity in cell extracts using primers from telomeric regions. The protocol includes incubation of the extracts with a primer with sequences similar to those of telomeres, deoxyribonucleotide triphosphates (d-NTPs) of adenine, thymine, guanine and cytosine, T4 gene 32 protein, thermostable Taq DNA polymerase, and [a -32P] d-CTP and [a -32P] d-TTP. Subsequently, the reaction is subjected to PCR amplification by adding a second primer and applying cycles with alternating temperatures. The PCR result is subjected to agarose gel electrophoresis and telomeres are detected by gel staining.

Modifications of this method have been described such as that of Taga S. et al. ((1999) Prognostic Impact of Telomerase Activity in Non-Small Cell Lung Cancers. Annals of Surgery 230(5): 715-720)) that use the same forward primer but a different reverse primer and some of the nucleotides labeled with phosphorus 32 ( 32P). Subsequently, they subject the PCR product to electrophoresis in a 10% polyacrylamide gel and identify the telomeres by autoradiography, considering, as active telomerase, the one that presents a 6 bp DNA band in the electrophoresis.

Gelmini S. et al. ((1998). Rapid, quantitative nonisotopic assay for telomerase activity in human tumors. Clin. Chem. 44 (10) 2133-2138)) use the same technique as Taga S. et al., but without using radioactive d-NTPs or electrophoresis. These authors use a blank obtained after subjecting the extract to RNase to eliminate telomerase, thereby inactivating it, and an extract without denaturing the enzyme. They then subject the reaction to PCR. Next, they take a quantity of the reaction product, both blank and test, and dilute it with an appropriate buffer containing Pico Green, a dye that fluoresces when bound to DNA. Fluorescence is measured at 480 nm excitation and 520 nm emission, in the blank and in the problem. The enzyme activity is calculated by subtracting the arbitrary fluorescence units (AFU) of the problem and those of the blank and the amount of DNA is obtained using a DNA standard curve between 0-100 μg/L.

Gollahona LS. and Holt SE. ((2000). Alternative methods of extracting telomerase activity from human tumor samples. Cancer Lett. 159(2): 141-149), to confirm that PCR amplification occurs on the substrate added to assess telomerase, they make a series of previous reactions consisting of recovering said product using a magnetic method, this product being the one that is subjected to PCR and then to electrophoresis and exposure to phosphoimaging.

Almost all known methods require a PCR step, including the method performed through the Kit called TeloTAGGG™ Telomerase PCR ELISAPLUS, which is the most used today.

Patent application WO0063429A2 relates to primers and probes for amplifying and detecting the mRNA of the human catalytically active telomerase (hTC) subunit. By applying PCR amplification techniques, in situ hybridization and labeling of the specific hTC protein, the presence of telomerase is detected. Although it is indicated that the detection can be carried out without amplification by PCR, the document does not include examples that do not require amplification.

Document W02011106671A1 relates to a method for measuring telomerase activity, which comprises isolating the telomerase enzyme on a solid support (for example, a Petri dish) with an anti-telomerase antibody (Ac) and measuring the level of activity of the bound enzyme to support. The method comprises: a suitable primer as a substrate for telomerase, the telomerase enzyme complex active and linked to the solid support by Ac and d-NTPs labeled with fluorescence or radioactivity, with which to carry out an extension reaction and detect the product of the extension. Immobilization of the enzyme allows the catalytic unit to be exposed to the substrate and facilitates the reaction. The invention also includes the PCR method, which is the preferred way to carry it out, as can be seen in the examples, for this they need a second primer with a suitable sequence and the presence of DNA polymerase.

In the state of the art there are some cases in which PCR is dispensed with. The patent application JP2012205568A describes a method in which the telomerase reaction and the RNA degradation reaction using RNase H are combined. The method is based on incubating the extract containing the desired telomerase. measured with a substrate consisting of a primer with a suitable sequence, a telomeric DNA and d-NTPs so that telomerase expands the substrate. To measure the extension of the substrate, they add RNase and an RNA labeled with a fluorescent substance and with a sequence complementary to that of the DNA. The reaction product is measured by fluorescence.

US2005244907A1 describes a method to detect the activity of an enzyme that is based on including substrates in liposomes capable of producing a detectable light signal; Enzymes whose activity can be detected include telomerase.

US2008153085A1 describes a method for determining an analyte in a sample, including telomerase, based on the use of semiconductor nanoparticles through the formation of hybrid systems that contain agents that recognize, either by immobilization or by reaction, the single-chain chain of the DNA. In the case of telomerase, the nanoparticles contain a primer. When telomerase is present in the test extract, and after the addition of d-NTPs, the substrate begins to elongate and the spectrum changes its characteristics, which allows measuring the concentrations of the reaction product. The detection is based on FRET (fluorescence resonance energy transfer) by electromagnetic irradiation of the system to cause the transfer of resonance energy from the nanoparticles to the acceptor, thus detecting the signal.

US2010105048A1 describes a method and a composition to detect telomerase in a sample, in which fluorescent nanocrystals are used without the need to perform PCR. Nanocrystals have a high fluorescence intensity, are soluble in water, have physical and chemical stability and have spectra that change depending on whether one or another functional group is attached to their surface. In the case of telomerase, the group with fluorescent labeling is the d-NTPs, at least one, and the intensity of the spectrum depends on the number of functional groups that bind to the substrate.

The interest in detecting telomerase activity lies in its possible application in both basic research and clinical research. Telomeres could maintain their length during cell division by telomerase, which is made up of proteins and RNA. This enzyme would lengthen the G strand of the telomere and DNA polymerase would complete the formation of tDNA by lengthening the C strand, which would lead to maintaining the length allowing cells to divide eternally, avoiding senescence, but would also allow defective cells to or malignant ones lived eternally, causing deformations or cancer. This means that telomerase activity must be highly regulated. Therefore, having a sufficiently suitable, precise, fast and cheap method to measure the enzyme could serve to control it effectively.

EXPLANATION OF THE INVENTION

Method and kit for the quantitative assessment of telomerase activity.

To simplify the procedures for assessing telomerase activity used until now in the state of the art, a method has been developed that does not require amplification by polymerase chain reaction (PCR) or ligase chain reaction (LCR) or use of complex and/or economically expensive resources.

One aspect of the present invention relates to a method for the quantitative assessment of telomerase activity that includes the following steps:

a) obtain extracts of the cells you want to study;

b) denature the proteins of an aliquot part of the extract obtained in step a), which will be used as a blank;

c) mix the extract from step a) with a suitable buffer for the telomerase reaction to take place, the first TS external substrate: 5'-TTAGGG TTAGGG TTAGGG-3' (SEQ ID NO: 1) and the first CX primer: 5'-AATCCC AATCCC AATCCC-3' (SEQ ID NO: 2), DNA polymerase, MgCh, d-NTPs and a double-stranded DNA-specific intercalating agent;

d) mix, on the other hand, the aliquot part of the extract whose proteins have been denatured in step b) with the same ingredients that are added to the extract in step c);

e) measure the fluorescence produced by the intercalating agent incorporated into the double-stranded DNA of step c) and step d) at time zero (T=0) and 30 minutes (T=30) after the reaction starts;

f) calculate the difference between the fluorescence values at T=30 and T=0 of the extract analyze (step c) and the blank (step d) in which the DNA polymerase does not have to be thermostable since it is not necessary to apply high temperatures since it is not required for the quantitative assessment of telomerase activity by this method.

The cells from which the extract in step a) is obtained may belong to cell cultures, tissues or organs that are of interest to study. On the other hand, the denaturation of proteins can be carried out by any procedure, such as the application of heat or the incubation of the extract with RNase under conditions such (for example, 20 minutes at room temperature) that telomerase is denatured and all enzymes that have RNA to be active.

In this specification, blank means the part of the sample that does not contain the analyte of interest (i.e., telomerase) but does contain all the reagents used in the method for the quantitative assessment of telomerase activity that is described here and, in addition, is subject to the same conditions and the same procedure as the extracts that you want to study. Likewise, in this specification, thermostable DNA polymerases are understood to mean DNA polymerases that are not easily altered by the action of heat, that is, they are not easily altered either by being subjected to high temperatures or by being subjected to frequent changes. Of temperature. Therefore, DNA polymerases that do not need to be thermostable include those DNA polymerases that may cease to be functional at high temperatures or ideally function isothermally, that is, at a constant temperature.

The buffer of step c) and step d) preferably has a pH equal to 8.3 and the reaction is carried out at a temperature between 25 and 350C, preferably at 300C.

The double-stranded DNA-specific intercalating agent can be selected from various fluorophores, and among them, SYBR-Green is preferably selected.

On the other hand, the fluorescence produced by the intercalating agent incorporated into the DNA formed by the action of telomerase during incubation can be measured at different time intervals, for example, every 15 minutes for 90 minutes. With this, it is possible to verify the proper functioning of the telomerase enzyme. Measurement of telomerase activity can be performed directly with a spectrofluorimeter.

Another aspect of the invention refers to a kit for assessing the telomerase activity of a sample that includes the primers defined by SEQ ID NO:1 and SEQ ID NO: 2.

The kit may also include DNA polymerase, MgCL, d-NTPs and/or a double-stranded DNA-specific intercalating dye. The dye is preferably a fluorogenic dye and, more preferably, is SYBR-Green I. On the other hand, the DNA polymerase may not be thermostable, since it will not have to withstand elevated temperatures or cyclic temperature changes.

The invention also relates to the use of the method and/or kit described above to study the proliferation capacity of cells, to identify drugs and/or molecules capable of acting as activators or inhibitors of telomerase activity, to study regenerative therapies. against myocardial infarction, identify tumor proliferation associated with cancer, detect the malignancy status of different types of cancer, detect the degree of proliferation of stem cells or study the possibility of extending life.

The examples presented in this report support that the present telomerase assessment method is effective, simple, reproducible, and fast.

BRIEF DESCRIPTION OF THE DRAWINGS

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of graphs is attached as an integral part of said description in which, with an illustrative and non-limiting nature, the following has been represented. following:

Figure 1. Assessment of the presence of DNA in rat liver extracts.

Figure 2. Effect of time on telomerase activity in rat liver.

Figure 3. Effect of extract concentration on telomerase titer in rat liver.

PREFERRED EMBODIMENT OF THE INVENTION

The present invention is illustrated by the following examples, which are not intended to be limiting of its scope.

Reagents used in the following examples:

- Tissue extraction buffer (lysis buffer):

10 mM Tris-HCl buffer

1mM MgCl2

1mM EGTA

5 mM dithiothreitol

0.1 mM protease inhibitor (Aprotinin) (Therm oFisher Scientific, MA, USA)

0.5% Triton X-100

Set the buffer to pH = 7.5

- Telomerase measurement buffer (10X):

The 10-fold concentrated buffer was prepared with:

- 200mM Tris-HCl

- 15mM MgCh

- 630mM KCI

- 10mM EGTA

Adjust the pH to 8.3

- Quantimix Easv Reagent:

To facilitate the work, in the following examples the commercial reagent called Quantimix Easy (Biotools, Madrid, Spain) was used, which contains a set made up of DNA polymerase, the four d-NTPs (adenine, cytosine, guanine and thymine), MgChy SYBR™ Green I as a specific intercalating agent for double-stranded DNA at the optimal concentration (4 mM final). This reagent is easier to handle than the separate use of d-NTPs, DNA polymerase and SYBR-Green I dye, however, the following examples can also be carried out with a thermostable or non-thermostable DNA polymerase, since it is not They will reach temperatures above 300C.

- Primers (substrate and primer):

- PrimerTS external substrate: (5'-TTAGGG TTAGGG TTAGGG-3'; SEQ ID NO:1), at 0.1 μM concentration in H ₂ O Mili Q.

- First CX primer: (5'-AATCCC AATCCC AATCCC-3'; SEQ ID NO:2), at 0.1 μM concentration in H ₂ O Mili Q.

- Dye to identify the DNA in the extract

- DAPI (Sigma, Darmstadt, Germany)

Example 1. Preparation of extracts from tissues

The organs to be tested, from 1-month-old female Wistar rats (Animalario de la Faculty of Pharmacy, UCM, Madrid. Registration number: ES280790000086), and cows, collected at the Madrid slaughterhouse, were frozen in liquid nitrogen. immediately after obtaining them and they were kept in a freezer at -80ºC until use. The storage of all these organs was done in sterile containers.

At the time of use, the following steps were followed:

- Remove the organs from the freezer, weigh them and let them thaw on ice. - Add 10 times its weight of extraction buffer and 5 Ul of RNase inhibitor per 500 μL of extract, to protect telomerase.

- Disintegrate the organ into ice with ULTRA-TURRAX®, 3 times, shaking each time for 3 seconds.

- Keep for 30 min on ice, shaking occasionally on a magnetic stirrer. - Centrifuge the lysate at 16,000 x g for 20 min at 4°C.

- Collect the supernatant in sterile tubes avoiding contamination with the pellet. - Keep the supernatant on ice during use.

- Use the supernatant to measure telomerase.

- If the extracts are not used at the moment, freeze them quickly with liquid nitrogen, and store them in a freezer at -80ºC until use.

- Carry out both the obtaining of the extract and the storage of the samples under sterile conditions.

- Quantify the protein content present in the extract using the Lowry method (Lowry, OH et al. (1951) Protein measurement with the Folin Phenol Reagent. J. Biol.

Chem. 193(1): 265-275) before measuring telomerase activity.

Example 1.1. Denaturation of the extract for use as blank The extract was heated for 10 min at 90ºC and centrifuged for 10 min at 15,500 g to eliminate proteins precipitated by heat.

Example 2. Telomerase assessment

Two examples of measurements to assess telomerase activity are shown, one with the undenatured extract and the other with the denatured extract.

Example 2.1. Preparation of the reaction with the undenatured substrate.

In a final volume of 50 μl, the following were mixed, in this order:

x μL sterile H20 Mili-Q® until completing 50 μL total reaction;

5 μL of 10 mM Tris-HCl buffer pH = 8.3 (10X);

2 μL of 0.1 μM substrate primer (SEQ ID NO:1);

2 μL of 0.1 μM primer primer (SEQ ID NO:2);

2 μL of Quantimix Easy;

x μL of undenatured extract obtained as described in example 1, containing 30-50 μg of protein;

Immediately after preparing the mixture, the fluorescence was read at 485 nm excitation (exc) and 550 nm emission (emis), with a gain of 1000. For this, a spectrofluorimeter (FLUOstar Omega, BMG Labtech, Ortenberg, Germany) was used. It will be time zero (T=0) of the reaction.

The mixture was incubated at 30ºC and, again, the fluorescence was read at 485 nm of excitation and 550 nm of emission, with a gain of 1000, 30 minutes after the reaction (T=30).

The difference between the fluorescence after the incubation time (T = 30 min) and the initial fluorescence (T = 0 min) is the fluorescence due to the enzyme activity in the undenatured extract, which is expressed in UAF/Time/ protein concentration.

Example 2.2. Preparation of the reaction corresponding to the denatured extract.

It was carried out exactly as in example 2.1, but using the denatured extract, as described in example 1.1. T =0,T =30 min.

This experiment was carried out to check whether the telomerase assay carried out in example 2.1. had been correct or had there been some artifact. If the assessment is correct, the activity values with the denatured extract should tend towards zero.

Example 2.3. Measurement of telomerase activity

Telomerase activity was calculated by measuring the difference between UAF (T=30) and UAF (T=0) of the extract to be analyzed (as described in example 2.1) and the blank (as described in example 2.2). The result is the fluorescence signals due to the amount of DNA formed by the reaction during 30 min. Enzyme activity is measured in UAF/time/protein.

Specifically, the activity calculations that were carried out were:

- Calculate the UAF (T=30) - UAF (T=0) of the problem = A (example 2.1.)

- Calculate the UAF (T=30) - UAF (T=0) of the target = B (example 2.2)

The measurement of telomerase activity was obtained as:

(A-B) = UAF/Time/amount of proteins

Example 3. Study of telomerase activity in different tissues

Several tissues were tested for the assessment of telomerase activity. It was evaluated in the heart and liver of one-month-old rats, and in cow bone marrow. The results are indicated in Table 1.

Table 1. Telomerase activity in different tissues.

The measurement was carried out with a gain of 1000. The activity is expressed in UAF/ 30 min/mg protein. The results of these data are the average of two different experiments, each performed in triplicate.

Example 4. Measurement of telom erase activity in the presence and absence of primers

To demonstrate that the first external substrate TS (SEQ ID NO:1) used in the telomerase assay works, the measurement of the telomerase enzyme was carried out in the absence and in the presence of the specific primers that serve as external substrate (SEQ ID NO: 1) and primer (SEQ ID NO:2). The results obtained in liver are indicated in Table 2.

Table 2. Telomerase activity without the primers and with the primers in rat liver.

The liver was chosen to be used because it was the tissue that presented the highest telomerase activity. The conditions of the telomerase measurement were the same as those indicated in Example 2.1. with the difference that the lack of primers was replaced by sterile H20 MiliQ. The amount of extract used was 15 μL with a total protein content of 50 μg/15 μL. Table 2 shows the results of the telomerase measurement at three times: 14, 30 and 60 min. Data are expressed in UAF/Time/mg of protein. The data are done in triplicate. The statistical significances have been made using the SigmaPlot program. The values are statistically significant from p<0.05.

The increase in DNA in the absence of primers mediated by telomerase could be explained because tissue extracts contain DNA and, therefore, also tDNA, which is the substrate of telomerase, which could generate lengthening of these telomeres. In any case, it can be observed that the activities with primers are greater than in the reactions that do not contain primers, the results being statistically significant, so it is concluded that tDNA has been formed from the external substrate (SEQ ID NO: 1) .

Example 5. Presence of DNA in the rat liver extract

To demonstrate that the liver extract used had DNA, the presence of DNA in said extracts was assessed. The results are presented in Table 3 and Figure 1. The tests were performed in triplicate.

Table 3. DNA assessment in rat liver extract

A curve was prepared with different concentrations of rat liver extract and 2 μL of DAPI (20 μg/mL) was added, the volume was completed to 50 μL with sterile H20 MiliQ and the fluorescence was measured at 360 nm excitation and 460 nm. emission, with a gain of 1000. DAPI is a fluorescent marker, which binds strongly to regions of DNA rich in adenine and thymine.

Example 6. Effect of time on telomerase activity in rat liver The effect of time on the measurement of telomerase in rat liver samples was analyzed. The data are shown in Table 4 and Figure 2; They are expressed in UAF/Time/mg of proteins versus time in minutes (min). Three experiments were done, each in triplicate.

Table 4. Effect of time on telomerase assessment in rat liver.

It was proven that rat liver telomerase presents a linear behavior versus incubation time (see Figure 2).

Example 7. Effect of the concentration of rat liver extract on telomerase activity

The effect of the concentration of rat liver extract on telomerase activity was assessed. The results are presented in Table 5 and Figure 3.

The experiment was carried out with an extract whose protein concentration was 1.5 μg of protein/μL of extract; Different amounts of extract (X μL) were used: 15, 20, 25 and 30 p L and the activity was assessed in UAF/40 minutes/X μL (Figure 3).

Table 5. Effect of extract concentration on liverderata telomerase titration.

As can be seen, telomerase activity is proportional to the concentration of rat liver extract, which means that it is proportional to the enzyme concentration (Figure 3).

Claims (14)

1. Method for the quantitative assessment of telomerase activity that includes the following steps: a) obtain extracts of the cells that you want to study; b) denature the proteins of an aliquot part of the extract obtained in step a), which will be used as a blank; c) mix the extract from step a) with a suitable buffer for the telomerase reaction to take place, the primers described in SEQ ID NO: 1 and SEQ ID NO: 2, DNA polymerase, MgCE d-NTPs and a specific intercalating agent double-stranded DNA; d) mix the aliquot part of the extract whose proteins have been denatured in step b) with a suitable buffer for the telomerase reaction to take place, the primers described in SEQ ID NO: 1 and SEQ ID NO: 2, DNA polymerase, MgCE d-NTPs and the same double-stranded DNA-specific intercalating agent as in step c); e) measure the fluorescence of step c) at time zero (T=0) and 30 minutes after the reaction starts (T=30), and measure the fluorescence of step d) at time zero (T=0) now 30 minutes after the reaction starts (T=30); f) calculate the difference between the fluorescence at T=30 and T=0 minutes of the extract to be analyzed (step c) and the blank (step d); where no amplification of the telomerase reaction product is performed by chain reaction techniques. 2. Method according to claim 1 wherein the double-stranded DNA-specific intercalating agent is a fluorogenic dye. 3. Method according to claim 2 wherein the double-stranded DNA-specific intercalating agent is SYBR-Green I. 4. Method according to any of the preceding claims in which the reaction in step e) is carried out at a temperature between 25 and 350C. 5. Method according to claim 4 wherein the reaction temperature is 300C. 6. Method according to any of the preceding claims wherein the pH of the buffer suitable for the telomerase reaction to take place is 8.3. 7. Method according to any of the preceding claims in which the reaction product is directly titrated using a spectrofluorimeter. 8. Method according to any of the preceding claims in which the DNA polymerase is not thermostable. 9. Kit to assess the telomerase activity of a sample that includes the primers defined by SEQ ID NO: 1 and SEQ ID NO: 2. 10. Kit according to claim 9, which further includes DNA polymerase, MgCE d-NTPs and a double-stranded DNA-specific intercalating agent. 11. Kit according to any of claims 9-10 wherein the double-stranded DNA-specific intercalating agent is a fluorogenic dye. 12. Kit according to claim 11 wherein the fluorogenic dye is SYBR-Greenl. 13. Kit according to any of claims 9-12 wherein the DNA polymerase is not thermostable. 14. Use of the method defined in claims 1-8 and/or the kit defined in claims 9-13 in the study of the proliferation capacity of cells, the identification of drugs and/or molecules capable of acting as activators or inhibitors of telomerase activity, the study of regenerative therapies against myocardial infarction, the identification of tumor proliferation associated with cancer, the detection of the malignancy status of different types of cancer, the detection of the degree of proliferation of stem cells and/ or the study of life extension.