Method to detect activity of a polymerase

Abstract

The present invention relates to methods for detection of nucleotide polymerase activity and methods of detecting compounds that modulate nucleotide polymerase activity, by detecting product formation of the nucleotide polymerase to be tested based on determination of close proximity of two labeled nucleotide probes able to bind the product of the nucleotide polymerase. It is preferred that proximity dependent energy transfer, such as forster resonance energy transfer, between said labeled nucleotide probes is determined. The invention further provides kits comprising components for carrying out the inventive methods for detection of nucleotide polymerase activity.

Claims

1. A method for detection of nucleotide polymerase activity comprising the following steps: a) providing a reaction mixture comprising at least said polymerase, an initiator, and nucleoside triphosphates, b) providing conditions sufficient to allow the polymerase an assembling of nucleotides, c) stopping polymerase assembling of nucleotides, d) after the step of stopping polymerase assembling of nucleotides, adding a donor nucleotide probe and an acceptor nucleotide probe wherein said donor nucleotide probe is conjugated to a first non-radioactive label and said acceptor nucleotide probe is conjugated to a second non-radioactive label, wherein said labels allow determination of close proximity of said nucleotide probes, e) providing conditions sufficient to allow the nucleotide probes to hybridize with the product of step b) and f) determining if said donor and acceptor nucleotide probes are in close proximity wherein close proximity correlates with the activity of the nucleotide polymerase wherein said initiator is a template oligonucleotide or a primer for a template-independent nucleotide polymerase, and wherein said donor nucleotide probe and said acceptor nucleotide probe are complementary to the product of the nucleotide polymerase assembled at step b).

2. The method according to claim 1, wherein the label of the donor nucleotide probe and the label of the acceptor nucleotide probe allow energy transfer which can be determined in step f).

3. The method according to claim 1, wherein the reaction mixture further comprises a candidate substance to test its effect on the nucleotide polymerase activity.

4. The method according to claim 3, wherein providing a reaction mixture of step a) comprises the following: 1) contacting said polymerase with the initiator, at least one transcription factor and an appropriate co-factor of the nucleotide polymerase resulting in a first reaction mixture 2) incubating said first reaction mixture 3) adding to said first reaction mixture the candidate substance resulting in a second reaction mixture 4) incubating said second reaction mixture 5) adding to said second reaction mixture nucleoside triphosphates.

5. The method according to claim 1, wherein the reaction mixture of step a) comprises further at least one transcription factor, replication factor or co-factor of the nucleotide polymerase.

6. The method according to claim 1, wherein the nucleotide polymerase is selected from the group consisting of DNA dependent DNA polymerase, RNA dependent DNA polymerase, DNA dependent RNA polymerase, RNA dependent RNA polymerase, independent RNA polymerase, and independent DNA polymerase.

7. The method according to claim 1, wherein the donor nucleotide probe or the acceptor nucleotide probe is conjugated to a label or fluorophore by covalent bonding or by non-covalent interaction.

8. The method according to claim 1, wherein the donor nucleotide probe or the acceptor nucleotide probe is conjugated to a label or fluorophore by non-covalent interaction mediated by biotin covalently coupled to the donor nucleotide probe or respectively the acceptor nucleotide probe and streptavidin covalently coupled to the label or fluorophore.

9. The method according to claim 8, wherein unspecific binding of the streptavidin covalently coupled to the label or fluorophore is blocked using random oligonucleotides before the streptavidin covalently coupled to the label or fluorophore is conjugated to the biotin covalently coupled to the donor nucleotide probe or the acceptor nucleotide probe.

10. The method according to claim 1, wherein the donor nucleotide probe and the acceptor nucleotide probe are used in a ratio between 1:2 and 2:1.

11. The method according to claim 1, wherein the method is adapted to be carried out as a homogenous high throughput screen.

12. The method according to claim 1, wherein the method is carried out at room temperature.

13. A method for detection of nucleotide polymerase activity comprising the following steps: a) providing a reaction mixture comprising said polymerase, an initiator, and nucleoside triphosphates, b) providing conditions sufficient to allow the polymerase an assembling of nucleotides, c) stopping polymerase assembling of nucleotides, d) after the step of stopping polymerase assembling of nucleotides, adding a donor nucleotide probe and an acceptor nucleotide probe wherein the donor nucleotide probe is conjugated to a fluorophore being a donor for frster resonance energy transfer and the acceptor nucleotide probe is conjugated to a fluorophore being a fluorescence quencher or an appropriate acceptor for frster resonance energy transfer, e) providing conditions sufficient to allow the nucleotide probes to hybridize with the product of step b) and f) determining fluorescence wherein said fluorescence correlates with the activity of the nucleotide polymerase; wherein said initiator is a template oligonucleotide or a primer for a template-independent nucleotide polymerase, and wherein said donor nucleotide probe and said acceptor nucleotide probe are complementary to the product of the nucleotide polymerase assembled at step b).

14. The method according to claim 13, wherein the reaction mixture further comprises a candidate substance to test its effect on the nucleotide polymerase activity.

15. The method according to claim 14, wherein providing a reaction mixture of step a) comprises the following: 1) contacting said polymerase with the initiator, at least one transcription factor and an appropriate co-factor of the nucleotide polymerase resulting in a first reaction mixture 2) incubating said first reaction mixture 3) adding to said first reaction mixture the candidate substance resulting in a second reaction mixture 4) incubating said second reaction mixture 5) adding to said second reaction mixture nucleoside triphosphates.

16. The method according to claim 13, wherein the reaction mixture of step a) comprises further at least one transcription factor, replication factor or co-factor of the nucleotide polymerase.

17. The method according to claim 13, wherein the nucleotide polymerase is selected from the group consisting of DNA dependent DNA polymerase, RNA dependent DNA polymerase, DNA dependent RNA polymerase, RNA dependent RNA polymerase, independent RNA polymerase, and independent DNA polymerase.

18. The method according to claim 13, wherein the donor nucleotide probe or the acceptor nucleotide probe is conjugated to a label or fluorophore by covalent bonding or by non-covalent interaction.

19. The method according to claim 13, wherein the donor nucleotide probe or the acceptor nucleotide probe is conjugated to a label or fluorophore by non-covalent interaction mediated by biotin covalently coupled to the donor nucleotide probe or respectively the acceptor nucleotide probe and streptavidin covalently coupled to the label or fluorophore.

20. The method according to claim 19, wherein unspecific binding of the streptavidin covalently coupled to the label or fluorophore is blocked using random oligonucleotides before the streptavidin covalently coupled to the label or fluorophore is conjugated to the biotin covalently coupled to the donor nucleotide probe or the acceptor nucleotide probe.

21. The method according to claim 13, wherein the donor nucleotide probe and the acceptor nucleotide probe are used in a ratio between 1:2 and 2:1.

22. The method according to claim 13, wherein the method is adapted to be carried out as a homogenous high throughput screen.

23. The method according to claim 13, wherein the method is carried out at room temperature.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: shows the principle of detection of nucleotide polymerase activity based on FRET. Thereby bio represents biotin and Strep means streptavidin. E.sub.x and E.sub.M represent the energy used for extinction and the emitted energy (light at emission wavelength), respectively. A shows an embodiment wherein the fluorophores of the donor nucleotide probe are conjugated to the oligonucleotide using biotin/streptavidin. B shows an embodiment wherein the fluorophores of the donor and the acceptor nucleotide probe are covalently conjugated to the oligonucleotide. C shows an embodiment wherein the fluorophores of the acceptor nucleotide probe are conjugated to the oligonucleotide using biotin/streptavidin.

(2) FIG. 2: shows the principle of detection of nucleotide polymerase activity based on Alpha technology. Thereby DIG means digoxygening, bio represents biotin and Strep means streptavidin. E.sub.x and E.sub.M represent the energy used for extinction and as the emitted energy (light at emission wavelength), respectively.

(3) FIG. 3: shows a summary of the assay robustness during a HT-screening of a compound library. The z values of individual plates, as a measure of assay robustness, were calculated and plotted for the 183 1536-well plates required to screen >250.000 compounds. The lower assay acceptance criterion is 0.4. The observed average z value of 0.88 (white line) is indicated.

(4) FIG. 4: shows a plot of resulting dose-dependent activity data obtained in example 2, an inventive method in 384-well microtiter plate format, yielding the concentration at which the enzymatic activity is reduced to 50% relative to the positive control sample (IC.sub.50).

(5) FIG. 5: shows resulting dose-dependent activity data of the nucleotide analogue TTTP in example 4 plotted and fitted to a four-parameter non-linear regression model, resulting in a sigmoidal curve, yielding the concentration at which the enzymatic activity is reduced to 50% relative to the positive control sample (IC.sub.50).

(6) FIG. 6: shows assay results of a substance test in the 1536-well microtiter plate format. The table shows a representation of the ratio of donor-(620 nm) and acceptor-fluorescence (665 nm). The rows of positive (C+) and negative control samples (C) are indicated, further some primary hits are indicated in bold and in italics.

(7) FIG. 7: is displaying the result obtained by example 5, describing IC.sub.50-determination of a small molecular weight substance, starting from a maximal compound concentration of 200 M to a lower limit of 90 nM. The substance had initially been identified as an inhibitor of the human POLRMT (mitochondrial DNA-directed RNA polymerase), with an IC.sub.50<10 M. It is apparent that the substance does also inhibit the bacteriophage T7 enzyme. These results exemplify that the general assay format is ideally suited to precisely differentiate effects of modulators on enzymes originating from different species.

(8) FIG. 8: is displaying the result of a comparative study of the effect of adding TEFM to the in vitro transcription reaction, as described in example 6. Two transcription reactions were set up either with or without the addition of TEFM (100 nM) and product formation quenched after 2 h. The specific FRET signal of the reaction without TEFM was used as a reference and normalized to 100%, while for the reaction including TEFM a signal increase of around 18% (10%) was measured under the chosen conditions.

EXAMPLES

Example 1: Determination of Activity of Mitochondrial RNA-Polymerase in a High-Throughput Screening in the 1536-Well Plate Format

(9) The inventive method used monitors the activity of mitochondrial RNA-polymerase via detection of the formation of its product, a 407 bp long RNA sequence (SEQ ID No. 1). Detection of the product is facilitated by hybridization of two DNA-oligonucleotide probes to specific and adjacent sequences within the RNA product sequence. Upon annealing of the probes, two fluorophores that are coupled directly to an acceptor nucleotide probe (ATTO647, 5) or introduced via a coupled streptavidin interacting with a biothinylated donor nucleotide probe on the other side (Europium cryptate, 3) are brought into sufficient proximity to serve as a fluorescence-donor-acceptor pair. Thus, a FRET signal at 665 nm is generated upon excitation at 340 nm.

(10) TABLE-US-00001 TABLE1 Sequenceofinitiatorandnucleotide probesusedinexample1and2: Name Sequence HumanLSP AAAGATAAAATTTGAAATCTGGTTAGGCTGGTGTTAGG (topstrand) GTTCTTTGTTTTTGGGGTTTGGCAGAGATGTGTTTAAG TGCTGTGGCCAGAAGCGGGGGAGGGGGGGTTTGGTGGA AATTTTTTGTTATGATGTCTGTGTGGAAAGTGGCTGTG CAGACATTCAATTGTTATTATTATGTCCTACAAGCATT AATTAATTAACACACTTTAGTAAGTATGTTCGCCTGTA ATATTGAACGTAGGTGCGATAAATAATAGGATGAGGCA GGAATCAAAGACAGATACTGCGACATAGGGTGCTCCGG CTCCAGCGTCTCGCAATGCTATCGCGTGCATACCCCCC AGACGAAAATACCAAATGCATGGAGAGCTCCCGTGAGT GGTTAATAGGGTGATAGACCTGTGATC ProbeNo.11 ATTO47N-5-ACAAAGAACCCTAACACCAG-3 ProbeNo.8 bio-5-AACACATCTCT(-bio)GCCAACCCCA- bio-3

(11) The following Equipment and apparatus were used: Thermo Multidrop+Labcyte Echo Thermo Vario Teleshaker or Eppendorf Mixmate Thermo Scientific Multifuge 1S-R+plate-rotor (384 well) Perkin Elmer EnVision plate reader+TRF light unit Assay Plate: Corning 3729 (1536, flat-bottom, non-binding, white) or Corning 3673 (384, low vol., round bottom, non-binding, white)

(12) Proteins used as transcription factors (POLRMT: NP_005026.3, TFAM: NP_003192.1, TFB2M: NP_071761.1) are diluted from their stocks to working concentrations of 150 nM, 1.8 M and 330 nM respectively, in a dilution buffer containing 100 mM TrisHCl pH 8.0, 200 mM NaCl, 10% (v/v) glycerol, 2 mM glutathione (GSH), 0.5 mM EDTA and 0.1 mg/mL bovine serum albumin (BSA). Protein dilutions and initiator (template DNA), comprising a pUC18 plasmid encoding the mitochondrial light strand promotor restriction linearized proximal to the promotor 3-end (pUC-LSP), are mixed at the twofold final assay-concentration in a reaction buffer, containing 10 mM TrisHCl pH 7.5, 10 mM MgCl.sub.2, 40 mM NaCl, 2 mM GSH, 0.01% (w/v) Tween-20 and 0.1 mg/mL BSA.

(13) 5 L of this mix are dispensed, depending on the chosen microtiter plate format, using multi-channel pipettes or a Thermo Multidrop dispenser into the wells of a microtiter plate and incubated at room-temperature (RT) for 10 minutes. Candidate substances in the assay are applied using contact-free acoustic droplet-dispensing (Labcyte Echo) from 10 mM compound stocks in 100% DMSO, to a final concentration of 10 M. Equal amounts of DMSO without any compound are added to positive control samples. An incubation step at RT for 10 minutes is applied to allow binding of compounds without having to compete with the natural nucleotide substrates.

(14) The enzymatic reaction of the polymerase is started by the addition of 5 L of a mix of dNTPs in reaction buffer to a final concentration of 500 M each. No nucleotide triphosphate mix is added to negative control samples. The content of the wells is mixed using a Thermo Vario Teleshaker at 1500 rpm for 45 sec after which the microtiter plate is centrifuged at 500 g for 1 min. The enzymatic reaction samples are incubated for 2 hours at room-temperature. Plates may be incubated in a humidifying chamber at 100% humidity, to avoid evaporation.

(15) In the meantime, a mix of the detection reagents is prepared in a buffer that is composed, such that the enzymatic reaction is terminated due to chelating of Mg-ions and increased ionic strength, containing 50 mM TrisHCl pH 7.5, 700 mM NaCl, 20 mM EDTA, and 0.01% (W/v) Tween-20. Eu-cryptate-coupled streptavidin is pre-incubated with a 100-fold molar excess of a random sequence oligonucleotide to block unspecific binding of single strand mRNA to the protein, for 10 min at RT in the dark. Afterwards, the blocked streptavidin(-Eu) is mixed with the nucleotide probes on ice and kept away from light until use.

(16) At the end of the enzymatic reaction time 10 L detection reagent mix is added, such that the final concentration of fluorescent-labeled donor nucleotide probe, fluorescent-labeled acceptor nucleotide probe in each assay well is 1 nM, and 3 nM respectively. Assay plates are again mixed and centrifuged as above and are stored at RT, protected from light for at least 2 h or until binding of the nucleotide probes to mRNA product of the polymerase leads to the development of the maximal FRET signal. A schematic overview of the assay workflow is depicted in FIG. 1.

(17) The generated signal is measured with a suitable microtiter plate reader (Perkin Elmer EnVision plate reader, including TRF light unit), using excitation at 320 nm, an integration time of 200 s and a delay time of 100 s, before detection at 620 nm and 665 nm. The ratio of donor- and acceptor-fluorescence is used to assess the specific FRET as a measure of the generated polymerase product (i.e. enzymatic activity). Usually, the signal-to-background ratio (S:B) between positive and negative control samples is >10 and the signal homogeneity of positive and negative control was lower than 10% (CV %<10) resulting in z values >0.7.

(18) According to this method small molecular weight substances have been tested for their effects on the mitochondrial transcription machinery in a high-throughput screen (HTS), at 10 M final candidate substance concentration.

(19) FIG. 6 is displaying a tabular representation of the results obtained during a screen of a library of small molecular weight compounds, for an exemplary 1536-well microtiter plate, comprising 1408 test wells and 64 wells each for positive and negative control samples. Based on the chosen hit-criteria (30% residual activity, relative to positive control). Primary hits on this plate were found in R15, X27, A44, B48 and T48. Activity data could be calculated based on fluorescence ratio values and normalized to positive (100%) and negative control samples (0%). One compound displaying inhibitory effects was identified with 13.7% activity on this plate. This finding is also representative for the generally observed hit-rate (0.05-0.1%) using larger substance libraries. As can be seen in FIG. 6, in addition to inhibition of the polymerase reaction, the assay format and specific conditions also allow for detection of apparently increased product formation, facilitating the identification of putative activators.

(20) During a screen of 183 1536-well microtiter plates the calculated z-values, as a measure of the assay robustness, were found to be consistently >0.75, with a mean value of 0.88 (see FIG. 3), giving reference of the observed homogeneity of positive and negative control samples.

(21) A plot of the distribution of measured activity data of all sample wells during the exemplary high throughput screen yields the expected Gaussian curve around the positive control, with a slight shift towards lower activity. The latter can be explained, by the specific tuning of the assay conditions, which had been performed to suit the requirements of a search for inhibitors.

Example 2: Inhibitor Activity Determination in the 384-Well Plate Dose-Response Format

(22) The assay protocol largely corresponds to the setup described in example 1 (Determination of activity of mitochondrial RNA-polymerase using the same initiator and probes than in example 1). For determination of the activity, however, the reaction mix always contains the nucleotide triphosphate mix, to enforce potential competition between inhibitor compound and substrate. Negative control samples are set up such that no template DNA (pUC-LSP) is included in the reaction mixture.

(23) Pre-diluted protein samples are mixed with the dNTP-mixture in a reaction buffer, containing 10 mM TrisHCl pH 7.5, 10 mM MgCl.sub.2, 40 mM NaCl, 2 mM GSH, 0.01% (w/v) Tween-20 and 0.1 mg/mL BSA, to yield final assay concentrations of 7.5 nM (POLRMT), 90 nM (TFAM), 20 nM (TFB2M) and 500 M (dNTPs). 5 L of this mix are dispensed, depending on the chosen microtiter plate format, using multi-channel pipettes or a Thermo Multidrop dispenser into the wells of a microtiter plate and incubated at room-temperature (RT) for 10 minutes.

(24) Candidate substances in the assay are applied using contact-free acoustic droplet-dispensing (Labcyte Echo) from 10 mM compound stocks (in 100% DMSO) in an appropriate 8-point dilution series to ensure accurate fitting of the resulting activity data (i.e. upper and lower asymptote reached). Equal amounts of DMSO without any compound are added to positive control samples. An incubation step at RT for 10 minutes is applied to allow binding of compounds.

(25) The enzymatic reaction is started by the addition of 5 L of a mix of template DNA (pUC-LSP) in reaction buffer to a final concentration of 0.5 nM. The following mixing, incubation and detection steps correspond to the steps described in example 1. The resulting dose-dependent activity data is plotted and fitted to a four-parameter non-linear regression model, resulting in a sigmoidal curve, yielding the concentration at which the enzymatic activity is reduced to 50% relative to the positive control sample (IC.sub.50).

(26) Table 2 is displaying an overview of the results obtained during IC.sub.50-determination of a series of inhibitors of different activity, starting from a maximal compound concentration of 200 M to a lower limit of 5 M. Based on the chosen concentrations of the test enzymes and scrutinized compounds the IC.sub.50-values observed for this assay format typically span four to five orders of magnitude, indicating the wide dynamic range of the assay system. Other tests have successfully been performed using reduced enzyme concentrations, allowing for a lower limit of detection of inhibition at 3 nM.

(27) TABLE-US-00002 TABLE 2 Assay results obtained for an IC.sub.50 determination of different compounds in the 384-well microtiter plate format. The calculated z-value during this experiment was 0.88, signal-to-background (S:B) = 10.2, signal-to-noise (S:N) = 49.7. Highest res. activity res. activity assayconc. Hill IC.sub.50 at highest at lowest Sample ID [M] slope (M) conc. (%) conc. (%) R.sup.2 2methyl/OH-GTP 200 2.05 56.2 65.5 96.5 0.9076 2methyl/OH-O-UTP 200 0.85 84.5 8.6 102 0.9988 2methyl/F-GTP 200 2.15 73.4 46.9 88.32 0.9962

Example 3: Inhibitor Activity Determination, Using Mouse Mitochondrial RNA-Polymerase, in a 384-Well Plate Dose-Response Format

(28) The assay protocol largely corresponds to the setup described in example 2 for determination of the IC.sub.50/EC.sub.50 for modulators of the activity of the human enzyme. However, proteins utilized in this embodiment are NM_172551.3 (mPOLRMT), NM_008249.4 (mTFB2M) and NM_009360.4 (mTFAM). Furthermore the initiator, or DNA transcription template corresponds to the murine mitochondrial light-strand promotor (mLSP), encoded in a pCRY-TOPO plasmid and restriction linearized proximal to the 3-end of the promotor, to allow for a run-off transcription reaction. As a consequence, the sequences of the employed DNA-probes had to be adjusted for complementarity to the promotor-transcript sequence.

(29) TABLE-US-00003 TABLE3 Sequenceofinitiatorandnucleotideprobes usedinexample3: Name Sequence MurineLSP TTTGGTTCACGGAACATGATTTTGTAAAATTTTTACAA (topstrand) GTACTAAAATATAAGTCATATTTTGGGAACTACTAGAA TTGATCAGGACATAGGGTTTGATAGTTAATATTATATG TCTTTCAAGTTCTTAGTGTTTTTGGGGTTTGGCATTAA GAGGAGGGGGTGGGGGGTTTGGAGAGTTAAAATTTGGT ATTGAGTAGCATTTATGTCTAACAAGCATGAATAATTA GCCTTAGGTGATTGGGTTTTGCGGACTAATGATTCTTC ACCGTAGGTGCGTCTAGACTGTGTGCTGTCCTTTCATG CCTTGACGGCTATGTT ProbeNo.6 bio-5-TAACT(bio)ATCAAACCCTATGT-3-bio ProbeNo.7 5-AACTTGAAAGACATATAAT-3-ATTO647N

(30) For determination of the activity, however, the reaction mix always contains the nucleotide triphosphate mix, to enforce potential competition between inhibitor compound and substrate. Negative control samples are set up such that no template DNA (pUC-LSP) is included in the reaction mixture.

(31) Pre-diluted protein samples are mixed with the dNTP-mixture in a reaction buffer, containing 10 mM TrisHCl pH 7.5, 10 mM MgCl.sub.2, 40 mM NaCl, 2 mM GSH, 0.01% (w/v) Tween-20 and 0.1 mg/mL BSA, to yield final assay concentrations of 40 nM (mPOLRMT), 80 nM (mTFAM), 80 nM (mTFB2M) and 500 M (dNTPs). This setup accommodates for the lower enzymatic activity of the employed preparation of the mouse enzyme compared to the human homolog. However, this observation might be related to the production of the enzyme samples and determination of the optimal polymerase concentration is necessary for any enzyme tested. Also, for this particular embodiment it was found that a ratio of polymerase and transcription factors of 1:2:2 yields the highest productivity and, thus best assay performance.

(32) 5 L of this reaction mixture are dispensed, depending on the chosen microtiter plate format, using multi-channel pipettes or a Thermo Multidrop dispenser into the wells of a microtiter plate and incubated at room-temperature (RT) for 10 minutes.

(33) Candidate substances tested in this example are applied using contact-free acoustic droplet-dispensing (Labcyte Echo) from 10 mM compound stocks (in 100% DMSO) in an appropriate 8-point dilution series to ensure accurate fitting of the resulting activity data (i.e. upper and lower asymptote reached). Equal amounts of DMSO without any compound are added to positive control samples. An incubation step at RT for 10 minutes is applied to allow binding of compounds.

(34) The enzymatic reaction is started by the addition of 5 L of a mix of template DNA (mLSP) in reaction buffer to a final concentration of 5 nM. The following mixing, incubation and detection steps correspond to example 2.

(35) The resulting dose-dependent activity data is plotted and fitted to a four-parameter non-linear regression model, resulting in a sigmoidal curve, yielding the concentration at which the enzymatic activity is reduced to 50% relative to the positive control sample (IC.sub.50).

(36) Table 4 is displaying the result obtained during IC.sub.50-determination of a small molecular weight substance, starting from a maximal compound concentration of 1 M to a lower limit of 0.25 nM. The substance had initially been identified as an inhibitor of the human POLRMT enzyme with an IC.sub.50-human 1 nM. Despite the higher enzyme concentration applied in this assay format, it is apparent that the substance does also inhibit the mouse enzyme, however with 20-fold higher IC.sub.50. These results exemplify that the general assay format is ideally suited to precisely differentiate effects of modulators of the enzymatic activity, even at very low substance concentrations.

(37) TABLE-US-00004 TABLE 4 Assay result obtained for an IC.sub.50 determination in the 384-well microtiter plate format The calculated z-value during this experiment was ~0.6, signal-to-background (S:B) = 3.1, signal-to-noise (S:N) = 13.7. Highest assay res. activity at res. activity at conc. Hill IC.sub.50 highest conc. lowest conc. Sample ID [M] slope (M) (%) (%) Compound 1 0.058 0.02 31.85 95.54 197459

Example 4: Inhibitor Activity Determination, Using Human Mitochondrial DNA-Polymerase, in a 384-Well Plate Dose-Response Format

(38) The assay protocol largely corresponds to the setup described in example 1 (Determination of activity of mitochondrial RNA-polymerase using the same initiator and probes than in example 1). For determination of the activity of the mitochondrial replication, however, the two-subunit complex of the mitochondrial DNA-polymerase (POLG, NM_002693.2, NM_007215.3), the transcription factor TWINKLE (NM_021830.4) and mitochondrial single-strand binding protein (SSBP1, NM_001256510.1) are used. Also, the reaction mix always contains the substrate nucleotide mix, to enforce potential competition between inhibitor compound and substrate.

(39) For DNA-replication the template is generated using a circular, single-stranded DNA formed from pBlueskript SK+. To this circular, ssDNA, a primer with 42-nucleotide poly-T is annealed to form a fork structure. To make dsDNA, the primer is elongated with KOD polymerase, which synthesizes DNA around the entire circle, until again it reaches the primer. At this point DNA-synthesis stops and a double-stranded DNA template of about 3000 bp with an artificial replication fork is formed. After this, the template is purified and used in the assay for a rolling circle DNA replication, which yields a product of repetitive stretches of the sequence displayed in Table 2.

(40) For activity determination, negative control samples are set up such that no template DNA is included in the assay mixture. Assay components are mixed in a buffer containing 25 mM TrisHCl pH 8.0, 5 mM MgCl.sub.2, 2 mM GSH, and 0.1 mg/mL BSA, to yield final assay concentrations of 30 nM POLG, 15 nM TWINKLE, 20 nM SSBP1, 250 M dNTPs, 4 mM ATP as well as the two DNA-probes at 3 nM concentration each.

(41) 5 L of this mix are dispensed, depending on the chosen microtiter plate format, using multi-channel pipettes or a Thermo Multidrop dispenser into the wells of a microtiter plate and incubated at room-temperature (RT) for 10 minutes.

(42) Chemical compounds under scrutiny in the assay are applied using contact-free acoustic droplet-dispensing (Labcyte Echo) from 10 mM compound stocks (in 100% DMSO) in an appropriate 8-point dilution series to ensure accurate fitting of the resulting activity data (i.e. upper and lower asymptote reached). Equal amounts of DMSO without any compound are added to positive control samples. An incubation step at RT for 10 minutes is applied to allow binding of compounds.

(43) The enzymatic reaction is started by the addition of 5 L of a mix template DNA in reaction buffer to a final concentration of 5 nM. Following incubation for 1 h at 37 C., the reaction is stopped by addition of a detection mix, containing 3 nM streptavidin-coupled Eu-cryptate pre-incubated with an excess of an unspecific random oligonucleotide, in 20 mM Tris-HCl pH 8.0, 400 mM NaCl and 50 mM EDTA. HTRF-signals are measured following over-night incubation at room-temperature.

(44) The resulting dose-dependent activity data is plotted and fitted to a four-parameter non-linear regression model, resulting in a sigmoidal curve, yielding the concentration at which the enzymatic activity is reduced to 50% relative to the positive control sample (IC.sub.50).

(45) Table 5 is displaying the results obtained during IC.sub.50-determination of a nucleotide-analogue inhibitor of POLG, starting from a maximal compound concentration of 200 M to a lower limit of 5 M. Based on the chosen concentrations of the nucleotide substrate (C.sub.(dNTP)=250 M) and the scrutinized compound, the resulting IC.sub.50-value is well within the expected range, indicating direct competition between the substrate nucleotides and the nucleotide-analogue inhibitor.

(46) TABLE-US-00005 TABLE 5 Assay results obtained for an IC.sub.50 determination of the nucleotide analogue TTTP The calculated z-value during this experiment was 0.7, signal-to-background (S:B) = 9.2. Highest res. activity res. activity assay conc. Hill IC.sub.50 at highest at lowest Sample ID [M] slope (M) conc. (%) conc. (%) R.sup.2 4-Thiothymidine-5- 200 0.9462 54.07 22.6 92.79 0.8693 Triphosphate

(47) TABLE-US-00006 TABLE6 Sequenceofinitiatorandnucleotideprobes usedinexample4: Name Sequence rolingcircle 5-ATAGGGGTATGAAATTTGAAATCTGGTT replicationproduct AGGCTGGTGTTAGGGTTCTTTGTTTTTGGGG TTTGGCAGAGATGTGTTTAAGTGCTGTGGCC ACATACCCCTC-3 ProbeNo.11 ATTO647N-5- ACAAAGAACCCTAACACCAG-3 ProbeNo.13 bio-5-AACACATCTCT(-bio)GCCAAA CCCCA-bio-3TGGGGTTTGGCAGAGAT

Example 5: Assay Protocol for Inhibitor Activity Determination, Using Bacteriophage T7 RNA-Polymerase, in the 384-Well Plate Dose-Response Format

(48) Assay principle, background and equipment correspond to the setup described in example 1. The assay protocol largely corresponds to the setup of example 2 describing the general application in determination of the IC.sub.50/EC.sub.50 for modulators of the activity of the human enzyme. However, the polymerase utilized in this assay version is the bacteriophage T7 RNA-polymerase, which can be acquired from diverse commercial sources.

(49) The present assay does not require the presence of any ancillary factors. Instead, a linear DNA transcription template which is shown by SEQ ID No. 21 and SEQ ID No. 22 as well as in table 7 is generated (e.g. via primer hybridization), which enables transcription initiation by the polymerase alone. Thus, monitoring the effect of small molecular modulators on the activity of the RNA-polymerase is achieved, free from artifacts that might be caused by compound binding to transcription factors. In the specific case of the bacteriophage T7 enzyme, a single-stranded overhang is not even required as transcription activity of this polymerase relies on a double-stranded DNA template.

(50) TABLE-US-00007 TABLE7 SequenceoflinearDNAtranscriptiontemplate usedinexample5: Name Sequence linear 5-GGCGGGAGAAGAATTTGAAATCTGGTTAGGCTGGTG transcription 3-CGGCGGCCCTTTTTTCCGCCCTCTTCTTAAACTTTAGACCAATCCGACCAC template TTAGGGTTCTTTGTTTTTGGGGTTTGGCAGAGATGTGTTTAAGTGCTGTGGC AATCCCAAGAAACAAAAACCCCAAACCGTCTCTACACAAATTCACGACACCG CAGAAGCGGGG-3(SEQIDNo.21) GTCTTCGCCCC-5(SEQIDNo.22)

(51) For determination of the activity, the reaction mix always contains the substrate nucleotide mix, to enforce potential competition between the tested inhibitor compound and the substrate. Negative control samples are set up such that no template DNA is included in the assay mixture. Pre-diluted enzyme samples are mixed with the dNTP-mixture in a reaction buffer, containing 10 mM TrisHCl pH 7.5, 10 mM MgCl.sub.2, 40 mM NaCl, 2 mM GSH, 0.01% (w/v) Tween-20 and 0.1 mg/mL BSA, to yield final assay concentrations of 50 nM (T7 RNA-polymerase) and 500 M (dNTPs). 5 L of this mix are dispensed, depending on the chosen microtiter plate format, using multi-channel pipettes or a Thermo Multidrop dispenser into the wells of a (low protein-binding) microtiter plate and incubated at room-temperature (RT) for 10 minutes.

(52) The chemical compound under scrutiny in the assay was 4-methyl-5-[2-(pyrimidin-2-ylamino)thiazol-4yl]thiazol-2-amine (available at ChemDiv) and is applied using contact-free acoustic droplet-dispensing (Labcyte Echo) from 10 mM compound stocks (in 100% DMSO) in an appropriate 8-point dilution series to ensure accurate fitting of the resulting activity data (i.e. upper and lower asymptote reached). Equal amounts of DMSO without any compound are added to positive control samples. An incubation step at RT for 10 minutes is applied to allow binding of compounds. The enzymatic reaction is started by the addition of 5 L of a mix of template DNA in reaction buffer to a final concentration of 2 nM. The following mixing, incubation and detection steps correspond to example 2.

(53) The resulting dose-dependent activity data is plotted and fitted to a four-parameter non-linear regression model, resulting in a sigmoidal curve, yielding the concentration at which the enzymatic activity is reduced to 50% relative to the positive control sample (IC.sub.50).

Example 6: Determination of Activity of Mitochondrial RNA-Polymerase with and without TEFM

(54) To enhance the enzymatic turnover and, thus, specific signal intensity of the mitochondrial in vitro transcription reaction, the mitochondrial transcription elongation factor (TEFM, NP_078959.3.) can be added to the assay reagent mixture.

(55) The general workflow of the assay and composition of assay reagents are described in Posse et al (Nucl. Acids Res. 2015). A linearized pUC18 plasmid comprising the human mitochondrial light-strand promotor sequence (LSP) is used as transcription plasmid.

(56) Adaptations made to the original protocol relate to the different detection method applied and correspond to the set-up described in example 1. Hence, instead of the addition and later scintillation detection of radio-isotope labeled nucleotide, only non-radioactive nucleotides are used and the detection of product formation is again based on the addition and specific binding of DNA oligonucleotide probes, labeled with fluorescent dyes to constitute a FRET pair.