WO2004035818A1

WO2004035818A1 - Use of osmolytes to heat stabilise luciferase and/or apyrase

Info

Publication number: WO2004035818A1
Application number: PCT/SE2003/001627
Authority: WO
Inventors: Pål NYRÉN
Original assignee: Biotage Ab
Priority date: 2002-10-21
Filing date: 2003-10-21
Publication date: 2004-04-29
Also published as: AU2003272174A1

Abstract

The present invention refers to the use of at least one osmolyte to heat stabilise at least one enzyme, such as luciferase and/or apyrase, that is used in an in-vitro bio-luminometric assay, such as a sequencing-by-synthesis reaction, especially Pyrose-quencingTM, during the enzymatic reaction. Moreover, the invention refers to a composition and a kit for use in the bioluminometric assay. Hereby, the temperature sensitive enzyme is heat stabilised, whereby the bioluminometric assay may be per-formed at a temperature that is higher and therefore more suitable for the other en-zymes of the assay.

Description

New use

Technical field

The invention refers to bioluminometric assays, for example involving luciferase, or an analogue thereof showing luciferase-like activity, as well as a composition and a kit for use in such a bioluminometric assay.

Technical background

Some organisms are capable of producing light in exergonic reaction, leading to bio- luminescence. The generic name for the enzyme involved in bioluminescence reactions is luciferase. Different variants of this enzyme have been found in various organisms such as bacteria, insects and dinoflagellates.

Firefly luciferase (EC 1.13.12.7) catalyses the oxidation of D-luciferin in the presence of ATP, magnesium and oxygen. The product, oxyluciferin, is generated in an exited state which decays to the ground state with the emission of a photon. Firefly luciferase has been extensively used in molecular and cell biology, in particular for efficient detection and quantification of ATP, and as a reporter enzyme for studies of gene regulation and expression (Gould, S.J., and Subramani, S. (1988) Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 175, 5-13). All enzymes and metabolites involved in ATP-converting reactions can be analyzed by the firefly luciferase system (Kricka, L.J. (2000) Application of bioluminescence and chemilumines- cence in biomedical sciences. Methods Enzymol. 305, 333-345; Kricka, L.J. (1988) Clinical and biochemical applications of luciferase and luciferins. Anal. Biochem. 175, 14-21; Kricka, L.J. (1991) Chemiluminescent and bioluminescence techniques. Clin. Chem. 37, 1472-1481). Bioluminometric assays have been limited to the optimal temperature of firefly luciferase (22-28°C); the enzyme is rapidly inactivated at temperatures above 30°C (Ueda, I., Shinoda, F., and Kamaya, H. (1994) Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase. Biophys. J. 66, 2107-2110; Ford, S.R., and Leach, R. (1998) Improvements in the application of firefly luciferase assays. Methods Mol. Biol. 102, 3-20). Thermal denaturation of firefly luciferase has been reported to occur at about 39°C (Matsuki, H., Suzuki, A., Kamaya, H., and Ueda, I. (1999) Specific and nonspecific binding of long-chain fatty acids to firefly luciferase: cutoff at octanoate. Biochim. Biophys. Acta 1426, 143-150). To date the practical application of luciferase for clinical diagnostics has been limited, due to its insufficient thermal stability. In addition, many coupled enzymatic reactions, using the firefly luciferase system for continuous detection, must be performed at sub-optimal temperatures.

To improve the thermostability of firefly luciferase, chemical mutagenesis and site- directed mutagenesis (Kajiyama, N., and Nakano, E. (1993) Thermostabilization of firefly luciferase by a single amino acid substitution at position 217. Biochemistry 32, 13795-13799; Kajiyama, N., and Nakano, E. (1994) Enhancement of thermostability of firefly luciferase from Luciola lateralis by a single amino acid substitution. Biosci. Biotechnol. Biochem. 58, 1170-1171; White, P. J., Squirrell, D J., Arnaud, P., Love, C. R., Murray, J. A. H. (1996) Improved thermostability of the North American firefly luciferase. Biochemical J. 319. 343-350) have been used. There are thermostable firefly luciferase preparations commercially available from Kikkoman Corp. (Japan) and Promega Corp. (USA). However, these preparations have been selected for storage stability and not for activity stability at higher temperatures.

Moreover, luciferase systems have been used in various bioluminescence assays. For example, various fusion proteins comprising luciferase and another protein, such as a RNA-binding protein for RNA identification, or a fusion conjugate of firefly lucifer- ase and a biotin acceptor peptide or a single chain antibody for an immunoassay, are known.

Further, Mackey and Park (Thermostability of bacterial luciferase expressed in different microbes, J Appl Bact 1994, 77:149-154) and Pinto et al. (Denaturation of proteins during heat shock, J Biol Chem 1991, 266:13941-13946) discloses the storage stabilisation of luciferase by xylitol and glycerol, respectively, and the subsequent luciferase activity measurement in vivo.

Also, trehalose is known to be able to stabilise luciferase during storage. Trehalose has a relatively high viscosity and thus has a high potency as a storage stabilising substance for various enzymes. However, trehalose is not known to stabilise the enzyme during an assay.

Thus, bioluminometric assays are today encumbered with several problems. Generally, the luciferase-coupled reaction must be performed at a temperature that often is sub- optimal for other involved enzymes. Accordingly, the quality and the reliability of the results, and the activity of these other enzymes in the reaction mixture, are decreased. Also, the known means for enhancing the stability of luciferase enzymes, are focused on stability during storage and subsequent in-vivo use (without the stabilising substance). This limits the use and the functionality of bioluminescence assays. Thus, it would be beneficial if means could be provided that solves the problems and drawbacks of the prior art.

Summary of the invention

The object of the invention is to provide means solving these problems. In one aspect of the invention, this is achieved by the use of at least one osmolyte to heat stabilise at least one enzyme that is used in an in-vitro bioluminometric assay, during the enzymatic reaction. In a preferred embodiment the in-vitro bioluminometric assay is a sequencing-by- synthesis reaction, especially a Pyrosequencing reaction.

Hereby, in one embodiment, in the presence of an osmolyte, luciferase, or an analogue thereof showing luciferase-like activity, can be used at a higher temperature, which is beneficial for many bioluminometric assays. Specifically, this is beneficial for the Pyrosequencing™ reaction, which in the presence of at least one osmolyte may be performed at 37 °C, instead of 28 °C. Hence, the activity of other enzymes of the reaction is enhanced, and thus the quality of the reaction, including the number of sequential nucleotides to be analysed, and the strength of light signals.

Moreover, the inventor has shown that another enzyme that is used in the Pyrosequencing™ reaction, namely apyrase, is also stabilised by the use of an osmolyte. Hence, in one preferred embodiment, at least one osmolyte is used for heat-stabilising apyrase during an in vitro bioluminometric assay, such as a sequencing-by-synthesis reaction.

In another aspect the invention refers to a method for performing a sequencing-by- synthesis reaction, wherein at least one osmolyte is used to heat stabilise the luciferase enzyme.

Also, in another aspect of the invention, a composition comprising luciferase, or an analogue thereof showing luciferase-like activity, and/or apyrase, and at least one osmolyte, in solution or freeze-dried, is provided. This composition is specifically adapted for use in a bioluminometric assay.

Moreover, in yet another aspect of the invention a kit is provided, comprising luciferase, or an analogue thereof showing luciferase-like activity, and/or apyrase, and at least one osmolyte, as well as possibly other necessary reagents, for use in a Pyrose- quencing TM reaction.

Short description of the drawings

Figure 1. The effect of different concentrations of trehalose, glycine betaine and pro- line on the firefly luciferase activity (measured at room temperature and at pH 7.75). The reactions were started by the addition of 10 pmol ATP. The luminescence was measured using a LKB 1250 (proline and trehalose) or 1251 (glycine betaine) luminometer. All measurements were done in at least triplicates. The experimental conditions were as described under Examples. See the text for details.

Figure 2. Effect of pH on the luciferase activity. Enzymatic activity was assayed at 22°C (in the absence of glycine betaine) or at 37°C (in the presence of 1.6 M glycine betaine). The reactions were started by the addition of 10 pmol ATP. The luminescence was measured using a LKB 1251 luminometer. The experimental conditions were as described under Examples. See the text for details.

Figure 3. Effect of glycine betaine on the firefly luciferase reaction at 37°C. Enzymatic activity was assayed at 37°C at different time intervals in the presence of different concentrations of glycine betaine. The reactions were started by the addition of 10 pmol ATP. The luminescence was measured using a LKB 1251 luminometer. The experiments were performed as described under Examples. See the text for details.

Figure 4. Temperature effect on the firefly luciferase activity. Enzymatic activity was assayed at different temperatures, in the presence of 1.6 M glycine betaine. The reactions were started by the addition of 10 pmol ATP. The luminescence was measured using a LKB 1251 luminometer. The experiments were performed as described under Examples. See the text for details. Figure 5. The effect of increasing the temperature from 28°C to 37°C on a self-looping template in the presence of sequence primer. Analysis, using Pyrosequencing™ technology, was performed on 50 μl PCR-generated 222-base-long (pLC19 304- IS) single-stranded template in the presence of 10 pmol sequence primer (TN-LOOP- SEQ) at 28°C (A) and 37°C (B). At 28°C (A) signals are obtained from both specific priming of the sequence primer and unspecific self-priming (arrows) of the free 3'-end of the ssDNA template. At 37°C (B), the unspecific self-priming signals decreased (arrows) and the signals from the sequence primer were dominant. The correct sequence is indicated above trace B. The order of nucleotide addition is indicated on the bottom of the traces. The reaction was performed as described under Examples.

Figure 6. The effect of increasing the temperature from 28°C to 37°C on a self-looping template in the absence of sequence primer. Analysis, using Pyrosequencing™ technology, was performed on 50 μl PCR-generated 222-base-long (pUC19 304-1 S) single-stranded template at 28°C (A) and 37°C (B). The signals observed are due to unspecific self-priming of the free 3'-end of the ssDNA template. The arrows indicate the expected height of a signal from incorporation of one correct base if the sequence primer (TN-LOOP-SEQ) would have been present (see Fig. 5). At 37°C (B), much lower signals were observed, indicating destabilization of the loop structure at the higher temperature. The order of nucleotide addition is indicated on the bottom of the traces. The reaction was performed as described under Examples.

Figure 7. The effect of increasing the temperature from 22°C to 37°C on a single- stranded template in the presence of a primer-dimer. Analysis, using Pyrosequencing™ technology, was performed on both strands of a PCR-generated 320-base-long template in the presence of 5 pmol sequence primer SEQ-AS-D (A, B, C) or PCR-AS- Up (D, E, F) at 22°C (A, D), 28°C (B, E), and 37°C (C, F). Both sequencing primers formed primer-dimers. The arrows indicate some of the background signals that were due to the primer-dimer. The correct sequence is indicated above traces C and D. The order of nucleotide addition was T, C, A, G. The reaction was performed as described under Examples.

Figure 8. Pyrosequencing⁷ reaction data from two different primer-dimers. Pyrosequencing™ technology was performed on 50 pmol of SEQ-ASD (A) and 10 pmol PCR-AS-UP (B), respectively. The experiments were performed at 28°C. The order of nucleotide addition is indicated on the bottom of the traces. The experimental conditions were as described under Examples. See the text for details.

Figure 9. The effect of increasing the temperature from 28°C to 37°C on a primed single-stranded template in the presence of a primer-dimer. Analysis, using Pyrosequencing™ technology, was performed on 2 pmol E3PN NUSPT template in the absence (A, B) and in the presence (C, D) of 0.8 pmol primer PCR-AS-UP at 28°C (A, C) and 37°C (B, D). The correct sequence is indicated above trace A. The order of nucleotide addition was T, C, A, G. The reaction was performed as described under Examples.

Figure 10. Effect of glycine betaine on apyrase activity.

Definitions

By an "osmolyte" is generally meant any organic or inorganic species whose concentration is regulated during the process of cell volume regulation in parallel with the osmotic stress imposed on the cell. However, in the context of the invention is by osmolyte meant any species, organic or inorganic, such as a compound or the like, which has the ability to increase the thermal stability of various protein structures. Examples of various osmolytes are given below. By "heat stabilizing" an enzyme, such as luciferase, is meant to increase the thermal stability of the enzymatic structure in such a way that the activity of the enzyme is maintained at a higher temperature than normally.

By "luciferase, or an analogue thereof showing luciferase-like activity" is meant an enzyme having the capacity to catalyse an energy-dependent reaction, such as an ATP- or FMNH₂-converting reaction resulting in the emission of light (i.e. a bioluminescence reaction). The presence of the reaction, and thus of the activity of the enzyme, is monitored by detecting the presence of a light signal (bioluminometric assay) (see for example the Example section of this disclosure).

A "bioluminometric assay" or a "bioluminescence assay" is an assay in which the activity of an enzyme, or the level of a compound, is monitored in a light-emitting reaction, whereby the light-emission is an enzymatic reaction (luciferase enzyme).

By an "ATP-assay" is in the context of the invention meant a bioluminometric assay, which uses ATP as substrate, and wherein the amount of bioluminescence is proportional to the ATP-concentration.

By "heat-stabilising at least one enzyme during the enzymatic reaction" is meant that an enzyme is heat-stabilised by e.g. the osmolyte at the same time it performs it catalytic action, i.e. in contrast to heat-stabilisation during storage, or before the enzymatic reaction. Hence, the osmolyte is present in the reaction solution in an amount necessary to heat-stabilise the enzyme at the time the enzymatic reaction is performed.

Detailed description of the invention

Firefly luciferase-based assays have several advantages, such as speed, simplicity, sensitivity, and specificity. However, the practical application of luciferase for clinical diagnostics has been limited, due to its insufficient thermal stability. In addition, many coupled enzymatic reactions, using the firefly luciferase systems for detection, must be performed at sub-optimal temperatures due to the limited thermal stability of the luciferase enzyme. In order to solve the above problems, the present inventor, as a first aspect of the invention, used osmolytes to heat-stabilise luciferase, or an analogue thereof showing luciferase-like activity, during a bioluminometric assay.

In biological systems, different ways to protect and adapt proteins against thermal changes and osmotic stress have been evolved. Many organisms produce or import molecules known as osmolytes. Osmolytes that protect proteins can be divided into three classes (Somero, G. N., Yancey, P. H. (1997) in "Handbook of Physiology, Section 14: Cell Physiology" (Hoffman, J. F., Jamieson, J. D., Eds.), pp. 441-484. Oxford University Press): i) sugars and polyhydric alcohols (polyoles), ii) amino acids and amino acid derivatives, and iii) methylated ammonium and sulfonium compounds.

Osmolytes of group (1) comprise for example trehalose, floridoside, glycerol, pinitol, sorbitol and myo-inisitol. Group (2) comprises for example alanine, β-alanine, proline, ectoine, taurine, N_ε-acetyl-β-lysine, N_α-carbamoyl-L-glutamine-l -amide. Group (3) comprises for example trimethylamine-N-oxide, glycerophosphoryl choline, proline betaine, β-alanine betaine, glycine betaine, choline-O-sulfate, homarine and dimethyl- sulfoniopropionate. All these compounds are included in the scope of the invention. Preferably, glycine betaine is used in the present invention. The suitable concentration for the osmolyte that is used is readily determined by a person skilled in the art. However, for the purposes of the invention the concentration of the osmolyte(s) is for example in the interval from 0.1 to 10 M, preferably from 0.5 to 2M. The upper concentration limit that is possible, is determined by the viscosity of the osmolyte and the solution in which the bioluminescent reaction occurs, i.e. if the osmolyte concentration is too high the solution may be too viscous. Also, a combination of different osmolytes may be used, whereby different osmolytes may contribute with different physical properties. The viscosity of the used osmolyte is important, in the sense that a too high viscosity may effect the kinetics of the reaction solution negatively. The osmolytes of group (1), sugars and polyhydric alcohols, may generally show a higher viscosity than the osmolytes of group (2) and (3), and thus osmolytes of group (2) and (3) are preferred. For a reaction like the Pyrosequencing™ reaction, it is important that the various reaction steps are allowed to be rapidly performed, and thus a fast mixing of components and a fast kinetics is an important parameter.

Various osmolytes are for example known to be able to decrease incidence of DNA polymerase stops (US-A-6270962) and to increase the thermal stability of DNA poly- merase (US-A-6428986). However, the use of osmolytes in in-vitro bioluminescence assays is not previously known.

With the stabilised luciferase it is possible to run coupled enzymatic reactions, such as sequencing-by-synthesis and particularly Pyrosequencing™ technology, at more optimal temperatures. Two different enzymes, with temperature optimums above 28°C, Klenow DNA polymerase and ATP sulfurylase, were tested and found to work approximately two times faster at 37°C compared to at room temperature. The rate of the firefly luciferase reaction was also increased at higher temperature.

The use of osmolytes therefore opens new possibilities for luciferase-based assays, and especially firefly luciferase-based assays, at elevated temperatures. The method is particularly useful for increasing the speed and quality of DNA sequencing using the Pyrosequencing™ technology. This method has so far been limited for use at 28°C. By increasing the temperature to 37°C, it is possible to improve the quality of the sequence data; false signals due to unspecific priming, primer-dimers and loop- structures were decreased. In the presence of osmolytes the luciferase activity can be followed in real-time at 37°C. The application area for the method is broad and opens up possibilities to study many ATP coupled reactions at more optimal temperatures.

The best results were obtained with glycine betaine, which increased the available temperature span for luciferase-based assays with about 10 degrees Celsius. Moreover, glycine betaine may be used for prolonging and improving the storage of luciferase. Also, an advantage with glycine betaine is that is not as viscous as for example trehalose.

Due to viscosity reasons, it is preferable, in case osmolyte sugar solutions according to group (1) are used in the present invention, that they are used at a saturation below 50 %, more preferably below 5 % (50 % saturation of trehalose corresponds to 1M, and 100 % saturation corresponds to about 1.25 M (at the reaction conditions used in the examples of this disclosure)).

In one embodiment of the invention the activity of luciferase, or an analogue thereof showing luciferase-like activity, or apyrase is maintained at at least 10 %, preferably at least 30 %, more preferably at least 50 %, even more preferably at least 70 % and most preferably at least 90 % of its normal activity at a temperature in the interval from 32 to 40 °C, preferably from 36 to 38 °C.

Accordingly, included in the invention as examples of luciferase are all known lucifer- ases, such as luciferases from firefly (Phorinus pyralis) (EC 1.13.12.7), and bacteria. Examples of various luciferases are the North American firefly (Phorinus pyralis), the Japanese fireflies Luciola cruciata and Luciola lateralis and the Russian firefly Luciola mingelica. Further examples include Beneckea hanegi (marine bacterial luciferase, EC 1.14.14.3, 39- and 42-kDa heterodimer), Pyrophorus plagiophthalamus (click beetle), Pyrocelia miyako (firefly) Ragophthalamus ohbai (railroad worm), Pyrearinus ter- mitilluminans (click beetle), Phrixothrix hirtus (railroad worm), Phrixothrix vivianii, Hotaria parvula and Photuris pensilvanica. All these luciferases are possible to use in connection with the invention. Since luciferase substrates (luciferin) vary among luciferases, the optimal substrate and the optimal conditions must of course be found from case to case. The firefly luciferase Phorinus pyralis (EC 1.13.12.7) is preferred. Also included are analogues of luciferase showing luciferase-like activity. Such analogues may be variants of wild type luciferase comprising mutations, such as one or more substitutions, deletions and insertions, as long as the luciferase-like activity is maintained, i.e. as long as it has the capacity to catalyse the conversion of luciferin under the emission of light. Examples of known firefly luciferase mutations are E354 K, E354R, D357Y mutations in the region around residues 209-300 and 300-452, Thr217Ile, substitutions in amino acid 286, and Arg218, Lys 529, engineering of the C-terminus and substitution of cysteine residues. These are only examples of known mutations in the firefly luciferase. The luciferase analogues of the invention showing luciferase-like activity include analogues comprising one or more of these mutations as well as any other possible mutation or change, as long as the mutated variant shows luciferase-like activity. The luciferase-like activity is measured in accordance with the methods disclosed in the example section of this disclosure.

A preferred mutated variant of luciferase is obtainable from Lucigene, and comprises two mutations: E354R and D357Y. This variant is more temperature resistant than normal luciferase (see Example 6).

Also included in the invention are luciferases from all bioluminescent organisms. Representatives of these are bacteria (genera: Photobacterium, Vibrio, Xenorrhabdus); mushrooms (genera: Panus, Armillaria, Pleurotus); dinoflagellates (genera: Gonyau- lax, Pyrocystis, Noctiluca); cnidaria, such as jellyfish (Aequorea), hydroid (Obelia), sea pansy (Renilla); ctenophores (genera Mnemiopsis, Beroe); annelids, such as earthworms (Diplocardia), chaeopterid worm (Chaeopterus), sullid fireworm (Odonto- syllis), scale worm (Acholoe); molluscs, such as limpet (Latia), clam (Pholas), squid (Heteroteuthis); Crustacea, such as ostracod (Nargula, Cypridinia), shrimp (Meganyc- tiphanes), copepods and others (Gaussia), insects, such as coleopterids (beetles), such as firefly (Photinus, Photuris), click beetles (Pyrophorus), railroad worm (Phengodes, Phrixothrix), diptera (flies) (Arachnocampa); echinoderms, such as brittle stars (Ophiopsila); chordates, such as tunicates (Pyrosoma); and fishes, such as cartilaginous fishes (Isistius), bony fishes, such as ponyfish (Leiognathus), flashlight fish (Photoblepharon), angler fish (Cryptopsaras), midshipman (Porichthys) and midwater fishes (Cyclothone, Νeoscopelus, Tarletonbeania).

Examples of various bioluminescence assays comprise assays for enzymes, such as alcohol dehydrogenase, ATPase, β-D-Galactosidase, creatine kinase, enolase, isocitrate dehydrogenase and phosphoenolpyruvate kinase; assays for substrates and cofactors, such as L-alanine, androgens, ATP, bile acids, creatine phosphate, creatinine, dehy- droepiandrosterone sulfate, estrogens, ethanol, FAD, free fatty acids, glucose, L- glutamate, glutathione, lactate, myo-inositol, NAD, NADH, oxalate and pheromones. Also, rapid microbiological tests may be based on bioluminescent ATP-assays. For example such a test may be used for susceptibility testing, bacteriuria, activated sludge, detection of organisms and food testing. Moreover bioluminescence assays may be used for toxicity and mutagenicity testing, for immunoassays, protein blotting and DNA probe assays as well as for photographic assays. Also included are methods involving sequencing of RNA, such as the use of RNA-dependent polymerase, such as RT-polymerase. Preferably, the assay is an ATP-assay, more preferably a sequencing- by-synthesis reaction, especially the Pyrosequencing™-reaction. All these assays are included in the scope of the invention.

Apyrase is, like luciferase, a temperature sensitive enzyme that is used in the sequencing by synthesis reaction. In order for apyrase to be used at an increased temperature, the inventor have shown that the presence of an osmolyte is beneficial. Thus, in a preferred embodiment of the invention, at least one osmolyte is used to heat- stabilise apyrase during the enzymatic reaction.

In another preferred embodiment, both luciferase and apyrase is heat-stabilised. Moreover, in yet another embodiment, a mutated, heat-insensitive variant of luciferase may be used, together with apyrase, whereby apyrase is heat stabilised by at least one osmolyte. Thus, apyrase may be used in connection with a thermostable luciferase, such as a mutated variant of luciferase comprising the mutations E354R and D357Y.

In a second aspect the invention refers to a method for performing a sequencing-by- synthesis reaction, comprising the steps of:

(a) providing a reaction solution comprising sample nucleic acid, luciferase enzyme, and other necessary components;

(b) adding at least one osmolyte to the reaction solution in an amount necessary to heat stabilise the luciferase enzyme, so that the activity of the luciferase during the reaction is maintained at at least 10 %, preferably at least 30 %, more preferably at least 50 %, even more preferably at least 70 % and most preferably at least 90 % of its normal activity at a temperature in the interval from 32 to 40 °C, preferably from 36 to 38 °C;

(c) performing the reaction.

In a preferred embodiment apyrase is included in the reaction solution. Other necessary components are necessary enzymes, such as DNA-polymerase and sulphurylase, and reagents as outlined in the Example section of this disclosure.

Hereby, the sequencing-by-synthesis is possible to perform at a temperature that is optimal for the polymerase enzyme and the sulphurylase enzyme.

In a third aspect, the invention provides a composition comprising luciferase, or an analogue thereof showing luciferase-like activity, and/or apyrase, and at least one os- molyte, chosen from amino acids and amino acid derivatives, or methylated ammonium and sulfonium compounds, in solution or freeze-dried. This composition is used in a bioluminometric assay, as specified above, for heat-stabilizing luciferase (or its analogue) or apyrase, or both.

In a fourth aspect, the invention provides a kit for performing a Pyrosequencing reaction comprising in separate compartments at least one osmolyte chosen from amino acids and amino acid derivatives, or methylated ammonium and sulfonium compounds, and at least one of (i) a luciferase, or an analogue thereof showing luciferase- like activity, and (ii) apyrase, and optionally other necessary reagents and/or enzymes, such as ATP-sulfiirylase and polymerase (DNA or RNA). These other reagents and enzymes include those necessary for performing a Pyrosequencing™ reaction, such as necessary solvents and buffer systems (see Example section), nucleotides (A, C, G and T), and enzymes (ATP-sulfiirylase and DNA-polymerase). Hereby, luciferase (or its analogue) is heat-stabilised and the Pyrosequencing™ reaction is possible to perform at a temperature that is more suitable for other enzymes in the assay. In a preferred embodiment, the kit comprises at least one osmolyte chosen from amino acids and amino acid derivatives, or methylated ammonium and sulfonium compounds, and a luciferase, or an anlaogue thereof showing luciferase-like activity, and apyrase. In an even more preferred embodiment, the kit further comprises ATP-sulfiirylase and DNA-polymerase

Pyrosequencing™ is a sequencing method developed at the Royal Institute of Technology in Stockholm (Nyren, P. (2001) Method for sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation; Patent: US-B1-6258568, EP-B1-946752; Ronaghi, M., Uhlen, M., and Nyren, P. (1998) A sequencing method based on real-time pyrophosphate. Science 281, 363-365. Ronaghi et al.,1998, Alderborn et al.,2000). The method is based on "sequencing by synthesis" in which, in contrast to conventional Sanger sequencing, the nucleotides are added one by one during the sequencing reaction. An automated sequencer, the PSQ96™ instru- ment, has recently been launched by Pyrosequencing™ AB (Uppsala, Sweden). The principle of the Pyrosequencing™ reaction: A single stranded DNA fragment (optionally attached to a solid support), carrying an annealed sequencing primer acts as a template for the Pyrosequencing™ reaction. In the first two dispensations, substrate and enzyme mixes are added to the template. The enzyme mix consists of four different enzymes; DNA polymerase, ATP-sulfurylase, luciferase and apyrase. The nucleotides are sequentially added one by one according to a specified order dependent on the template and determined by the user. If the added nucleotide is matching the template, the DNA polymerase will incorporate it into the growing DNA strand. By this action, pyrophosphate, PP;, will be released. The ATP-Sulfurylase converts the PP_; into ATP, and the third enzyme, luciferase, transforms the ATP into a light signal. Following these reactions, the fourth enzyme, apyrase, will degrade the excess nucleotides and ATPs, and the template will at that point be ready for the next reaction cycle, i.e. another nucleotide addition. Since no PPj is released unless a nucleotide is incorporated, a light signal will be produced only when the correct nucleotide is incorporated. The PSQ 96 Instrument has been developed by Pyrosequencing AB (Uppsala, Sweden) in order to automate the sequencing reaction and to monitor the light release. The PSQ 96 Instrument software presents the results as peaks in a pyrogram™, where the height of the peaks corresponds to the number of nucleotides incorporated. Dedicated softwares have been developed for SNP analysis, sequencing of shorter DNA stretches (30-50 bases), and assessment of allele frequencies.

Compared to other techniques used for SNP analysis, for example hybridisation techniques, minisequencing, RFLP and SSCP, sequencing-by-synthesis presents some strong advantages. One is its ability to confirm that the correct SNP position is examined, by presenting the surrounding sequence and not only the polymorphic positions. Another advantage is the flexibility in primer design, i.e. the primer can be situated up to 15 nucleotides from the polymorphism, where it in minisequencing has to lay adjacent to the polymorphic site. Furthermore, it is a rapid technique, which is a benefit compared to SSCP and RFLP. The rapidity is also the main advantage compared to Sanger sequencing, when this technique is used for sequencing of shorter DNA stretches. Another advantage with sequencing-by-synthesis versus Sanger sequencing is that the first base directly after the extension primer can be read with high accuracy.

Pyrosequencing™ is a real time DNA sequencing method based on sequencing-by- synthesis. The method is proved to be a fast and accurate method for SNP (single nucleotide polymorphism) scoring, sequencing of shorter DNA stretches (signature tags), and assessment of allele frequencies. Pyrosequencing AB (Sweden) manufactures the PSQ™ 96 for genotyping, as well as dedicated softwares for automatic delivery of genotype and a quality assessment for each sample. A major advantage with those systems is the combination of accuracy, speed and ease-of-use.

Examples

Experimentals

Chemicals and solutions

ATP and dTTP were from New England Biolabs, Inc. (MA, USA). Exonuclease- deficient Klenow DNA polymerase, natural deoxynucleoside triphosphates (dNTP) and poly(dA)p(dT)12-18 (A260unit = 50 μg) were from Amersham Biosciences (Uppsala, Sweden). The homopolymeric template was supplied in 1 :1 ratio with equal number of As and Ts. Pure 2'-deoxyadenosine-5'-O'-(l-thiotriphosphate) Sp-isomer was from Biolog Life Science Institute (Bremen, Germany). D(+)trehalose dihydrate. anhydrous glycine betaine, L-proline, adenosine 5'-phosphosulfate (APS), polyvinyl- pyrrolidone (360;000), and apyrase were from Sigma Chemical Co. (St. Louis, MO, USA). Bovine serum albumin, D-luciferin and purified firefly luciferase were from BioThema AB (Dalaro, Sweden). Recombinant ATP sulfiirylase was produced according to an earlier described procedure (Karamohamed, S., Nilsson, J., Nourizad, K., Ronaghi, M., Pettersson, B., and Nyren, P. (1999) Production, purification, and lumi- nometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphos- phate sulfurylase expressed in Escherichia coli. Protein Expr. Purif. 15, 381-388). Trehalose and proline were dissolved in 0.1 M Tris-acetate (pH 7.75) to a final concentration of 1.25 M and 2.4 M, respectively (no pH-effect was observed). Glycine betaine was dissolved in 0.1 M Tris-acetate (pH 7.75) to a final concentration of 5 M (the pH shifted to 8.5 M and was adjusted to 7.75 with acetic acid). At room temperature, in 0.1 M Tris-acetate (pH 7.75), the trehalose, proline and glycine betaine solutions were saturated at final concentrations of 1.25 M, 2.4 M and 5.4 M. respectively. The pH of all buffers used in this study was measured at room temperature. For buffers used at higher temperatures (except for buffers used at 28°C) the pH was adjusted at room temperature to compensate for a pH decrease of 0.028 pH units per degree Celsius (Dawson, M.C., Elliott, D.C., Elliott, W.H., and Jones, K.M. (1986) in "Data for biochemical research". Oxford University Press).

Synthesis and purification of oligonucleotides

The oligonucleotides:

pUC- 1641UP (5 ^λ -CCCTCCCGTATCGTAGTTAT), pUC- 1843Do (5 ' - ACGTTAAGGGATTTTGGTC A),

PCR-AS-UP (5'-GCCTGGTGGTGGGTTCGAGCC),

PCR-AS-UP(B) (5 '-(biotin)-GCCTGGTGGTGGGTTCGAGCC),

SEQ-AS-D (5'-GGGCGCCCTAGGCACAGCTG-3'),

SEQ-AS-D(B) (5'-(biotin)-GGGCGCCCTAGGCACAGCTG-3'),

E3PN (5'-GTCGGAATTCGTCAGACTGGCCGTCGTTTTACAAC),

NUSPT (5'-GTAAAACGACGGCCAGT), and

TN-LOOP-SEQ (5'-GTG AGG CAC CTA TCT CAG CG) were synthesized and

HPLC purified by Interactiva (Ulm, Germany). Luciferase assay

The standard assay volume was 0.5 ml and contained the following components: 0.1 M Tris-acetate (pH 7.75); 0.5 mM EDTA: 5 mM magnesium acetate; 0.1 % bovine serum albumin; 0.4 mg/ml polyvinylpyrrolidone; 100 μg/ml D-luciferin; variable amounts of proline, trehalose or glycine betaine. After pre-heating of the buffer for 10 minutes, 0.4 μg purified luciferase and 0.5 μmol dithiothreitol (DTT) were added. The reaction was started by addition of 1 μl 10 μM ATP. The luminescence was measured using a LKB 1250 (proline and trehalose) or 1251 (glycine betaine) luminometer (Bio- Orbit Oy, Turku, Finland) connected to a potentiometric recorder. The temperature was controlled in the measuring chamber (LKB 1251 luminometer) within (+/-) 0.1 °C for the interval 20 to 45°C. The reaction conditions for the firefly luciferase reaction was optimized to give an almost constant light emission (decay < 1%/min) for concentrations of ATP up to 1 μM.

ATP sulfurylase assay

The ATP sulfurylase assay was performed as described earlier (Karamohamed, S., Ny-ren, P. (1999) Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach. Anal. Biochem. 271, 81-85). Briefly, 0.5 ml of the same reaction buffer as described above for the luciferase assay was used with the addition of 0.8 mmol glycine betaine; 125 nmol APS and 250 nmol inorganic pyrophosphate (PPj). The reaction was started (after preheating for 10 minutes and subsequent addition of 0.4 μg luciferase and 0.5 μmol DTT), by the addition of 95 μU recombinant ATP sulfurylase. The luminescence was measured as described above. The luminescence output was calibrated by addition of a known amount of ATP.

DNA polymerase assay The DNA polymerase assay was performed as described earlier (Nyren, P. (1987) Enzymatic method for continuous monitoring of DNA polymerase activity. Anal. Biochem. 167, 235-238). Briefly, 0.2 ml of the same reaction buffer as described above for the luciferase assay was used. The following components were added: 47 ng luciferase: 0.2 μmol DTT; 28 mU recombinant ATP sulfurylase: 2 nmol APS: 0.32 mmol glycine betaine; 2.5 mU exonuclease-deficient Klenow DNA polymerase; 20 mU poly(dA)p(dT)12-18. The solution was pre-heated for 10 minutes. The reaction was started (after calibration with 10 pmol ATP) by addition of dTTP to a final concentration of 10 μM. The luminescence was measured as described above.

In vitro amplification

One PCR reaction was performed on the plasmid pUC19 304- IS (New England Bio- labs, New Heaven. USA) using primer pair pUC-lC41UP/pUC-1843Do for amplification of a 222 bp fragment. Amplification was performed in a total volume of 50 μl containing: 20 ng pUC19 304- IS plasmid; 2 units Taq polymerase; 2 mM magnesium chloride; 0.2 mM dNTPs; and 10 pmol of each primer. The PCR was carried out in a PTC-200 PCR system (MJ Research Inc., Watertown, MA, USA). The thermocycler temperature program consisted of denaturation at 95°C for 45 sec, annealing at 50°C for 45 sec, and extension at 72°C for 1 min during 34 cycles. The PCR was initiated with a 45 sec activation step at 95°C and finished by a 5 min extension step at 72°C.

Amplification of the human glutathione peroxidase gene was performed by seminested PCR. The first PCR reaction was performed in a total volume of 50 μl, using 100 ng human genomic DNA, 0.15 mM dNTP, 1 unit Taq polymerase, 75 mM Tris-HCl (pH 9), 20 mM ammonium sulphate, 1.5 mM magnesium chloride. 0.01 % Tween 20, and 10 pmol of each primer (PCR-AS-UP and PCR-AS-D). The temperature cycle used, after 2 min denaturation at 94°C, consisted of: 94°C for 45 sec, 50°C for 45 sec. 72°C for 1 min (30 times). The product from the first PCR was diluted 100-fold and 1 μl was used as template for the second PCR and another 1 μl for the third PCR. The sec- ond PCR was performed with 5 pmol of each PCR primer (SEQ-AS-D and PCR-AS- UP(B1). Amplification was performed in a volume of 50 μl containing: 1 unit of Taq polymerase, PCR buffer (50 mM KC1, 10 mM Tris-HCI, 2 mM magnesium chloride, 0.1 % Tween 20) and 0.2 mM dNTPs. The thermal cycle condition for the inner PCR were as follow: 30 cycles at 94°C for 10 sec, 60°C for 20 sec. 72°C for 60 sec, ending with 10 min at 72°C. The third PCR was performed as the second PCR with the exception that the primer pair SEQ-ASD(B)/PCR-AS-UP was used.

Preparation of single-stranded DNA templates

Streptavidin-coated super paramagnetic beads (100 μg) (Dynabeads™ M280- Streptavidin, Dynal A.S., Oslo. Norway) were washed three times by washing buffer supplied from the manufacturer. The biotinylated PCR product (50 μl) was added to the washed beads and the solution was incubated for 30 min at room temperature. Nonbound DNA was removed by washing three times with 100 μl of washing buffer. NaOH (10 μl 0.15 M) was added to the beads and the solution was incubated for 3 min. Subsequently, single-stranded DNA (ssDNA) was obtained by removing the supernatant. After a washing step, the immobilized ssDNA was dissolved in 10 μL 0.1 M Tris-acetate buffer, pH 7.75. Hybridization of oligonucleotides to the respective template was carried out as described earlier (Ronaghi, M., Karamohamed, S., Petters- son, B., Uhlen, M., and Nyren, P. ( 1996) Realtime DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242, 84-89).

Pyrosequencing™

Analyses were performed at 22°C, 28°C or 37°C in a volume of 50 μl on an automated PSQTM96 System (Pyrosequencing AB, Uppsala, Sweden). Ten microliter of the PCR reaction product (template prepared by the strategy described above) with or without sequence primer was used for each assay. Alternatively, 2 pmols of E3PN NUSPT with or without a primer-dimer was used as template. Primed target DNA was added to the Pyrosequencing™ reaction mixture (final volume 50 μl), containing: 5 U exo- nuclease deficient (exo-) Klenow DNA polymerase; 50 mU apyrase. 1 μg purified luciferase; 28 mU purified recombinant ATP sulfurylase (12); 0.1 M Tris-acetate pH 7.75 for analyses performed at 22°C and 28°C, and 0.1 M Tris-acetate with a final concentration of 1.6 M glycine betaine pH 8.25 for analyses performed at 37°C; 0.5 mM EDTA; 5 mM magnesium acetate: 0.1% bovine serum albumin: 2 mM DTT; 5 μM APS; 0.4 mg/ml polyvinylpyrrolidone (360 000); and 100 μg/ml D-luciferin. The sequencing procedure was carried out by stepwise elongation of the primer strand upon sequential addition of the different dNT'Ps and pure 2'-deoxyadenosine-5'-O^,-(l- thiotriphosphate) Sp-isomer, with simultaneous degradation of nucleotides by apyrase.

Example 1

Effect of osmolytes on the firefly luciferase reaction at room temperature

Figure 1 shows the effect of trehalose, glycine betaine and proline on the firefly luciferase activity (measured at room temperature). Trehalose inhibited the luciferase activity negligible (less than 7% inhibition) at the maximum concentration tested (1 M). In the presence of 1.6 M glycine betaine 35% inhibition was observed, and at 2 M, 50% of the activity was lost. Proline had a stronger influence on the luciferase activity at room temperature: 50% inhibition was observed at a final concentration of 0.9 M and 80% at 1 .8 M.

Example 2

The optimum pH for firefly luciferase

The optimum pH for the firefly luciferase reaction was 7.75 (Fig. 2). The optimum pH was the same at 37°C as at room temperature ('22°C). Fifty-percent activity was observed at pH 6.75. It is worth noting that a change in temperature from room temperature to 37°C gave a change in pH of nearly 0.5 pH units for a 0.1 M Tris-acetate buffer (pH 7.75 at room temperature). This effect must be corrected for when reac- tions are performed at higher temperatures. The pH of all buffers used in this study was measured at room temperature. For buffers used at higher temperatures the pH was adjusted at room temperature to compensate for a pH decrease of 0.028 pH units per degree Celsius (13).

Example 3

Effect of osmolytes on the firefly luciferase reaction at elevated temperatures

Glycine betaine was found to have a strong stabilizing effect on the firefly luciferase reaction at 37°C (Fig. 3). The stabilization of the luciferase activity was concentration- dependent. In the presence of 1.6 M glycine betaine the enzyme was active for more than 60 minutes at 37°C (90% of the activity was left). In the absence of glycine betaine, 60% of the activity was lost within 10 minutes. Higher concentrations than 1.6 M of glycine betaine was not tested due to the inhibition observed at room temperature (Fig. 1). At higher temperatures, the effect of glycine betaine was less pronounced, although nearly 85% of the activity was left after 30 minutes at 40°C, and 40% at 42°C (Fig. 4). In the absence of glycine betaine, there was no activity left after 8 minutes at 42°C (not shown). When the temperature was increased from 22°C to 37°C, and the standard luciferin concentration was used (100 μg/ml), a 20% increase of the luciferase activity was observed (Table 1). However, if the luciferin concentration was increased to 500 μg/ml the activity increased with 166%, indicating a temperature effect on the K_M for luciferin.

TABLE 1

Effect of temperature on the activity of three different enzymes

Enzyme Rate

22°C 37°C

DNA polymerase ^l 100% 204%

ATP sulfurylase ² 100% 210%

Luciferase 100% 120% lOOμg / ml D-lucifeπn

Luciferase ⁴ 100% 166%

500μg / ml D-lucifeπn

Note. Experimental conditions were as described under Materials and Methods.

¹ 100% activity corresponds to 2.4 pmol Ppi/min.

¹ 100% activity corresponds to 18 pmol ATP/min

¹ 100% activity corresponds to a light output giving a signal of 120 mV

¹ 100% activity corresponds to a light output giving a signal of 218 mV.

Both proline and trehalose stabilised the luciferase activity at elevated temperatures (not shown). However, due to the inhibitory effect of proline on the luciferase activity (Fig. 1), and the high viscosity in the presence of trehalose, detailed studies of these osmolytes were not performed.

Example 4

Enzymatic assays at elevated temperatures

The stabilised luciferase was used for continuous detection of both ATP sulfurylase and DNA polymerase activity at 37°C. When the optimized luciferase system is used for analysis of a coupled enzymatic reaction, the resulting change is proportional to the rate of conversion of ATP. The ATP sulfurylase assay used was based on detection of the ATP formed in the ATP sulfurylase-catalyzed reaction by the firefly luciferase- system (14). At room temperature (22°C), the rate 18 ATP/min was detected, whereas at 37°C the rate increased to 38 ATP/min (Table 1).

The DNA polymerase assay used was based on detection of the PP; formed in the DNA polymerase-catalyzed reaction by a two-step process (15). In the first step, PPj was converted to ATP by the ATP sulfurylase, and in the second step, the ATP formed was detected by the firefly luciferase-system. The rate of the DNA polymerase- catalyzed reaction increased with more than 100% (from 2.4 to 4.9 PPj/min) when the temperature was increased from room temperature to 37°C (Table 1).

Example 5

Pyrosequencing™ at elevated temperatures

Traditionally, Pyrosequencing™ has been performed at 28°C due to the low thermostability of the firefly luciferase. However, for DNA sequencing it is an advantage to use higher temperatures to avoid disturbing background signals due to unspecific priming, self-priming and priming from primer-dimers.

Self-priming occur when the free 3 '-end of a single stranded DNA template binds to the template and form an extendable substrate for the polymerase. In figure 5, the effect of increasing the temperature from 28°C to 37°C on self-priming template in the presence of sequence primer is shown. Analysis, using Pyrosequencing™ technology, was performed on a 222 base-long single-stranded template in the presence of sequence primer at 28°C and 37°C.

At 28°C (Fig 5 A) signals are obtained from both specific priming of the sequence primer and unspecific self-priming (arrows) of the free 3'-end of the ssDNA template. At 37°C (Fig. 5B), the unspecific self-priming signals decreased (arrows) and the signals from the sequence primer were dominant. At the higher temperature, the self- binding effect is decreased, whereas the sequence primer still binds strongly. Self- priming will be less of a problem if a sequence primer that binds close to the 3'-end of the template is chosen.

In figure 6, the effect of increasing the temperature from 28°C to 37°C on a self- looping template in the absence of sequence primer is shown. Analysis was performed at 28°C and 37°C on the template that was used in the above-described experiment. The signals observed in figure 6A are due to unspecific self-priming of the free 3'-end of the ssDNA template. The arrows indicate the expected height of a signal from incorporation of one correct base if the sequence primer would have been present (see also Fig. 5). At 37°C (Fig. 6B), much lower signals were observed, indicating destabi- lization of the loop structure at the higher temperature.

Another problem that might occur is that the excess amount of sequence primer form primer-dimers. If a primer-dimer function as substrate for the polymerase, background signals that disturb the correct reading of the DNA sequence, might turn out. In figure 7, the effect of increasing the temperature from 22°C to 37°C on a single-stranded template in the presence of a primer-dimer is shown. Analysis was performed on both strands of a PCR-generated 320-base-long template in the presence of 5 pmol of respectively sequence primer. Both sequencing primers formed primer-dimers. The arrows indicate some of the background signals that were due to the primer-dimer. At higher temperatures both primer-dimers are destabilised and at 37°C only very low background signals are observed and the correct sequence can easily be read (Fig. 7C and D). The correct sequence is indicated above traces C and D.

Pyrosequencing™ data from the above-described primer-dimers, without template, is shown in figure 8. Pyrosequencing™ was performed on 50 pmol of SEQ-ASD (Fig. 8 A) and 10 pmol PCR-AS-UP (Fig. 8B), respectively. The experiments were performed at 28°C. Strong signals were observed from both primer-dimers. The symmetric nature of the signals is probably due to priming from both 3' ends of the primer- dimers. At 37°C, the signals were strongly decreased (not shown), indicating destabili- zation of both primer-dimers at the higher temperature.

To analyze an unknown primer for possible primer-dimer formation it is useful to use an internal control for evaluation of the signal-strength of the background signals. This is important for evaluation of the possibility that the background signals might disturb the correct sequence determination. In figure 9, the effect of increasing the temperature from 28°C to 37°C on a primed single-stranded template in the presence of a primer-dimer is shown. Analysis was performed on 2 pmol E3PN/NUSPT template in the absence (Fig. 9 A and B) and in the presence (Fig. 9C and D) of primer PCR-AS- UP (the primer used in Fig. 8B) at 28°C and 37°C. The correct sequence is indicated above the trace in figure 9A. The data clearly show, that the background-signals from this primer-dimer disturb the possibility to determine the correct sequence of the template if the sequencing is performed at 28°C (Fig. 9C), but not at 37°C (Fig. 9D).

Example 6

Effect of glycine betaine on apyrase activity

In this example, a mutated luciferase variant was used (from Lucigene, two mutations: E354R and D357Y), which variant is more heat-insensitive than normal luciferase. The experiment was performed at a pH of 7.75. Experimental conditions are, if nothing else is stated, analogous to the experiments described above.

Analysis, using Pyrosequencing™ technology, was performed on 2 pmol E3PN NUSPT template in the (a) absence and (b) presence of 1.6 M glycine betaine at 37°C. In the absence of glycine betaine, a continuous decrease of the apyrase activity was observed (a). The inhibition of apyrase is observed as a broadening of the peaks. In the presence of glycine betaine the apyrase activity was stabilised (b). The order of nucleotide addition was A, G, T, C. In the absence of glycine betaine, apyrase loses more than 50 % of its activity within an hour at 37°C.

Claims

Claims:

1. Use of at least one osmolyte to heat stabilise at least one enzyme that is used in an in-vitro bioluminometric assay, during the enzymatic reaction . 2. Use according to claim 1, wherein the enzyme is a luciferase, or an analogue thereof showing luciferase like activity.

3. Use according to claim 1 or 2, wherein the enzyme is apyrase.

4. Use according to claim 3, wherein apyrase is used in connection with a thermostable luciferase, such as a mutated variant of luciferase comprising the muta- tions E354R and D357Y.

5. Use according to claim 1-4, whereby the osmolyte is chosen from (1) sugars and polyhydric alcohols, (2) amino acids and amino acid derivatives, or (3) methylated ammonium and sulfonium compounds.

6. Use according to claim 5, whereby the osmolyte is chosen from the group com- prising: trehalose, floridoside, glycerol, pinitol, sorbitol and myo-inisitol, ala- nine, β-alanine, proline, ectoine, taurine, N_ε-acetyl-β-lysine, N_α-carbamoyl-L- glutamine-1 -amide, trimethylamine-N-oxide, glycerophosphoryl choline, proline betaine, β-alanine betaine, glycine betaine, choline-O-sulfate, homarine and di- methylsulfoniopropionate. 7. Use according to claim 6, wherein the osmolyte is chosen from the group comprising trimethylamine-N-oxide, glycerophosphoryl choline, proline betaine, β- alanine betaine, glycine betaine, choline-O-sulfate, homarine and dimethylsul- foniopropionate. 8. Use according to claim 7, wherein the osmolyte is glycine betaine. 9. Use according to claim 1-8, wherein the activity of luciferase, or an analogue thereof showing luciferase-like activity, or apyrase is maintained at at least 10 %, preferably at least 30 %, more preferably at least 50 %, even more preferably at least 70 % and most preferably at least 90 % of its normal activity at a temperature in the interval from 32 to 40 °C, preferably from 36 to 38 °C.

10. Use according to claim 1-9, wherein the luciferase, or analogue thereof showing luciferase activity, is chosen from North American firefly (Phorinus pyralis) (EC 1.13.12.7), Luciola cruciata (Japanese firefly), Luciola lateralis (Japanese firefly), Luciola mingelica (russian firefly), Beneckea hanegi (marine bacterial luciferase, EC 1.14.14.3), Pyrophorus plagiophthalamus (click beetle), Pyrocelia miyako

(firefly) Ragophthalamus ohbai (railroad worm), Pyrearinus termitilluminans (click beetle), Phrixothrix hirtus (railroad worm), Phrixothrix vivianii, Hotaria parvula and Photuris pensilvanica, and mutated variants thereof.

1 l.Use according to claim 10, whereby the luciferase is firefly luciferase Phorinus pyralis (EC 1.13.12.7).

12. Use according to any one of the preceding claims, wherein the in-vitro bioluminometric assay is an ATP-assay.

13. Use according to claim 12, wherein the ATP-assay is a sequencing-by-synthesis reaction. 14. Use according to claim 13, wherein the sequencing-by-synthesis reaction is a Pyrosequencing reaction.

15. Method for performing a sequencing-by-synthesis reaction, comprising the steps of:

(a) providing a reaction solution comprising sample nucleic acid, luciferase en- zyme, and other necessary components;

(b) adding at least one osmolyte to the reaction solution in an amount necessary to heat stabilise the luciferase enzyme, so that the activity of the luciferase during the reaction is maintained at at least 10 %, preferably at least 30 %, more preferably at least 50 %, even more preferably at least 70 % and most preferably at least 90 % of its normal activity at a temperature in the interval from 32 to 40 °C, preferably from 36 to 38 °C

(c) performing the reaction.

16. Method according to claim 15, wherein the reaction solution further comprises apyrase.

17. Composition comprising luciferase, or an analogue thereof showing luciferase- like activity, and/or apyrase, and at least one osmolyte chosen from amino acids and amino acid derivatives, or methylated ammonium and sulfonium compounds in solution and/or freeze-dried.

18. Kit for performing a Pyrosequencing reaction comprising in separate compartments at least one osmolyte chosen from amino acids and amino acid derivatives, or methylated ammonium and sulfonium compounds, and at least one of (i) a luciferase, or an analogue thereof showing luciferase-like activity, and (ii) apyrase, and optionally other necessary reagents and/or enzymes.