(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 22 April 2010 (22.04.2010) WO 2010/044101 Al
(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 9/90 (2006.0 1) C12Q 1/68 (2006.0 1) kind of national protection available): AE, AG, AL, AM, C12Q 1/18 (2006.01) GOlN 21/64 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C12Q 1/533 (2006.01) GOlN 33/68 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/IN2009/000561 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 8 October 2009 (08.10.2009) NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, (25) Filing Language: English TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 2159/MUM/2008 8 October 2008 (08. 10.2008) IN GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): V. B. TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, MEDICARE PVT. LTD. [IN/IN]; 141 Walchand Hirac- ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, hand Marg, Mumbai 400 001, Maharashtra (IN). MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventors; and ML, MR, NE, SN, TD, TG). (75) Inventors/Applicants (for US only): GUPTA, Munish- war, Nath [IN/IN]; Chemistry Department, Indian In sti Declarations under Rule 4.17: tute of Technology Delhi, Hauz Khas, New Delhi 110 016 — of inventorship (Rule 4Λ 7(iv)) (IN). SINGH, Pradeep, K. [IN/IN]; Chemistry Depart ment, Indian Institute of Technology Delhi, Hauz Khas, Published: New Delhi 110 016 (IN). RAGHAVA, Smita [IN/IN]; — with international search report (Art. 21(3)) Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016 (IN). — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of (74) Agents: SAVANGIKAR, Vasant, Anantrao et al; Kr amendments (Rule 48.2(h)) ishna & Saurastri Associates, 74/F, Venus, Worli Sea Face, Mumbai 400 018, Maharashtra (IN).
(54) Title: PURIFICATION AND ASSAY FOR TOPOISOMERASES AND USE OF THE ASSAY FOR SCREENING MODU LATORS OF TOPOISOMERASES (57) Abstract: This invention comprises a process of isolation and purification of Topoisomerases adsorbed on an immobilized metal affinity chromatography column charged with a metal ions by sequential elution by imidazole solution of different molar strengths. This invention also comprises a novel method of assay of Topoisomerase activity in presence or absence of a Topoiso- merase modulator and a kit for same wherein unreacted ScDNA separated from the product of topoisomerase action are removed using a chelating matrix charged with metal ions and fluorescence is reacted with a dye to result in a fluorescent DNA dye com plex, and measuring the florescence The assay may be performed in a multi-well plate for assay of Topoisomerase activity, for de tecting chemical agents for control of microorganisms or tumours or microbial spoilage of edible products by determining topoiso merase inhibiting or enhancing activity of a potential compound. The dye used may be a cyanine dye. TITLE
PURIFICATION AND ASSAY FOR TOPOISOMERASES AND USE OF THE
ASSAY FOR SCREENING MODULATORS OF TOPOISOMERASES.
TECHNICAL FIELD
This invention relates to isolation, -purification, and quantitative assay of DNA
topoisomerase and screening of topoisomerase modulators including the protein
CcdB using the assay
BACKGROUND OF INVENTION
DNA topoisomerases interconvert topoisomers of DNA. DNA topoisomerase I
(Tl) and DNA topoisomerase Il [(TII) DNA gyrase] are enzymes which regulate
the degree of supercoiling of intracellular DNA. There are other topoisomerases,
e.g., Ill and IV which are related to I and Il and have similar function. These
ubiquitous enzymes play a significant role in replication, transcription and
recombination of DNA [1]. These enzymes are now well established as the
targets for some of the antibiotics and anticancer drugs [2-5]. DNA topoisomerase I change the helical pitch of the genetic material by allowing
passage of a DNA single strand through a transient nick created in the
complementary strand of the double helix. Thus, a convenient assay for these enzymes for the purpose of screening antineoplastic drugs, kits for the assays
and methods for isolation and purification of topoisomerases have become areas for work that serve a current need. PRIOR ART
In some presently available methods for assay of enzyme activity of topoisomerases is measured in ε semiquantitative fashion by following the relaxation of supercoiled plasmid DNA (ScDNA) by agarose gel electrophoresis
[6]. U.S. patent no. 5,998,152 describes both solid phase and liquid phase assay formats for measuring the activity of a topoisomerase in the presence of a potential topoisomerase activity modulator. This patent provides high-throughput screening methods, compositions, kits and integrated systems for performing the assays. In general, the assays are performed by contacting a nucleic acid with a topoisomerase in an appropriate reaction mixture that also includes a potential modulator of topoisomerase activity, a denaturant is added to the reaction mixture, resulting in the stabilization of any covalent complexes that are present, the presence or absence of topoisomerase-nucleic acid complex is then detected to ascertain whether a modulator of topoisomerase activity was present in the reaction mixture.
WO2006/015369 deals with detection and continuous assay of topoisomerase activity spectroscopically wherein a fluorescent moiety is attached to the one strand of the duplex nucleic acid and fluorescent quencher is covalently attached to the complimentary strand of the duplex nucleic acid. The topoisomerase activity results in fluorescence from fluorescence moiety, which is measured spectroscopically. This principle is used to determine whether a compound is anti-viral, anti-cancer, antitrypanosome, anti-malarial, or antibacterial and the patent application also provides a kit for determining if a compound is a topoisomerase inhibitor comprising a duplex nucleic acid molecule with above mentioned properties, and instructions for use. In view of their biological importance, specifically in DNA replication and transcription, the enzymology of topoisomerases is an area of considerable interest. The purification of these enzymes has been a challenging task. For example, in case of Escherichia coli DNA gyrase [7] the subunits of the enzyme are separately isolated and the enzyme is obtained by reconstitution.
Topoisomerases can either be isolated and purified from natural source or they can be isolated by cloning in a variety of host cells followed by ov3r-expression in the cloned cells and isolation there from.
Topoisomerase I has often been purified by multistep purification protocol from various sources [8, 9], which often includes phosphocellulose/hydroxyapatite column chromatography followed by affinity chromatography on heparin affinity media [8, 9]. Topoisomerase I from a recombinant E. coli [10] has been purified by a shorter protocol using phosphocellulose chromatography.
Topoisomerase Il is a dimer of heterodimer AB. Often B subunit has been purified on a novobiocin or monoclonal antibody affinity column and ieconstituted with gyrase A subunit which is purified again by a multistep protocol which includes ion exchange chromatography and phosphocellulose chromatography
U].
In two cases, it has been possible to purify topoisomerase I and topoisomerase Il simultaneously [11,12]. Both protocols require fast protein liquid chromatography or high performance liquid chromatography.
Above mentioned prior art methods of isolating topoisomerases are expensive and cumbersome approaches. SUMMARY OF INVENTION:
In one embodiment this invention comprises a process of isolation and
purification of a Topoisomerase from a cell free extract from lysed cells
comprising the steps of (a) removing the nucleic acids, (b) adsorbing some
proteins comprising Topoisomerases selectively on an immobilized metal affinity
chromatography column charged with a metal ion, and (c) selectively and
sequential elution of at Topoisomerases. The said Topoisomerases illustrated
here are Topoisomerase I and Topoisomerase II. However, it is clear that it may
be possible to separate otherTopoisomerases too by this method. In the
illustrated embodiment of the process, the said metal ion is Zn2+ , however other
metal ions such as Cu2+ may also give good results. In another aspect of the said
process the said elution is done by using imidazole solution of different molar strengths selected such that each molar strength capable of dissolving one
Topoisomerase.
The process disclosed here of isolation and purification of a topoisomerase comprises steps of (a) lysing the bacterial cells with sonication, (b) centrifuging to get a crude lysate, (c) treating with streptomycin sulphate to remove nucleic acids, (d) loading the supernatant to an immobilized metal affinity chromatography column charged with Zn2+, (e) eluting with 25 mM imidazole to get Topoisomerase II, and (f) eluting with 75 mM imidazole to get
Topoisomerase.
This inventio i also comprises a method of assay of Topoisomerase activity further comprising the steps of (a) bringing topoisomerase in contact with a composition of super-coiled DNA free from relaxed form of DNA for a period of time sufficient to lead to transforming at least a portion of the said super-coiled- DNA into a relaxed DNA and optionally adding either a substance whose
Topoisomerase activity is to be detected or assayed, or a substance that is a known modulator of Topoisomerase activity whose concentration is to be assyed,
(b) separating unreacted ScDNA from the products produced by topoisomerase action, (c) adding a dye to the supernatant that intercalates in or binds with double strand DNA to result into a DNA_dye complex that emits fluorescence, and (d) mtasuring the florescence emitting from the DNA-dye complex. The said step of separating unreacted ScDNA from the products of produced by topoisomerase action is illustrated by applying the reaction mixture to a chelating matrix charged with metal ions for a period of time sufficient to adsorb all the relaxed form of DNA. The Toposiomerases illustrated in this invention of method of assay are Topoisomerase I and Topoisomerase II. However, any other
Topoisomerase can be assyed using this method. In one aspect of this invention, the chelating matrix is charged with metal ion Zn2 . However, use of Cu2+ has also given good results. In another aspect, the said dye is a cyanine dye that is commercially available as a 200 times concentrated solution under trademark
PicoGreen™, Use of this dye for the assay can be made even if the metal ion used for charging could be any metal ion other than Zn2+.
The method of this invention for assay of Toposomerases comprises the steps of
(a) making a reaction mixture containing 50 µL of NEB buffer 4 (50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 1 mM DTT and 100 µg/mL BSA, pH 7.9), 10 µL of the purified topoisomerase I, und 1 µg of a supercoiled DNA in a 96-well plate, (b) carrying the reaction at 37°C, (c> taking out aliquots of 5 µL containing 100 ng ScDNA at different time intervals from the reaction mixture and diluting with 150µL of Immobilized Metal Affinity
Chromatography (IMAC) binding buffer, (d) applying the solution to the
Znz+charged chelating matrix for 45 minutes at 3O0C, (e) mixing the supernatant with 50 µl_ of a cyanine dye commercially available with trademark PicoGreen™
after 1:200 dilution of the commercial concentrated dye solution in anhydrous
dimethylsulfoxide with MiIIi Q grade water, and (f) taking fluorescence intensity
measurements. The said time interval in this method may be 0, 10, 20, 30, 40,
50, 60, 70, and 80 min, although other time interval may also be used as required
or appropriate. The said Immobilized Metal Affinity Chromatography buffer in this
method comprises 20 mM HEPES (Dear Prof. Gupta: please give long form of
HEPES here), pH 7.0, containing 250 mM NaCI. The said modulator of
Topoisomerase activity used in the metod of assay may either be an inhibitor or
an enhancer of Topoisomerase activity.
The invention comprises a kit for assaying Topoisomerase activity comprising a
ScDNA, topoisomerase enzyme, buffer solutions, a solution of a dye that can bind with a double stranded DNA (ScDNA) to emit fluorescence and instructions to use, the said instructions to use. The said solution of a dye is solution of a cyanine dye that is commercially available with trademark PicoGreen™. Said instructions to use comprise application of a reaction mixture comprising above components and test substance to chelating matrix charged with metal ions, addition of the said dye to the supernatant and measuring the intensity of the flourescenece of the DNA-dye complex. The kit may be suppleiod with a plate having wells, to the wells of which a chelating matrix is applied that can be charged with metal ions, or a plate with wells to wells of which chelating matrix charged with metal ions is applied, or a plate with wells, a chelating matrix solution and metal ion solution. The plate may be a 96 well plate.
The method of invention comprises detecting chemical agents for control of microorganisms or tumours or microbial spoilage of edible products by determining topoisomerase inhibiting or enhancing activity of a potential compound by using assay of Topoisomerase, by methiod of this invention comprising the steps of (a) bringing topoisomerase in contact with a composition
of super-coiled DNA free from relaxed form of DNA for a period of time sufficient
to lead to transforming at least a portion of the said super-coiled DNA into a
relaxed DNA and optionally adding either a substance whose Topoisomerase
activity is to be detected or assayed, or a substance that is a known modulator of
Topoisomerase activity whose concentration is to be assyed, (b) separating
unreacted ScDNA from the products produced by topoisomerase action, (c)
adding a dye to the supernatant that intercalates in or binds with double strand
DNA to ' result into a DNA_dye complex that emits fluorescence, and (d)
measuring the florescence emitting from the DNA-dye complex. The invention is illustrated with dye that is commercially available under tradename PicoGreen™.
The said microorganisms comprise viruses, fungi, bacteria and trypanosoma; and the said edible products comprises a food, a fruit, a vegetable or a natural product susceptible to microbial spoilage.
DETAILED DESCRIPTION OF INVENTION
This invention provides novel methods that are simpler to perform and less costly for assay of topoisomerases and for testing and quantitating topoisomerase modulating activity of compounds that are potential modulators of topoisomerases; and isolation and purification of topoisomerases.
This invention a so provides use of immobilized metal-ion affinity chromatography
(IMAC) directly 'or isolation and purification of both topoisomerases I and Il in a single protocol.
In ono embodiment of this invention, we describe a simple, convenient and quantitative assay for DNA topoisomerase based upon steady-state fluorescence measurements of PG-ScDNA (PicoGreen-supercoiled DNA) complexes. Upon reaction with DNA topoisomerase I the amount of ScDNA available decreases which can be quantified by adding PG after all other forms of DNA have been removed by IMAC. The assay can be carried out in multi-well pla es.
In another embodiment of this invention, we describe a process of isolation of topoisomerases from lysed cells comprising removing the nucleic acid as a precipitate, adsorbing the topoisomerases selectively on immobilized metal affinity chromatography column charged with a suitable metal ion including Zn2+ and using eluent suitable for selective elution of the topoisomerases sequentially, such as using 25 mM imidazole to elute out topoisomerase Il followed by elution with 75 mM imidazole to elute out topoisomerase I. By making appropriate variations in the eluent and the concentration of imidazole or an alternative eluent
(including a solution of a different pH), the same method possibly would serve as method for isolation and purification of other topoisomerases too.
In the following are described experiments conducted that serve as non limiting illustrations of how the invention is performed. Any modifications or variations in the parameters including but not limited to organisms used, enzymes isolated, chemicals and their concentrations used, isolation and purification steps used are merely illustrative and any equivalents of them obvious to a person skilled in the art and capable of achieving the same objective may be used in their place and they shall also be considered as included in the scope / cor tent of this specification.
Murphy et al [13] showed that RNA and damaged plasmid- DNA are effectively removed by Cu (ll)-charged IDA Sepharose (IMAC) without loss of closed circular plasmids, presumably through interactions with exposed single-stranded regions. Their work was aimed primarily at removal of damaged DNA from cell
lysates, which was claimed to be difficult to achieve by other methods.
The unsymmetric monomethine cyanine dye PicoGreen™ (PG), which has been
used here to detect changes in supercoiling of ScDNA, displays a high
fluorescence emission upon intercalation in DNA. This DNA-dye interaction has
found quite a few applications. Ahn et al. [14] as early as 1996 used this for
quantitation of DNA in pre- and post-PCR samples and showed that as little as
0.25 ng of DNA could be detected. Levy et al. [15] used this to estimate the
ScDNA content in plasmid preparations. Their method exploited reversible
denaturation of ScDNA upon heating at 95 0C. Quite a few studies have tried to
determine the ratio of single-to-double stranded DNA in the samples using the
Picogreen, which is a unsymmetric monomethine cyanine dye is commercially
available as a has CAS number 177571-06-1 ε nd no description on that. It is
available commercially under trademark PicoGreen™ as a concentrated dye
solution in anhydrous dimethylsulfoxide, and is advised to be used in 1:200
dilution.
In a search conducted on ChEBI database, PicoGreen is described as a fluorochrome that selectively binds double-stranded DNA and has characteristics similar to that of SYBR-Green I. Its SMILES are given as
[H]C(=C1C=C(N(CCCN(C)C)CCCN(C)C)N(c2ccccc2)c2ccccc12)c1sc2ccccc2[n+
]1C with a formula of C34H42N5S, charge of +1, mass of 552.79600, IPUAC names of 2-{[2-{bis[3-(dimethylamino)propyl]amino}-1-phenylquinolin-4(1/-/)- ylidene]methyl}-3-methyl-1,3-benzothiazol-3-ium and 2-{bis[3- (dimethylamino)propyl]amino}-4-[(3-methyl-1,3-ben2othiazol-2(3H)- ylidene)methyl]-1-phenylquinolinium with a synonym 2-[ Λ/-bis-(3- dimethylaminopropyl)-aminol-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methyϊ idene]-1-phenyl-quinolinium. This information is available on following link: http://www.ebi.ac.uk/chebi/searchld.do;isessionid=9D0C75EB22F7A1379EA857
9FF8B89337?chebild^CHEBI%3A51467
SHORT DESCRIPTION OF FIGURES AND LEGENDS
Fig.1. Schematic outline of the microtiter plate-based assay of DNA topoisomerase I
Fig. 2. Determination of the time required for exposure of nucleic acid with metal- charged streamline™ chelating matrix. (A) The total nucleic acid preparation
(150 ng) from boiling lysate in IMAC binding buffer (20 mM HEPES, pH 7.0, containing 250 mM NaCI) was applied to streamline™ chelating matrix (20 µL) charged with Cu (II), Zn (II) and Ni (II) separately in a 96-well plate at 3 O0C.
Supernatants (150 µL) at different time intervals were mixed with 50 µL of
PicoGreen (after 1:200 dilution of commercial solution of the dye with MiIIi Q grade water) for fluorescence intensity measurements. All the readings were taken in sets of four and difference in individual reading in the sets was less than
5%. Controls to measure PicoGreen fluorescence and buffer contributions were run and subtracted. (B) Agarose gel showing separation of ScDNA by Zinc (II)-
IMAC. Lane 1, nucleic acid preparation; Lane 2, supernatant obtained from Zn
(II) charged streamline™ chelating matrix after 45 min incubation at 3O0C.
Fig. 3. Time course of the fluorimetric topoisomerase assay. (A) The reaction mixture contained 50 µL of NEB buffer 4 (50. mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 1 mM DTT and 100 µg/mL BSA, pH 7.9), 2
U of topoisomerase I, and 1 µg , of ScDNA, either pTYB12 or pGEM ®-T Easy in a
96-well plate. The reaction was carried out at 37°C. Aliquots (5 µl_ containing 100 ng ScDNA) were taken out at different time intervals (0, 10, 20, 30, 40, 50, 60,
70, and 80 min) from the reaction mixture and diluted with 150µL of IMAC binding buffer (this was same as equilibrating buffer A described in the methods section).
The solution was then applied to the Zn (!') charged streamline™ matrix for 45 min at 3O0C. The supernatant (150 µL) wa mixed with 50 µl_ of PicoGreen (after
1:200 dilution of commercial solution of the dye with MiIIi Q grade water) for fluorescence intensity measurements. All the readings were taken in sets of four and difference in individual reading in the sets was less than 5%. Fluorescence of PicoGreen and buffer contributions were subtracted. The inset shows minimum amount of supercoiled DNA (as a substrate) and enzyme that could be used in the fluorimetric topoisomerase I assay. These experiments were done keeping the ratio of substrate (in terms of amounts in ng) to enzyme constant (as in the main figure). The enzyme units used were 0.04 [with 20 ng ScDNA (■)],
0.02 [with 10 ng ScDNA (A)] and 0.01 [with 5 ng ScDNA (D)]. (B) Agarose gel showing the supernatants containing ScDNA (pTYB12) obtained from Zn (II)-
IMAC at different time periods. Lane 1, 0 min; lane 2, 40 min; lane 3, 60 min.
Fig. 4. Effect of norfloxacin on topoisomerase I activity. The reaction mixtures contained 50 µL of NEB buffer 4 (50 mM potassium acetate, 20 mM Tris acetate,
10 mM magnesium acetate, 1 mM DTT and 100 µg/mL BSA, pH 7.9), 2 U of topoisomerase I, and 1 µg of ScDNA (pTYB12) and two different concentrations of norfloxacin (50 µg/mL, A and 150 µg/mL, α) in a 96-well plate. Control assay
( ) was carried out in absence of norfloxacin. The reaction was carried out at
37°C. Aliquots (5 µL containing 100 ng ScDNA) were taken out at different time acetate, 10 mM magnesium acetate, 1 mM DTT and 100 µg/mL BSA, pH 7.9), 2
U of topoisomerase I, and 1 µg of ScDNA, either pTYB12 or pGEM ®-T Easy in a
96-well plate. The reaction was carried out at 37°C. Aliquots (5 µL containing 100 ng ScDNA) were taken out at different time intervals (0, 10, 20, 30, 40, 50, 60,
70, and 80 min) from the reaction mixture and diluted with 150µL of IMAC binding buffer (this was same as equilibrating buffer A described in the methods section).
The solution was then applied to the Zn (II) charged streamline™ matrix for 45 min at 3O0C. The supernatant (150 µL) was mixed with 50 µL of PicoGreen (after
1:200 dilution of commercial solution of the dye with MiIIi Q grade water) for fluorescence intensity measurements. All the readings were taken in sets of four and difference in individual reading in the sets was less than 5%. Fluorescence of PicoGreen and buffer contributions were subtracted. The inset shows minimum amount of supercoiled DNA (as a substrate) and enzyme that could be used in the fluorimetric topoisomerase I assay. These experiments were done keeping the ratio of substrate (in teπn s of amounts in ng) to enzyme constant (as in the main figure). The enzyme units used were 0.04 [with 20 ng ScDNA (■)],
0.02 [with 10 ng ScDNA (A)] and 0 0 1 Iwith 5 ng ScDNA (D)]. (B) Agarose gel showing the supernatants containing ScDNA (pTYB12) obtained from Zn (II)-
IMAC at different time periods. Lane 1, 0 min; lane 2, 40 min; lane 3, 60 min.
Fig. 4. Effect of norfloxacin on topoisomerase I activity. The reaction mixtures contained 50 µL of NEB buffer 4 (50 mM potassium acetate, 20 mM Tris acetate,
10 mM magnesium acetate, 1 mM DTT and 100 µg/mL BSA, pH 7.9), 2 U of topoisomerase I, and 1 µg of ScDNA (pTYB12) and two different concentrations of norfloxacin (50 µg/mL, and 150 µg/mL, ■) in a 96-well plate. Control assay
( ) was carried out in absence of norfloxacin. The reaction was carried out at
37°C. Aliquots (5 µL containing 100 ng ScDNA) were taken out at different time EXPERIMENTS ON ASSAY OF TOPOISOMERASE
MATERIALS USED IN ILLUSTRATIVE EXPERIMENTS
PicoGreen™ (a product of Molecular Probes, Eugene, OR) was a kind gift from
Dr. Narayanasamy (The Centre for Genomic Application, New Delhi, India).
Streamline™ chelating matrix was procured from Amersham Pharmacia Biotech
AB, Uppsala, Sweden. Topoisomerase I from E. coli alongwith NEB buffer 4 and the plasmid pTYB12 were obtained from New England Biolabs, Beverly, USA.
The plasmid pGEM®-T Easy (Promega, Madison, Wl) was a kind gift from Dr. S.
Baskar Singh (Department of Biophysics, All India Institute of Medical Sciences,
New Delhi, India). Agarose was obtained from Sigma Chemical Co.. St. Louis,
USA. All other chemicals were of analytical grade.
PLASMID AND BACTERIAL GROWTH CONDITIONS
Large scale production of the plasmids was carried out by the cell culture of E. coli DH5α transformed with plasmids pTYB12 or pGEM®-T. Growth was carried out at 37°C in a shaking culture flask at 200 rpm with 1 litre of LB media (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCI) supplemented with 50 µg/mL ampicillin. Cells were harvested by centrifugation at 5000χg for 10 min at 4°C after Aβoo of approximately 4 was reached [18]..
CELL LYSIS AND PURIFICATION F ScDNA
Cells were lysed by boiling method following the protocol of Holmes and Quigley
[18]. Total nucleic acid was precipitated by 1/10 volume of 2.5 M sodium acetate
(pH 5.2) and 7/10 volume of isopropanol. After vortexing and incubation at room temperature for 5 min, the nucleic acid pellet was recovered by centrifugation at
12000χg for 5 min at 4°C. The isolated nucleic acid was washed with 1 mL of 70 % ethanol and centrifuged at 12000χ g for 2 min at 4°C. Finally the pellet was
dissolved in IMAC binding buffer [20 mM HEPES (N-2-hydroxy ethylpiperazine-
N'-2-ethanesulfonic acid), pH 7.0, containing 250 mM NaCI]. The ScDNA was
purified by the method as follows: total nucleic acid (0.75 µg) was applied to 100
µL of streamline™ chelating matrix charged with Cu (II) and incubated at 30+
0C for 1 h. The supernatant was collected and subjected to 0.7 % agarose gel
electrophoresis. Ethidium bromide (0.5 µg/mL) staining was used to analyze the
purity of isolated ScDNA.
MEASUREMENT OF DNA
DNA concentration was determined by measuring the sample absorbance at 260
nm with A o of 1.0 corresponding to 50 µg/mL of double stranded DNA in the
sample in a 10 mm path length cuvette. The 260 nm 28o nmwas also measured as
an indicator of sample DNA purity.
CHARGING OF STREAMLINE™ CHELATING MATRIX
IDA chelated streamline™ matrix was washed with water and equilibrated with
buffer A (20 mM HEPES, pH 7.0. containing 250 mM NaCI) and then incubated
with 50 mM metal sulfate solution in equilibration buffer A for 30 min at 25 0C to
immobilize metal ion on matrix [19].
AGAROSE GEL ELECTROPHORESIS
Horizontal gel electrophoresis -was performed using 0.7% agarose gel [20]. The gel was stained with ethidium bromide (0.5 µg/mL) and visualized on GelDoc
Mega-V 4.00 gel documentation system (Biosystematica, UK). INSTRUMENTATION FOR FLUORESCENCE INTENSITY MEASUREMENTS
Fluorescence intensity values were acquired using a BMG FLUOstar Optima multimode plate reader with accompanying BMG FLUOstar Optima Version 1.20-
0 software (BMG LabTechnologies, Durham, NC USA). Wavelengths of 485 and
520 nm were taken for fluorescence excitation and emission acquisition, respectively. Each well of the 96-well fluorescent plate was excited with three flashes per measurement.
The outline of the protocol for the assay of topoisomerase I is shown in Fig 1. A key step in the assay was separation of unreacted ScDNA from the products
(various relaxed forms of DNA produced by topoisomerase action). It was found that treatment with IMAC matrix decreased the fluorescence over a period of 1 h to a constant value (Fig 2A). The matrix charged with Cu (II) and Zn (II) gave better results. The analysis of the DNA sample before IMAC treatment and after
45 min exposure to IMAC matrix by agarose gel electrophoresis is shown in Fig
2B. It was seen that IMAC treatment with Zn (II) charged matrix completely removed all other forms of DNA and only ScDNA was left unbound in the supernatant. The optimization of this contact time to achieve complete removal of relaxed forms of plasmid DNA was a critical step in the design of this assay. The unreacted ScDNA could be then quantified as PG-DNA complex by fluorescence emission measurements. Fig 3 shows the time course of the reaction of DNA topoisc merase I with two plasmid preparations. It is worth noting that the reaction with plasmid with smaller s\±e (3 kb) was completed earlier (within 30 min) as compared to the reaction when large plasmid (7.4 kb) was used as the substrate.
In order to evaluate the sensitivity of this assay, the assay was repeated with much lower concentration of ScDNA by keeping the ratio of substrate (in terms of amounts in ng) to enzyme (in terms of units as given by the enzyme vendor, New
England Biolabs) constant. It was seen that the assay could be- carried out with
as little as 5 ng DNA and 0.01. U of topoisomerase I (inset of Fig 3A). It may be
mentioned that the conventional assay (using agarose gel electrophoresis) is
carried out with 500 ng DNA and about 4 U of topoisomerase [6].
The inhibition of DNA topoisomerases by various drugs is of considerable clinical
importance and has been extensively investigated [2,3,5]. Among other drugs,
antibiotic norfloxacin has been reported to inhibit E. coli DNA topoisomerase I
activity [9]. Fig 4 shows the time course of DNA topoisomerase I reaction with
pTYB12 ScDNA in the presence of two different concentrations of norfloxacin. It
is seen that the present assay can be usefully employed for screening chemical
compounds as possible modulatiors (inhibitors or enhancers) of DNA
topoisomerase activity.
While the assay described here has been initially developed for DNA
topoisomerase I, it was shown to be effective for topoisomerase Il too when
topoisomerse Il was assayed during its isolation protocol and in experiments with
inhibitor of topoisomerase Il shown in Fig 8; and in principle it should be useful
for all DNA topoisomerases. Also, PicoGreen™ here has been used as it shows
high fluorescence emission enhancement upon binding to DNA. The assay can
be adapted to any other dye serving the purpose of binding with and detection of
DNA. The sensitivity of the assay, of course, would vary depending upon the quantum yield of the emission fluorescence by the DNA-dye complex. It is a common practice to- define an international unit for enzymes by specifying rate of product formation (or substrate disappearance) with a specific substrate under specified conditions. In the case of DNA topoisomerases, different workers use whatever ScDNA preparations are available to carry out the gel electrophoresis assay. With the availability of this quantitative fluorescence- based assay, it
should be possible to define the unit of DNA topoisomerase as rate of decrease
of fluorescence with an easily available ScDNA preparation. This should also
facilitate better comparison of results reported by different workers. For example,
with 10 ng of pTYB12, one unit can be defined as the amount of enzyme activity,
which shows decrease of 2.5 fluorescence intensity (arbitrary units) per minute.
Also, this multi-well assay can be easily adapted to a robotic platform, which
should facilitate high throughput screening of various potential drugs for human
disea'ses [2,21].
METHOD FOR SCREENING AND DETERMINING ACTIVITY OF POTENTIAL
TOPOISOMERASE MODULATORS
Method of determining topoisomerase inhibiting or enhancing activity of potential
compounds shall offer chemical agents for control of micro-organisms including viruses, fungi, bacteria, trypanosoma, and tumours and a Kit for screening such
compounds. This invention makes it possible to screen all potential compounds that could -have topoisomerase modulating (inhibiting/enhancing/stimulating)
activity. Such compounds could be useful in controlling micro-organisms
including viruses, fungi, bacteria, trypanosoma, and tumours and the diseases, disorders or undesirable results caused from them including microbial spoilage of foods and fruits and vegetables in use and storage. A con- pound that decreases
topoisomerase activity when added to a ScDNA in a solution conducive for action
of topoisomerase on the ScDNA shall be a potential in vivo inhibitor of topoisomerase with a potential application for preventing disease or disorder
arising from the target activity. This method also opens up a scope for a kit that
may comprise a topoisomerase, a ScDNA, buffers solutions and pico-green solution (or other suitable intercalating dye) and instructions for use. The contents of the kit may vary as per the perceptions of need of a flexibility to user, such as option of using a topoisomerase of his choice, or a intercalating dye of his choice.
METHOD TO DETECT AND/OR ASSAY THE CONTENT OF MODULATORS
OF TOPOISOMERASES
As a converse of detecting or assaying the topoisomerases, the methods of this invention shall also be useful to detect presence of as well as assaying the content of any known modulator of topoisomerase by comparing the activity of the test sample with standard one. Thus, the assay of this invention can also be used to estimate/detect CcdB.
PROCESS FOR ISOLATION AND PURIFICATION OF TOPOISOMERASES
Fig 5 outlines the flow sheet of the purification process.
The E coli cells were sonicated on an ice bath in 6 cycles of 15 sec, at 30% of the maximum power (Sonics, model VC-505), with a 30 sec cooling time between cycles. The crude lysate obtained as supernatant (after centrifugation at 12000χg for 20 min at 4°C) was treated with 1 % (wv~1 ) streptomycin sulfate to remove nucleic acids. The supernatant containing various proteins /enzymes present in
E. coli were loaded on an IMAC column charged with Zn2+. The binding stage was selective and only 25% of the loaded proteins bound to the column. The unbound proteins (75 %) did not contain Tl and TII activity. The bound protein was eluted with stepwise elution with imidazole. The elution with 25 mM imidazole contained TII and Tl was eluted with 75 mM imidazole. TII was further purified by a second IMAC step using a stepwise elution by imidazole (10-30 mM) (Fig. 6). This step can be inbuilt in the first chromatography step itself. FLUORIMETRIC TOPOISOMERASE I ASSAY
The reaction mixture contained 50 µt of NEB buffer 4 (50 mM potassium acetate,
20 mM Tris acetate, 1C mM magnesium acetate, 1 mM DTT and 100 µg/mL BSA, pH 7.9), 10 µL of the purified topoisomerase I, and 1 µg of ScDNA, pTYB12 in a
96-well plate. The reaction was carried out at 37°C. Aliquots (5 µL containing 100 ng ScDNA) were taken out at different time intervals (0, 10, 20, 30, 40, 50, 60,
70, and 80 min) from the reaction mixture and diluted with 150µL of IMAC binding buffer. The solution was then applied to the Zn (II) charged streamline™ matrix for 45 min at 3O0C. The supernatant (150 µL) was mixed with 50 µL of
PicoGreen™ (after 1:200 dilution of commercial solution of the dye with MiIIi Q grade water) for fluorescence intensity measurements.
FLUORIMETRIC TOPOISOMERASE Il ASSAY
The reaction mixture contained 50 µL of NEB buffer (35 mM Tris-HCI, 24 mM
KCI, 4 mM magnesium chloride, 2 mM DTT, 5 mM spermidine, 6.5% glycerol and
100 µg/mL BSA, pH 7.5), 10 µL of purified topoisomerase II, and 1 µg of ScDNA, pTYB12 in a 96-well plate. The reaction was carried out at 37°C. Aliquots (5 µL containing 100 ng ScDNA) were taken out at different time intervals (0, 10, 20,
30, 40, 50, 60, 70, and 80 min) from the reaction mixture and diluted with 150µL of IMAC binding buffer. The solution was then applied to the Zn (II) charged streamline™ matrix for Ai> min at 3O0C. The supernatant (150 µL) was mixed with
50 µL of PicoGreen™ (alter 1:200 dilution of commercial solution of the dye with
MiIIi Q grade water) for fluorescence intensity measuremen s.
SDS-PAGE analysis is shown in Fig. 7. Fig. 7A (lane 6) shows single band of Tl.
Fig. 7B (lane 2) shows a broad band of TII around 95 kDa region of the two subunits of the protein A and B, molecular weight of 97 kDa and 90 kDa, respectively. As these are close, they did not resolve on SDS-PAGE.
Inhibition assay of opoisomerase with known inhibitor CcdB (controller of cell division or death B protein) for topoisomerase Il and Norfloxacin for topoisomerase I in the eluted fractions were described in fig. 8.
CcdB is a homodimeric protein encoded by F plasmid in E. coli. The protein is a known inhibitor of DNA gyrase and is a potent cytotoxin in E. coli.
REFERENCES
[1] J.C. Wang, DNA topoisomerases, Annu. Rev. Biochem. 65 (1996) 635-
692.
[2] F. Cortes, N. Pastor, S. Mateos, I. ϋ ominguez, Topoisomerase inhibitors as therapeutic weapons, Expert Opin. Therap. Pat. 17 (2007) 521-532.
[3] S.J. Froelich-Ammon, N. Osheroff, Topoisomerase poisons: Harnessing the dark side of enzyme mechanism, J. Biol. Chem. 270 (1995) 21429-21432.
[4] B. Montaner, W . Astillo-Avila, M. Martinell, R. ό llinger, J. Aymami, E.
Giralt, R. Perez-Tomas, DNA interaction and dual topoisomerase I and Il inhibition properties of the anti-tumor drug prodigiosin, Toxicol. Sci. 85 (2005)
870-879.
[5] L. Lotito, F. Feiri, A. Russo, G. Capranico, DNA topoisomerase I as a transcription protein and a lethal cellular toxin, Ital. J. B:ochem. 5& (2007) 122-
129.
[6] C. Bailly, DNA relaxation and cleavage assays to study topoisomerase I inhibitors, Methods Enzymol. 340 (2001) 610-623. [7] V. Higgins, CL. Peebles, A. Sugino, N.R. Cozzareli, Purification of sυbunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity,
Proc. Natl Acad. Sci. 75 (1978) 1773-1777.
[8] I. Alkorta, C. Park, J. Kong, C. Garbisu, M. Albert!, N. Pon, J.E. Hearst,
Rhodobacter capsυlatυs DNA Topoisomerase I: Purification and
Characterization, Arch. Biochem. Biophys. 362 (1999) 123-130.
[9] T. Bhaduri, V . Nagaraja, DNA topoisomerase I from Mycobacterium smegmatis, Ind. J. Biochem. Biophys. 3 1 (1994) 339-343.
[10] T. Mizushima, S. Natori, K. Sekimizu, Inhibition of Escherichia coli DNA topoisomerase I activity by phospholipids, Biochem. J. 285 (1992) 503-506.
[11] Y. Onishi, Y. Azuma, H. Kizaki, An assav method for DNA topoisomerase activity based on separation of relaxed DNA from supercoiled DNA using high- performance liquid chromatography, Anal. Biochem. 210 (1993) 63-68.
[12] U. Straufeld, A. Richter, Simultaneous purification of DNA topoisomerase I and Il from eukaryotic cells, Prep. Biochem. 19 (1989) 37-48.
[13] J.C. Murphy, D.L Jewell, K.I. White, G.E. Fox, R.C. Willson, Nucleic acid separations utilizing immobilized metal affinity chromatography, Biotechnol. Prog.
19 (2003) 982-986.
[14] S.J. Ahn, J. Costa, J.R. Emanuel, PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR, Nucl. Acids Res. 24 (1996)
2623-2625. [15] M.S. Levy, P. Lotfian, R. O'Kennedy, M.Y. Lo-Yim, P.A. Shamlou,
Quantitation of supercoiled circular content in plasmid DNA soiutions using a fluorescence-based method, Nucl. Acids Res. 28 (2000) 57.
[16} G. Cosa, K.-S. Focsaneanu, J.R.N. McLean, J.P. McNamee, J.C.
Scaiano, Photochemical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution, Photochem. Photobiol.
(2001) 585-599.
[17] L. Beach, C. Schweitzer, J.C. Scaiano, Direct determination of single-to- double stranded DNA ratio in solution using steady-state fluorescence measurements, Org. Biomol. Chem. 1 (2003) 450-451.
[18] D.S. Holmes, M. Quigley, A rapid boiling method for the preparation of bacterial plasmids, Anal. Biochem. 114 (1981) 193-197.
[19] R.H. Clemmitt, H.A. Chase, Immobilised metal affinity chromatography of
β-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption, J. Chromatogr. A 874 (2000) 27-43.
[20] J. Sambrook, E.F. Fritsch, T. Maniatis, T. (Eds.), Molecular cloning: A
Laboratory Manuel, 2nd ed., Cold Spring Harbour Laboratory Press, New York,
1989.
[21] J.A. Holden, DNA topoisomerases as anticancer drug targets: from the laboratory to the clinic, Curr. Med. Chem. Anticancer Agents, 1 (2001) 1-25. CLAIMS
1. A process of isolation and purification of a Topoisomerase from a cell free
extract from lysed cells comprising the steps of:
a. removing the nucleic acids,
b. adsorbing some proteins comprising Topoisomerases selectively
on an immobilized metal affinity chromatography column charged
with a metal ion, and
c. selective and sequential elution of at least Topoisomerase I and
Topoisomerase II.
2. A process of claim 1 wherein the said metal ion is Zn2 .
3. A process of claim 1 wherein the said elution is done by using imidazole
solution of different molar strengths, each molar strength capable of
dissolving one Topoisomerase.
4. A process of claim 1 of isolation and purification of a topoisomerase
comprising steps of:
a. lysing the bacterial cells with sonication,
b. centrifuging to get a crude lysate,
c. treating with streptomycin sulphate to remove nucleic acids,
d. loading the supernatant to an immobilized metal affinity
chromatography column charged with Zn2+,
e. eluting with 25 mM imidazole to get Topoisomerase II, and
f. eluting with 75 mM imidazole to get Topoisomerase I.
5. A method of assay of Topoisomerase activity comprising the steps of:
(a) bringing topoisomerase in contact with a composition of super-
coiled DNA free from relaxed form of DNA for a period of time sufficient
to lead to transforming at least a portion of the said super-coiled DNA
into a relaxed DNA and optionally adding either a substance whose Topoisomerase activity is to be detected or assayed, or a substance
that is a known modulator of Topoisomerase activity whose
concentration is to be assyed,
(b) separating unreacted ScDNA from the products produced by
topoisomerase action,
(c) adding a dye to the supernatant that intercalates in or binds with
double strand DNA to result into a DNA_dye complex that emits
fluorescence, and
a. (d) measuring the florescence emitting from the DNA-dye complex.
6. A method of claim 5 wherein separating unreacted ScDNA from the
products of produced by topoisomerase action isachieved by applying the
reaction mixture to a chelating matrix charged with metal ions for a period
of time sufficient to adsorb all the relaxed form of DNA.
7. A method of claim 5 wherein the said topoisomerase is Topoisomerase I
or Topoisomerase II.
8. A method of claim 5 wherein the said metal ion is Zn2+ or Cu2+.
9. A method of claim 5 where the said dye is a cyanine dye PicoGreen that is
commercially availaole as a 200 times concentrated solution under
trademark PicoGreen™.
10. A method of claim 5 comprising the steps of:
a. making a reaction mixture containing a buffer, purified
topoisomerase, a supercoiled DNA, and optionally adding a
substance (i) whose Topoisomerase activity is to be detected or
assayed, or (ii) is a known modulator of Topoisomerase activity
whose concentration is to be assayed,
b. carrying the reaction at a suitable temperasture, c. taking out aliquots of ScDNA at different time intervals from the
reaction mixture and diluting with Immobilized Metal Affinity
Chromatography (IMAC) binding buffer,
d. applying the solution to the 2 n2+.charged chelating matrix for a
[eriod of time at a suitable temeprature,
e. mixing the supernatant with a cyanine dye that is commercially
available with trademark PicoGreen™, and
f. taking fluores ence intensity measurements.
11.'A method of claim 5 comprising the steps of:
a. making a reaction mixture containing 50 µl_ of NEB buffer 4 (50 mM
potassium acetate, 20 mM Tris acetate, 10 mM magnesium
acetate, 1 mM DTT and 100 µg/mL BSA, pH 7.9), 10 µL of the
purified topoisomerase I, and 1 µg of a supercoiled DNA and
optionally adding a substance (i) whose Topoisomerase activity is
to be detected or assayed, or (ii) is a known modulator of
Topoisomerase activity whose concentration is to be assayed,
b. carrying the reaction at 37bC,
c. taking out aliquots of 5 µL containing 100 ng ScDNA at different
time intervals from the reaction mixture and diluting with 150µL of
Immobilized Metal Affinity Chromatography (IMAC) binding buffer,
d. applying the solution to the Zn2+.charged chelating matrix for 45
minutes at 3 O0C,
e. mixing the supernatant with 50 µL of a cyanine dye that is
commercially available with trademark PicoGreen™ after 1:200
dilution of the commercial concentrated dye solution in anhydrous
dimethylsulfoxide with MiIIi Q grade water, and
f. taking fluorescence intensity measurements. 12. A method of claim 11 wherein the said time interval is 0, 10, 20, 30, 40,
50, 60, 70, and 80 min.
13. A method of claim 1 1 wherein the sai l Immobilized Metal Affinity
Chromatography buffer comprises 20 mM HEPES, pH 7.0, containing 250
mM NaCI.
14. A method of claim 5 wherein the said modulator of Topoisomerase activity
is either an inhibitor or an enhancer or a stimulator.
15. A kit for assaying Topoisomerase activity comprising a ScDNA,
topoisomerase enzyme, buffer solutions, a solution of a dye that can bind
with a double stranded DNA to emit fluorescence and instructions to use.
16. A kit of claim 16 whereinr
a. the said solution of a dye is solution of a cyanine dye that is
commercially available with trademark PicoGreen™, and
b. the said instructions to use comprise application of a reaction
mixture comprising above components and test substance to a
chelating matrix charged with metal ions, addition of the said dye to
the supernatant and measuring the intensity of the flourescenece of
the DNA-dye complex.
17. A kit of claim 16 additionally provided with:
a. at least a plate having wells, to the wells of which a chelating matrix
is applied that can be charged with metal ions, or
b. at least a plate with wells, to wells of which chelating matrix
charged with metal ions is applied, or
c. at least a plate with wells, a chelating matrix solution and metal ion
solution.
18. A method of purification of Topoisomerase Il comprising 19. A method of detecting chemical agents for control of microorganisms or
tumours or microbial spoilage of edible products by determining
topoisorr erase inhibiting or enhancing activity of a potential compound by
method of claim 5.
20. A method of claim 19 where:
a. the said microorganisms comprise viruses, fungi, bacteria and
trypanosoma; or
b. the said edible products comprises a food, a fruit, a vegetable or a
natural product susceptible to microbial spoilage.
INTERNATIONAL SEARCH REPORT International application No. PCT/IN2009/000561
A . CLASSIFICATION OF SUBJECT MATTER Int. Cl. C12N 9/90 (2006.01) C12Q 1/533 (2006.01) GOlN 21/64 (2006.01) Cl 2Q 1/18 (2006.01) C12Q 1/68 (2006.01) GOlN 33/68 (2006.01)
According to International Patent Classification (IPC) or to both national classification and IPC
B . FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols)
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) EPODOC, WPI, CAPLUS, MEDLINE, BIOSIS: TOPOISOMERASE, PURIFICATION, ASSAY, IMAC, DYE and like terms C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category? Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. WO2006/051303 Al (PLANT BIOSCIENCE LIMITED [GB]) 18 May 2006 X See the abstract, Figure 1, and p. 11 in particular 5, 7, 14-15, 19-20 Y 16 MAXWELL, A. et al "High-throughput assays for DNA gyrase and other topoisomerases" Nucleic Acids Research, 2006, vol 34, pps elO4/l-elO4/7 [online], [retrieved on 25 February 2010]. Retrieved from the internet
* Special categories of cited documents: "A" document defining the general state of the art which is "T' later document published after the international filing date or priority date and not in not considered to be of particular relevance conflict with the application but cited to understand the principle or theory underlying the invention "E" earlier application or patent but published on or after the "X" document of particular relevance; the claimed invention cannot be considered novel international filing date or cannot be considered to involve an inventive step when the document is taken alone "L" document which may throw doubts on priority claim(s) "Y" document of particular relevance; the claimed invention cannot be considered to or which is cited to establish the publication date of involve an inventive step when the document is combined with one or more other another citation or other special reason (as specified) such documents, such combination being obvious to a person skilled in the art "O" document referring to an oral disclosure, use, exhibition "&" document member of the same patent family or other means "P" document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 02 March 2010 1 6 MAR 2019 Name and mailing address of the ISA/AU Authorized officer FELIXWHITE AUSTRALIAN PATENT OFFICE PO BOX 200, WODEN ACT 2606, AUSTRALIA AUSTRALIAN PATENT OFFICE E-mail address: [email protected] (ISO 9001 Quality Certified Service) Facsimile No. +61 2 6283 7999 Telephone No : +61 2 6283 2565
Form PCT/ISA/210 (second sheet) (July 2009) INTERNATIONAL SEARCH REPORT International application No. PCT/IN2009/000561 C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. Topoisomerase I Assay Kit Cat# 1015, TopoGen, Inc, 2000. [Retrieved on 25 February 2010.] Retrieved from the internet.
WO1999/049077 Al (THE PROCTER & GAMBLE COMPANY) 30 September 1999 Y- See abstract 16
SAHAI B.M. et al, "A Quantitative Decatenation Assay for Type II Topoisomerases", Analytical Biochemistry, 1986, vol 156, pps 364-379 See abstract 5-17, 19-20
WO1999/046595 Al (TULARIK, INC) 16 September 1999 See abstract 5-17, 19-20
BIERSACK, H et al, "Rapid purification of DNA topoisomerase II containing a hexahistidine tag by metal ion affinity chromatography" Methods in Molecular Biology (Totowa, New Jersey), 1999, vol 94 (DNA Topoisomerase Protocols, Vol. 1), pps 235- 242 See abstract 1-4
MURPHY, J.C. et al, "Nucleic acid separations utilizing immobilized metal affinity chromatography", Biotechnology Progress, 2003, vol 19 pps 982-986 Cited in application See abstract 5-17, 19-20
Form PCT/ISA/2 10 (continuation of second sheet) (July 2009) n erna ona app ca on o. PCT/IN2009/000561
Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)
This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:
1. Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:
2. X Claims Nos.: 18 because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be earned out, specifically: Claim 18 appears to be truncated as a result of a typographical error. Consequently its scope cannot be meaningfully understood and no search can be carried out.
3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a)
Box No. EQ Observations where unity of invention is lacking (Continuation of item 3 of first sheet)
This International Searching Authority found multiple inventions in this international application, as follows: Invention I: Claims 1-4 characterised by a method of purifying topoisomerase using immobilised metal affinity chromatography. Invention II: Claims 5-17, 19-20 characterised by methods of assaying topoisomerase activity using supercoiled DNA and an intercalating dye, and kits for use in these methods. The only shared feature between the two inventions is topoisomerase which is well known in the art and cannot therefore be considered a unifying special technical feature. These two groups of claims therefore lack unity. I I As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2 | χ I As all searchable claims could be searched without effort justifying additional fees, this Authority did not invite payment of additional fees. I I As only some of the required additional search fees were timely paid by the applicant, this international search report - covers only those claims for which fees were paid, specifically claims Nos.:
4. No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.:
Remark on Protest | | he additional search fees were accompanied by the applicant's protest and, where applicable, the payment of a protest fee.
The additional search fees were accompanied by the applicant's protest but the applicable protest fee was not paid within the time limit specified in the invitation.
No protest accompanied the payment of additional search fees.
Form PCT/ISA/210 (continuation of first sheet (2)) (July 2008) nternat ona app caton o. Information on patent family members PCT/EV2009/000561
This Annex lists the known "A" publication level patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.
Patent Document Cited in Patent Family Member Search Report
WO 2006/051303 AU 2005303599 CA 2586534 EP 1812592 US 2008026388 WO 1999/049077 AU 32699/99 CA 2324344 EP 1086242 NO 20004753 WO 1999/046595 AU 29964/99 US 5998152 US 6197527
Due to data integration issues this family listing may not include 10 digit Australian applications filed since May 2001. END OF ANNEX
Form PCT/ISA/210 (patent family annex) (July 2009)