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WO1998037241A1 - Methode de dosage d'allongement de telomerase - Google Patents

Methode de dosage d'allongement de telomerase Download PDF

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Publication number
WO1998037241A1
WO1998037241A1 PCT/US1998/003725 US9803725W WO9837241A1 WO 1998037241 A1 WO1998037241 A1 WO 1998037241A1 US 9803725 W US9803725 W US 9803725W WO 9837241 A1 WO9837241 A1 WO 9837241A1
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WO
WIPO (PCT)
Prior art keywords
telomerase
nucleic acid
sequence
acid sequence
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1998/003725
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English (en)
Inventor
Michael J. Lane
Albert S. Benight
Brian D. Faldasz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Luminex Molecular Diagnostics Inc
Original Assignee
TM Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by TM Technologies Inc filed Critical TM Technologies Inc
Priority to AU63395/98A priority Critical patent/AU6339598A/en
Publication of WO1998037241A1 publication Critical patent/WO1998037241A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • Telomeres are tandem repeats at the ends of eukaryotic chromosomes that appear to function in chromosome replication.
  • Telomerase is a ribonucleoprotein enzyme that synthesizes one strand of the telomeric DNA using as a template a sequence contained within the RNA component of the enzyme. See. e.g.. Blackburn (1992) Annu. Rev. Biochem. 61 : 1 13-129. incorporated herein by reference.
  • Human telomerase is known to synthesize telomeric repeat units with the sequence 5'-TTAGGG-3'. See. e.g.. Morin (1989) Cell. 59:521-529. and Morin ( 1991) Nature 353:454-456. incorporated herein by reference.
  • telomere shortening appears to dictate the number of cell divisions a normal somatic cell can undergo.
  • Telomerase activity has also been linked to senescence. Whereas normal cells with relatively long telomeres and a senescent phenotype may contain little or no telomerase activity, tumor cells with short telomeres may have significant telomerase activity. Thus, organismal senescence might be halted or at least slowed if an effective. non-toxic telomerase inhibitor can be found. Accordingly, the identification of compounds that affect telomerase activity provides important benefits to efforts at treating human diseases such as cancer.
  • the present invention relates to methods of measuring levels of telomerase. e.g., human telomerase activity, in a variety of samples, e.g., a biological sample.
  • the invention also includes methods for screening compounds for their ability to modulate. e.g., to enhance or inhibit, telomerase activity, e.g., as a high throughput drug screening assay for pharmaceuticals which affect telomerase activity.
  • the invention provides a method for screening for compounds that modulate telomerase activity.
  • the method can detect telomerase activity by incubating a sample that is suspected of having telomerase activity with a nucleic acid test sequence and detecting elongation of the test sequence by telomerase.
  • the comparison of the level of telomerase activity in the absence of a test compound with the level of activity in its presence can provide information regarding the inhibitory or the enhancing effects of the compound. For example, a compound shows inhibitory effects if the elongation occurs in its absence, but fails in its presence.
  • test nucleic acid sequences preferably DNA sequences, that are other than a preparation of genomic DNA or cDNA library.
  • test nucleic acid sequences preferably DNA sequences, that are other than a preparation of genomic DNA or cDNA library.
  • These embodiments utilize a substantially pure nucleic acid sequence that is bound to a solid substrate and is exogenously added to a sample whose telomerase activity is being investigated.
  • One aspect of the present assay relates to employing a hairpin nucleic acid sequence, having a single stranded region with a 3 '-terminus, that is affixed to a solid substrate.
  • Telomerase in presence of appropriate oligonucleotides can add telomeric repeat sequences to the 3 '-terminus of the hairpin.
  • the elongation of the test nucleic acid sequence is detected by adding a detector sequence that is selected to be complementary to a portion of the test sequence that includes at least one telomeric repeat sequence, thus hybridizing to this portion.
  • the assay detects the hybridized detector sequence by providing a label, e.g., a radiolabel. in the detector sequence or adding such a label to the detector sequence. For example, addition of reagents that can hybridize to a section of the detector sequence and cause chemiluminescence or color change of the sample results in detection of telomerase activity.
  • FIGURE 1 is a schematic depiction of the various steps of a preferred embodiment of the telomerase assay of the invention.
  • the invention relates to methods for detecting telomerase activity and for screening for compounds that modulate the activity of telomerase in vivo or in vitro.
  • the methods of the invention can identify compounds which may be useful for the treatment of conditions related to activity of telomerase.
  • the invention also relates to methods for detecting the presence or absence of telomerase activity in a sample such as a biological sample, a tissue biopsy sample or a biological fluid, e.g., blood, urine, saliva, and the like.
  • the telomerase assays of the invention are useful for detecting tissue (or other biological materials) which has telomerase activity, which may be an indicator of telomerase-linked conditions such as the presence of cancerous or precancerous cells.
  • the invention thus provides methods for rapidly screening biological samples for conditions such as cancer.
  • the invention provides a method for detecting telomerase activity in a sample.
  • the method includes the steps of contacting a test nucleic acid sequence with a sample suspected of containing telomerase activity, under conditions such that if telomerase is present in the sample, the nucleic acid sequence is elongated by telomerase to form an elongated nucleic acid sequence, and detecting the elongated nucleic acid sequence.
  • the sample can include cells, which can be lysed if desired. under conditions such that telomerase is not denatured, i.e.. such that telomerase activity is not destroyed.
  • the invention provides a method for screening for compounds capable of modulating telomerase activity.
  • the method includes the steps of contacting a nucleic acid sequence with a sample suspected of containing a telomerase. in the presence of a test compound, under conditions such that if telomerase is present in the sample, the nucleic acid sequence is elongated by telomerase to form an elongated nucleic acid sequence in the absence of the test compound, and detecting the presence or absence of the elongated nucleic acid sequence.
  • Compounds which suppress the activity of telomerase may be useful as inhibitors of telomerase activity in vitro, and may therefore be useful as inhibitors of carcinogenesis or cell senescence, as described above.
  • Compounds which enhance the activity of telomerase in the assays of the invention may be promoters of cancer or cell senescence. Accordingly, the assays of the invention provide the ability to screen for both potentially harmful and potentially therapeutic compounds.
  • methods of the invention typically employ a nucleic acid hairpin having a 3 '-terminus to which telomerase can add telomeric repeat sequences.
  • the nucleic acid sequence e.g., a hairpin: see W097/08183 and U.S. Patent Application No. 08/519.197
  • the nucleic acid sequence includes at least one telomeric repeat sequence as a terminal portion of the hairpin sequence (e.g., TTAGGG-3').
  • Telomeric repeat sequences include the human telomeric repeat sequence (5'-TTAGGG-3'), and other telomeric repeat sequences known to one of ordinary skill in the art.
  • FIGURE 1 shows schematically that the nucleic acid test sequence can be immobilized on a solid support.
  • the test sequence is a nucleic acid "hairpin", e.g., a nucleic acid molecule having self- complementary regions capable of hybridizing to each other to form a duplex "stem" region and, preferably, at least one dangling single-stranded portion.
  • the advantages of immobilization of the test sequence include ease of handling, and manipulation, e.g., washing, separating, etc. The immobilization is achieved by a variety of methods known in the art.
  • One embodiment incorporates commercially available biotinylated nucleic acid bases into the test sequence in order to attach the test sequence to a solid support that has been derivatized with avidin or streptavidin. e.g., streptavidin-coated 96-well plates, or streptavidin-coated beads such as Dynabeads. available for Dynal. Other methods for attaching or immobilizing a nucleic acid sequence on a solid support will be apparent to one of ordinary skill in the art.
  • a nucleic acid hairpin which has been affixed to a solid support, is contacted with a sample that is suspected of having telomerase activity.
  • telomerase is known to elongate a strand of nucleic acid by addition of telomeric repeat sequences.
  • presence of telomerase activity in the sample is detected by determining whether tandemly repeated telomeric sequences have been added to the test hairpin strand.
  • the elongation process requires the presence of appropriate oligonucleotide phosphates ("NTP"). i.e.. NTPs required for synthesis of a telomeric repeat sequence.
  • telomerase extension reaction can be readily purchased from commercial suppliers and are added to the reaction mixture to permit the telomerase extension reaction to proceed (e.g.. human telomerase requires ATP. TTP. and GTP). Accordingly, if telomerase is present in the sample, it recognizes the 3'- terminus of the test sequence as a substrate and extends it by addition of telomeric repeat sequences.
  • the test sequence includes a telomeric repeat sequence before addition of a sample which contains telomerase, e.g., a synthetic test sequence which is provided with a telomeric repeat sequence at the 3'-terminus by chemical synthesis.
  • a preferred 3'-terminal portion of the test sequence is 5'- CTGGGTTAGGG-3'.
  • the test nucleic acid sequence has the sequence 5'-CTAGT CGACG TGGTC CTTT(biotin)T T TGGAC CACGT CGACT AGCTG GGTTA GGG-3' .
  • T(biotin) denotes a biotinylated thymidine.
  • the methods of the invention can detect the presence of elongated nucleic acids with sufficient sensitivity such that no target replication steps such as Polymerase Chain Reaction ("PCR") or Ligase Chain Reaction (“LCR”) are required, and in a preferred embodiment no PCR or LCR steps are performed.
  • Application of such replication methods typically involve various steps including selection of appropriate primers and necessary enzymes, heating and cooling cycles and the like. The elimination of such steps by the preferred embodiments can render the methods of the invention more convenient to use and less costly in comparison with those that utilize replication procedures.
  • it can be advantageous to replicate the elongated nucleic acid (or its complement) or a portion thereof, e.g.. by PCR. LCR. or other known methods. For example, one may desire to incorporate such methods in the present invention if ultrasensivity of detection of telomerase is required.
  • the methods of the invention detect repeat sequences added by telomerase to the nucleic acid substrate (e.g., a hairpin) by employing detection sequences that are complementary to a portion of the single-stranded region of the hai ⁇ in that includes at least one repeat sequence.
  • a detector sequence which is complementary to a region of the hai ⁇ in as well as to at least a portion of a telomeric repeat sequence rather than to a single strand provides certain advantages.
  • a "nicked" duplex structure is formed, comprising contiguous regions of intramolecular hai ⁇ in ai ⁇ in duplex and intermolecular detector:hai ⁇ in duplex.
  • This arrangement provides base stacking between the intramolecular duplex (i.e.. the duplex "stem” of the hai ⁇ in) and the intermolecular duplex (i.e.. the detecto ⁇ hai ⁇ in duplex), which results in a greater sequence stringency than hybridization to a simple single strand. Further, the duplex region of the hai ⁇ in stabilizes, e.g., entropically, the detector-specific region of the hai ⁇ in and thereby favors formation of a detecto ⁇ hai ⁇ in duplex.
  • the length of the single stranded region of the hai ⁇ in can be selected to allow the use of a variety of detector sequences. In particular, if detection of only a few telomeric repeat sequences is desired, it may be advantageous to select a hai ⁇ in with a relatively long single stranded region to allow stable attachment of a detector sequence that has only a few sequences that are complementary to telomeric repeat sequences.
  • the detector sequence preferably contains at least a portion which is complementary to at least two telomeric repeat sequences, e.g.. the sequence 5'- CCCTAACCCTAA-3' or 5'- CCCUAACCCUAA-3'.
  • the detector sequence also can include a poly(T). e.g., T30, "tail" region that is not complementary to the hai ⁇ in.
  • T30 poly(T)
  • the detector sequence hybridizes to this segment. Excess detector sequences and non-specifically- bound detector sequences are removed by washing with a buffer having an appropriate stringency to remove unbound detector sequences while not removing specifically- bound detector molecules, as known in the art.
  • the hybridized detector sequences are then detected by well-known methods in the art for detecting nucleic acid sequences.
  • the detector sequence contains a label, e.g., radiolabel. that allows a direct detection of the sequence.
  • the sequence is detected indirectly by attaching a label to a portion of the detector sequence which can be subsequently detected by direct or indirect means.
  • Labels can include radioisotopes, fluorophores, chemiluminescent tags and the like.
  • European Patent Publication EP 128 332 herein inco ⁇ orated by reference, reports one such method that employs a "bridging moiety" which provides a bridge between an analyte and a "signaling moiety” which provides a detectable signal.
  • the "signal generating" portion of the signaling entity can encompass virtually any of the signal generating systems used in the prior art. In particular, it comprises a moiety which generates a signal itself, e.g., a radiolabel. or a moiety which . upon further reaction or manipulation, will give rise to a signal, e.g.. an enzyme-linked system.
  • Some other methods utilize radioactive nucleotides in a label and detect a nucleotide sequence to which such a label has hybridized by detecting the decay of the radioactive nucleotides.
  • a preferred embodiment of the present invention employs a detector sequence shown in FIGURE 1 that is selected to have a poly(T) or poly(dT) "tail" to which a complementary probe sequence, i.e.. poly d(A) ⁇ 500 * hybridizes.
  • the probe sequence is chosen to be long enough so as to allow attachment of various labels in the regions that are not hybridized to the detector sequence.
  • excess polyd(A) i.e.. unhybridized polyd(A).
  • FITC fluoreseinisothiocycanate
  • telomerase activity in the sample, e.g., by addition of a color- forming substrate for AP. followed by spectrophotometric detection of any color formation.
  • Example 1 provides further clarification of various steps of a preferred embodiment of the invention and is meant as illustrative and not in a limiting sense.
  • a synthetic nucleic acid "hai ⁇ in” is used as a substrate for elongation by telomerase.
  • Appropriate nucleic acid hai ⁇ ins can be designed and prepared by the skilled artisan (see, e.g., U.S. Patent Application Serial No. 08/519,197, filed August 25, 1995, which is hereby inco ⁇ orated by reference).
  • the synthetic hai ⁇ in is first affixed to a solid support in a reaction vessel (such as a 96-well plate), e.g., through a biotin-streptavidin linkage or other means known in the art.
  • a sample to be tested (which may contain telomerase) is then incubated with the solid-supported hai ⁇ in.
  • Telomerase present in the sample extends the dangling single strand of the hai ⁇ in at the 3'-terminus. attaching six base repeats of the sequence 5'-TTAGGG-3'.
  • the bound hai ⁇ ins are (optionally) washed to remove sample, and a "detector" sequence is supplied to the reaction vessel.
  • the detector sequence includes at least one sequence which is complimentary to the telomerase extension products, i.e..
  • the detector hybridizes to the bound hai ⁇ in only if two or more telomerase generated hexamer sequences (5'-TTAGGG-3') are present.
  • the detector sequence preferably includes the sequence 5'-CCCTAACCCTAA-3' (or 5'- CCCUAACCCUAA-3').
  • the detector further include a poly(T) (e.g., T 30 ) "tail", i.e.. a portion which is not complementary to the hai ⁇ in. If the bound hai ⁇ ins contain the sequence 5'-TTAGGGTTAGGG-3' (i.e. have been extended by telomerase). the detector sequence (or a portion thereof) will hybridize and bind to the hai ⁇ in. Excess detector, and non-specificallv-bound detector sequence, can be removed by washing with buffer of the appropriate stringency, as is known in the art.
  • a probe complementary to the poly(T) "tail” of the detector sequence e.g., polyd
  • polyd(A) 1500 is added, and hybridizes to the T 30 tail of the "detector" under appropriate conditions.
  • anti-FITC-AP conjuggate of anti-FITC antibody (commercially available) with alkaline phosphatase (AP)
  • AP alkaline phosphatase
  • the methods of the invention attain the above-mentioned objectives including detection of telomerase activity.

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Abstract

L'invention permet de détecter la présence d'une activité de télomérase, au moyen d'une séquence d'acides nucléiques d'essai employée comme substrat par la télomérase pour l'ajout d'une séquence répétée télomérique. La détection des séquences d'allongement permet de déterminer la présence d'une activité de télomérase. De plus, les procédés de l'invention peuvent être utilisés pour déterminer l'effet inhibiteur ou activateur d'un composé sur l'activité de télomérase.
PCT/US1998/003725 1997-02-24 1998-02-23 Methode de dosage d'allongement de telomerase Ceased WO1998037241A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU63395/98A AU6339598A (en) 1997-02-24 1998-02-23 Telomerase extension assay

Applications Claiming Priority (4)

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US3879897P 1997-02-24 1997-02-24
US60/038,798 1997-02-24
US5010997P 1997-06-18 1997-06-18
US60/050,109 1997-06-18

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000046601A1 (fr) * 1999-02-02 2000-08-10 Frank Larsen Detection d'activite telomerase

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4734363A (en) * 1984-11-27 1988-03-29 Molecular Diagnostics, Inc. Large scale production of DNA probes
EP0297379A2 (fr) * 1987-06-30 1989-01-04 Miles Inc. Procédé pour l'amplification des gènes
US5489508A (en) * 1992-05-13 1996-02-06 University Of Texas System Board Of Regents Therapy and diagnosis of conditions related to telomere length and/or telomerase activity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4734363A (en) * 1984-11-27 1988-03-29 Molecular Diagnostics, Inc. Large scale production of DNA probes
EP0297379A2 (fr) * 1987-06-30 1989-01-04 Miles Inc. Procédé pour l'amplification des gènes
US5489508A (en) * 1992-05-13 1996-02-06 University Of Texas System Board Of Regents Therapy and diagnosis of conditions related to telomere length and/or telomerase activity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MANIATIS T., ET AL.: "MOLECULAR CLONING A LABORATORY MANUAL, PASSAGE.", MOLECULAR CLONING., XX, XX, 1 January 1982 (1982-01-01), XX, pages 214/215., XP002910094 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000046601A1 (fr) * 1999-02-02 2000-08-10 Frank Larsen Detection d'activite telomerase

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AU6339598A (en) 1998-09-09

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