WO2002072885A1 - Detection d'interactions entre oligonucleotides et arn par transfert d'energie par resonance de fluorescence (fret) - Google Patents

Detection d'interactions entre oligonucleotides et arn par transfert d'energie par resonance de fluorescence (fret) Download PDF

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Publication number
WO2002072885A1
WO2002072885A1 PCT/GB2002/001016 GB0201016W WO02072885A1 WO 2002072885 A1 WO2002072885 A1 WO 2002072885A1 GB 0201016 W GB0201016 W GB 0201016W WO 02072885 A1 WO02072885 A1 WO 02072885A1
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Prior art keywords
rna
oligonucleotides
array
chromaphore
labelled
Prior art date
Application number
PCT/GB2002/001016
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English (en)
Inventor
Peter Estibeiro
Original Assignee
Expresson Biosystems Limited
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 Expresson Biosystems Limited filed Critical Expresson Biosystems Limited
Priority to EP02704918A priority Critical patent/EP1370688A1/fr
Publication of WO2002072885A1 publication Critical patent/WO2002072885A1/fr

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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
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • FRET Fluorescence Resonance Energy Transfer
  • a device for, and a method of, detecting interactions between oligonucleotides immobilised on a solid array and RNA that is added to the array relies on fluorescence resonance energy transfer (FRET) between two chromaphores .
  • FRET fluorescence resonance energy transfer
  • Antisense as a means of controlling gene expression for research or therapeutic purposes was first described in the 1970s. Since then, much effort has been put into understanding how antisense works and into developing methods to map effective antisense targets.
  • Antisense works by introducing a short synthetic nucleic acid, the antisense agent, that is complimentary to a target mRNA, into a cell.
  • the antisense agent binds to its target mRNA and prevents translation by mechanisms thought to involve both tagging for degradation by endogenous nucleases and a physical hindrance of translocation or translation.
  • antisense agents are complicated by the fact the mRNA has extremely complex secondary and tertiary structures. At least 90% of the nucleotide sequence of any given mRNA is involved in intra-molecular interactions within the secondary and tertiary structure of the molecule, and is thus unavailable to participate in inter-molecular interaction with an antisense agent.
  • the key to the design of a successful antisense agent is to identify the limited regions of a potential mRNA target that are available for inter-molecular hybridisation. Antisense agents targeted specifically to these accessible regions have a high probability of binding to the target mRNA in vivo, and effectively knocking down the level of expression of its encoded product .
  • the sequence of the target mRNA is represented in the oligonucleotide library, such that the first oligonucleotide may be complimentary to positions one to fifteen on the target mRNA, the second will be complimentary to two to sixteen, an the third three to seventeen, etc.
  • oligonucleotides immobilised on a glass or plastic support, such that all possible sequence combinations are represented by oligonucleotides of between four and eight nucleotide units.
  • This type of device is disclosed in International Patent No 098/15651 and also in US Patent No US006054270.
  • each oligonucleotide is proposed to be physically separate on the array and after hybridisation of the target mRNA and washing off of unbound material, the signal is detected from the bound oligonucleotides.
  • the sequence of target mRNA that is accessible to inter-molecular hybridisation can be inferred.
  • the inferred sequence is likely to be an effective target for antisense mediated gene knockdown.
  • RNA rather than the oligonucleotides is immobilised to the solid support and that the oligonucleotides are applied in solution to denatured RNA. Under these conditions, it is unlikely that the RNA would be folded into an authentic representation of its in vivo structure, and the method demonstrated might not map the structure of the RNA in a suitable manner to target antisense.
  • oligonucleotides of at least ten nucleotides in length usually it is necessary to use oligonucleotides of at least ten nucleotides in length to get a consistent signal under conditions that will map RNA structure.
  • Short, immobilised oligonucleotides will hybridise to full-length RNA transcripts under conditions where the RNA concentration is sufficiently high to drive the hybridisation reaction in favour of forming duplexes.
  • the kinetic change associated with diluting away the excess RNA generally favours the melting apart of short duplexes, such the genuine interactions, signalling a region of the RNA that may be accessible to antisense mediated knockdown, cannot be directly detected.
  • a device for use in detecting interactions between oligonucleotides and RNA comprising an array of oligonucleotides wherein one end of each oligonucleotide is anchored to a solid support and the other end of each oligonucleotide is conjugated to the first of a matched pair of chromaphores (CHI) .
  • CHI chromaphores
  • the solid support may be made of plastic.
  • the solid support may be made of glass.
  • the solid support is made of any appropriate material.
  • the oligonucleotides may be anchored to the solid support by chemical modifications at their 5' end.
  • the oligonucleotides may be anchored to the solid support by chemical modifications at their 3' end.
  • oligonucleotides may be synthesised in situ by standard procedures.
  • the oligonucleotides are spaced away from the solid support by a chemical spacer.
  • the chemical spacer is between six and forty carbon atom equivalents.
  • the chromaphore may be spaced away from the oligonucleotide using a chemical spacer.
  • oligonucleotides may be made of any naturally occurring nucleotide or deoxynucleotide or analogue of these.
  • the invention is not limited by the chemical composition of the oligonucleotides.
  • a method for detecting interactions between oligonucleotides and RNA using the device of the first aspect wherein:
  • the target RNA is labelled during transcription with a second chromaphore (CH2) that when excited is able to emit energy at a wavelength that will excite the first chromaphore (CHI) present on the oligonucleotides, when the two are in sufficiently close proximity; and
  • CH2 second chromaphore
  • CHI first chromaphore
  • step a) may be that the target RNA is labelled during transcription with a second chromaphore (CH2) that is excited by energy emitted by the first chromaphore (CHI) when it is excited.
  • CH2 second chromaphore
  • the RNA is in sufficiently high concentration to drive the formation of duplexes with the oligonucleotides on the array.
  • target RNA is transcribed in vitro under conditions that allow authentic folding.
  • the RNA is labelled with a chromaphore by incorporation of a modified nucleotide during transcription.
  • RNA is labelled with chromaphore post-transcriptionally by enzymatic or photochemical means .
  • RNA any appropriate means of labelling the RNA may be used.
  • the appropriate light source is a laser .
  • the light source is a diode.
  • the light source is a lamp.
  • RNA is added to the array in an RNA application buffer that supports maintenance of secondary structure, promoting the formation of duplexes between applied RNA and oligonucleotides on the array.
  • RNA application buffer is compatible with FRET and with maintenance of RNA secondary and tertiary structures.
  • a volume excluder may be used in the buffer.
  • the volume excluder is polyethylene glycol (PEG) .
  • volume excluder may be dextran sulphate.
  • duplexes formed between the applied RNA and immobilised oligonucleotides may be detected using a commercially available fluorescence plate reader or array reader fitted with appropriate emitters and filters. This will allow detection of the one of the matched pair chromaphores if it has been excited by the close proximity of other excited chromaphore in the matched pair, which signifies duplex formation.
  • Preferably interpretation of the signal from the array is by computer based algorithm.
  • an array of oligonucleotides where each oligonucleotide is six to eight or nine to fifteen nucleotides long, and where the array comprises all possible sequence combinations of these oligonucleotides at physically separate positions on the array.
  • the precise sequence of the oligonucleotide at each position is known. All of the oligonucleotides are anchored to the solid array support by chemical modifications at their 5' or 3' ends and may be spaced away from the surface of the array by inclusion of a nucleotide or other chemical linker.
  • the oligonucleotide may instead by synthesised in situ by standard procedures for example using the methods described in Marshall, A and Hodgson J. DNA chips: An array of possibilities. Nature Biotechnology 15:27- 31(1998) .
  • RNA molecules that are labelled with the second of the matched pair of chromaphores can then be added to the array to allow the detection of any duplex formations.
  • the abovementioned device is used in a method for detecting interactions between oligonucleotides and mRNA.
  • a copy of the target RNA is transcribed in vitro from a full length or partial cDNA clone under conditions in which the nascent RNA can fold in a manner that is an authentic representation of its in vivo structure. Once it has been synthesised, this RNA is maintained under conditions that will maintain its authentic secondary and tertiary structure.
  • RNA is labelled during transcription with a chromaphore (CH2) that, when excited is able to emit energy at a wavelength that will excite another chromaphore (CHI) , which has been conjugated to the immobilised oligonucleotides, when the two are in sufficiently close proximity.
  • CH2 chromaphore
  • CHI chromaphore
  • the target RNA is then applied to the array at sufficient concentration to drive the formation of duplexes between the RNA and the oligonucleotides immobilised on the array.
  • the target RNA will form duplexes with a subset of oligonucleotides that are complimentary to its accessible regions.
  • the high concentration of the applied RNA drives the kinetics of the hybridisation reaction, between said RNA and those oligonucleotides on the array that are complimentary to the accessible regions of the RNA, in the direction of duplex formations .
  • any duplexes that are formed between the two will enable FRET.
  • the FRET can be detected by shining an appropriate laser or other suitable controlled light source onto the array to excite one of the chromaphores. If a duplex is formed, the two chromaphores will be sufficiently close that the emissions from the first chromaphore will excite the second chromaphore and duplex formation can be inferred by detecting emissions from that second chromaphore.
  • Duplexes formed between the applied RNA and the immobilised oligonucleotides are detected using a commercially available fluorescence plate reader or array reader fitted with appropriate emitters and filters. Interpretation of the signals that are detected may be by computer based algorithms.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un dispositif et un procédé pour détecter des interactions entre des oligonucléotides immobilisées sur un ensemble solide et l'ARN ajouté à l'ensemble. Cette détection est effectuée par transfert d'énergie par résonance de fluorescence (FRET) entre les deux chromophores. La séquence précise des oligonucléotides immobilisées étant connue, il est possible de déterminer des paramètres structuraux des produits de la transcription d'ARN endogène et des zones susceptibles d'être des cibles efficaces pour le choc de gène d'origine antisens.
PCT/GB2002/001016 2001-03-08 2002-03-07 Detection d'interactions entre oligonucleotides et arn par transfert d'energie par resonance de fluorescence (fret) WO2002072885A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02704918A EP1370688A1 (fr) 2001-03-08 2002-03-07 Detection d'interactions entre oligonucleotides et arn par transfert d'energie par resonance de fluorescence (fret)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0105789.2 2001-03-08
GBGB0105789.2A GB0105789D0 (en) 2001-03-08 2001-03-08 Detecting interactions between oligonucleotides and RNA using fluorescence resonance energy transfer (FRET)

Publications (1)

Publication Number Publication Date
WO2002072885A1 true WO2002072885A1 (fr) 2002-09-19

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PCT/GB2002/001016 WO2002072885A1 (fr) 2001-03-08 2002-03-07 Detection d'interactions entre oligonucleotides et arn par transfert d'energie par resonance de fluorescence (fret)

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EP (1) EP1370688A1 (fr)
GB (1) GB0105789D0 (fr)
WO (1) WO2002072885A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0721016A2 (fr) * 1994-10-21 1996-07-10 Affymax Technologies N.V. Banque d'acides nucléiques immobilisées, procédés pour leur fabrication et procédés de séquençage et de screening les utilisant
WO1998015651A1 (fr) * 1996-10-04 1998-04-16 Brax Genomics Limited Identification de la liaison d'un oligonucleotide non codant
US5876930A (en) * 1994-11-16 1999-03-02 Perkin-Elmer Corporation Hybridization assay using self-quenching fluorescence probe
WO1999013105A1 (fr) * 1997-09-05 1999-03-18 Mikael Kubista Preparation d'une sonde en vue d'une hybridation d'acide nucleique
GB2335035A (en) * 1998-03-03 1999-09-08 Brax Genomics Ltd Screening for functional antisense agents
WO1999049293A2 (fr) * 1998-03-24 1999-09-30 Boston Probes, Inc. Procedes, kits et compositions se rapportant a des complexes de detection
US6080585A (en) * 1994-02-01 2000-06-27 Oxford Gene Technology Limited Methods for discovering ligands
WO2000042222A2 (fr) * 1999-01-15 2000-07-20 Gene Logic Inc. Reactif d'hybridation d'acide nucleique immobilise et procede associe

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6080585A (en) * 1994-02-01 2000-06-27 Oxford Gene Technology Limited Methods for discovering ligands
EP0721016A2 (fr) * 1994-10-21 1996-07-10 Affymax Technologies N.V. Banque d'acides nucléiques immobilisées, procédés pour leur fabrication et procédés de séquençage et de screening les utilisant
US5876930A (en) * 1994-11-16 1999-03-02 Perkin-Elmer Corporation Hybridization assay using self-quenching fluorescence probe
WO1998015651A1 (fr) * 1996-10-04 1998-04-16 Brax Genomics Limited Identification de la liaison d'un oligonucleotide non codant
WO1999013105A1 (fr) * 1997-09-05 1999-03-18 Mikael Kubista Preparation d'une sonde en vue d'une hybridation d'acide nucleique
GB2335035A (en) * 1998-03-03 1999-09-08 Brax Genomics Ltd Screening for functional antisense agents
WO1999049293A2 (fr) * 1998-03-24 1999-09-30 Boston Probes, Inc. Procedes, kits et compositions se rapportant a des complexes de detection
WO2000042222A2 (fr) * 1999-01-15 2000-07-20 Gene Logic Inc. Reactif d'hybridation d'acide nucleique immobilise et procede associe

Also Published As

Publication number Publication date
GB0105789D0 (en) 2001-04-25
EP1370688A1 (fr) 2003-12-17

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