EP1370688A1 - Detecting interactions between oligonucleotides and rna using fluorescence resonance energy transfer (fret) - Google Patents

Detecting interactions between oligonucleotides and rna using fluorescence resonance energy transfer (fret)

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Publication number
EP1370688A1
EP1370688A1 EP02704918A EP02704918A EP1370688A1 EP 1370688 A1 EP1370688 A1 EP 1370688A1 EP 02704918 A EP02704918 A EP 02704918A EP 02704918 A EP02704918 A EP 02704918A EP 1370688 A1 EP1370688 A1 EP 1370688A1
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EP
European Patent Office
Prior art keywords
rna
oligonucleotides
array
chromaphore
labelled
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.)
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Application number
EP02704918A
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German (de)
French (fr)
Inventor
Peter Expresson Biosystems Limited ESTIBEIRO
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Expresson Biosystems Ltd
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Expresson Biosystems Ltd
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Application filed by Expresson Biosystems Ltd filed Critical Expresson Biosystems Ltd
Publication of EP1370688A1 publication Critical patent/EP1370688A1/en
Withdrawn legal-status Critical Current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
    • 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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Abstract

There is provided a device and method for detecting interactions between oligonucleotides immobilised on a solid array and RNA that is added to the array. This detection of the interaction relies on Fluorescence Resonance Energy Transfer (FRET) between the two chromaphores. As the precise sequence of the immobilised oligonucleotides is known, it is therefore possible to determine structural parameters of native RNA transcripts and infer regions that may be effective targets for antisense mediated gene knock-down.

Description

Detecting Interactions Between Oligonucleotides and RNA Using Fluorescence Resonance Energy Transfer (FRET)
According to this invention there is provided a device for, and a method of, detecting interactions between oligonucleotides immobilised on a solid array and RNA that is added to the array. The detection of these interactions relies on fluorescence resonance energy transfer (FRET) between two chromaphores . As the precise sequence of the immobilised oligonucleotides is known, it is therefore possible to map mRNA transcripts and determine regions that may be effective targets for antisense mediated gene knockdown.
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.
The design of antisense agents is 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 .
There are a number of in vitro experimental methods available in the prior art that can be used to effectively target antisense agents. Generally these experimental methods rely on using libraries of oligonucleotides to mediate cleavage of in vitro RNA transcripts by RNaseH, or to bind to the RNA and cause retardation of a labelled species during gel electrophoresis or to bind to the RNA on an array of separate elements, such that the signal from each element can be detected and correlated with ability to bind in that position. Successful methods depend on knowing the sequence of the target mRNA and designing a library of overlapping oligonucleotides generally of up to twenty-five nucleotides in length. 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.
Methods have been proposed in the prior art, whereby antisense agents can be designed using a device that is not dependent on advanced knowledge of the target sequence. Such a device is proposed to comprise an array of 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. In this Patent and Patent Application 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. By knowing which sequences are present, and at which physically separate location on the array, 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.
Both 098/15651 and US006054270 describe arrays comprising all possible four to eight base sequence combinations. While this is technologically feasible, with four base sequence combinations requiring 256 elements on an array to represent all sequence combinations, and eight base sequence combinations requiring 65,536 elements on an array to represent all sequence combinations, there are difficulties with using such short four to eight base oligonucleotides as the basis for an array. The difficulty with using short four to eight base oligonucleotides as the basis for an array to map RNA structure and define regions that are effective antisense targets, is that the interaction between the oligonucleotides on the array and the labelled transcript applied to it under hybridising conditions is very weak. Under normal washing conditions, the transcript is washed off and no signal is detected. Increasing salt concentration or decreasing temperature tends to increase non-specific background, but does not improve the signal. In Patent Application W098/15651 it is demonstrated that a signal can be detected by hybridising RNA to four base oligonucleotides. However, under the conditions in the described protocol, there is a requirement that the 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.
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. Under normal washing conditions, 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.
It can therefore be seen that there is a requirement for a method that will enable detection of interactions between RNA and members of a combinatorial library of oligonucleotides of less than ten nucleotides long.
It is a first object of this invention to provide a device and method for the detection of interactions between oligonucleotides and RNA.
It is a further object of this invention to provide a device and method for detection of interactions between short (less than ten nucleotides in length) oligonucleotides and RNA.
It is a yet further object of this invention to provide a device and method for mapping mRNA transcripts and determining regions that may be effective targets for antisense mediated gene knockdown.
According to a first aspect of the present invention, there is provided 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) .
Preferably, all possible sequence combinations of a specific length or lengths of oligonucleotides are represented on the array.
Most preferably, all possible combinations of 6 to 8 or 9 to 15 nucleotide oligonucleotides are represented on the array.
Optionally, the solid support may be made of plastic.
A further option is that the solid support may be made of glass.
Alternatively, the solid support is made of any appropriate material.
Optionally, the oligonucleotides may be anchored to the solid support by chemical modifications at their 5' end.
Alternatively, the oligonucleotides may be anchored to the solid support by chemical modifications at their 3' end.
A further alternative is that the oligonucleotides may be synthesised in situ by standard procedures.
Preferably, the oligonucleotides are spaced away from the solid support by a chemical spacer. Most preferably the chemical spacer is between six and forty carbon atom equivalents.
Optionally, the chromaphore (CHI) may be spaced away from the oligonucleotide using a chemical spacer.
The 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.
According to a second aspect of the present invention, there is provided a method for detecting interactions between oligonucleotides and RNA using the device of the first aspect, wherein:
a) 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
b) where the labelled RNA is applied to the array and allowed to bind to complementary oligonucleotides ; and
c) an appropriate light source is used to excite one of the matched pair of chromaphores; and
d) emissions from the second of the chromaphores is detected. Alternatively 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.
Preferably, the RNA is in sufficiently high concentration to drive the formation of duplexes with the oligonucleotides on the array.
Preferably, target RNA is transcribed in vitro under conditions that allow authentic folding.
Optionally, the RNA is labelled with a chromaphore by incorporation of a modified nucleotide during transcription.
A further option is that the RNA is labelled with chromaphore post-transcriptionally by enzymatic or photochemical means .
Optionally any appropriate means of labelling the RNA may be used.
Most preferably the appropriate light source is a laser .
Alternatively the light source is a diode.
Alternatively the light source is a lamp.
Optionally any appropriate nature or wavelength of light source may be used. Preferably the 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.
Most preferably the RNA application buffer is compatible with FRET and with maintenance of RNA secondary and tertiary structures.
Optionally a volume excluder may be used in the buffer.
Preferably the volume excluder is polyethylene glycol (PEG) .
Alternatively, the volume excluder may be dextran sulphate.
Preferably, 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.
In order to provide a better understanding of the present invention, embodiments of the invention will now be described by way of example. According to the first aspect of the invention, there is provided 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. All are conjugated to one of a matched pair of chromaphores that will enable hybridisation detection by FRET. As an alternative to anchoring the oligonucleotides to the solid support by chemical modifications at their 5' or 3' end, 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.
According to the second aspect of this invention, 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. The 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.
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 .
As the target RNA is labelled with one chromaphore (CH2) in a matched pair and the oligonucleotides are labelled with the other chromaphore (CHI) in the same matched pair, 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.
The embodiments disclosed above are merely exemplary of the invention, which may be embodied in different forms. Therefore, the details disclosed herein are not to be interpreted as limiting, but merely as a basis for the Claims and for teaching one skilled in the art as to the various uses of the present invention in any appropriate manner.

Claims

1. A device for detecting interaction 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) .
2. A device as described in Claim 1, wherein all possible sequence combinations of a specific length or lengths of oligonucleotides are represented on the array.
3. A device as described in Claims 1 or 2, wherein all possible combinations of 6 to 8 or 9 to 15 nucleotide oligonucleotides are represented on the array.
4. A device as described in any of the previous Claims, wherein the oligonucleotides are anchored to the solid support by chemical modifications at their 5' or 3' end.
5. A device as described in Claims 1 to 3, wherein the oligonucleotides are synthesised in situ by standard procedures.
6. A device as described in any of the previous Claims, wherein the oligonucleotides are spaced away from the solid support by a chemical spacer.
7. A device as described in Claim 6, wherein the chemical spacer is between 6 and 40 carbon atom equivalents.
8. A device as described in any of the previous Claims, wherein the chromaphore (CHI) is spaced away from the oligonucleotide using a chemical spacer.
9. A device as described in any of the previous Claims, wherein the oligonucleotides are made of any naturally occurring nucleotide or analogue of those.
10. A method of detecting interactions between oligonucleotides and RNA using the device as described in any of the previous Claims, wherein:
a) 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
b) where the labelled RNA is applied to the array and allowed to bind to complementary oligonucleotides; and
c) an appropriate light source is used to excite one of the matched pair of chromaphores; and
d) emissions from the second of the chromaphores is detected.
11. A method as described in Claim 10, wherein step a) is amended so 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.
12. A method as described in Claims 10 or 11, wherein the RNA is in sufficiently high concentration to drive the formation of duplexes with the oligonucleotides on the array.
13. A method as described in Claim 10 to 12, wherein the target RNA is transcribed in vi tro under conditions that allow authentic folding.
14. A method as described in Claims 10 to 13, wherein the RNA is labelled with a chromaphore by incorporation of a modified nucleotide during transcription.
15. A method as described in Claims 10 to 13, wherein the RNA is labelled with chromaphore post- transcriptionally by enzymatic or photochemical means .
16. A method as described in Claims 10 to 15 wherein the appropriate light source may be a laser, a diode or a lamp.
17. A method as described in Claims 10 to 16, wherein the RNA is added to the RNA application buffer that supports maintenance of secondary structure, promoting the formation of duplexes between applied RNA and oligonucleotides on the array.
18. A method as described in Claim 17, wherein the RNA application buffer is compatible with FRET and with maintenance of RNA secondary and tertiary structures.
19. A method as described in Claims 17 or 18, wherein a volume excluder is used in the RNA application buffer.
20. A method as described in Claim 19, wherein the volume excluder is polyethylene glycol (PEG) .
21. A method as described in Claim 19, wherein the volume excluder is dextran sulphate.
22. A method as described in Claims 10 to 21, wherein 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.
23. A method as described in Claims 10 to 22, wherein the signal from the array is interpreted by computer based algorithm.
EP02704918A 2001-03-08 2002-03-07 Detecting interactions between oligonucleotides and rna using fluorescence resonance energy transfer (fret) Withdrawn EP1370688A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB0105789 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)
PCT/GB2002/001016 WO2002072885A1 (en) 2001-03-08 2002-03-07 Detecting interactions between oligonucleotides and rna using fluorescence resonance energy transfer (fret)

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Family Cites Families (8)

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Publication number Priority date Publication date Assignee Title
US5538848A (en) * 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe
GB9401833D0 (en) * 1994-02-01 1994-03-30 Isis Innovation Method for discovering ligands
US6974666B1 (en) * 1994-10-21 2005-12-13 Appymetric, Inc. Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA
GB9620749D0 (en) * 1996-10-04 1996-11-20 Brax Genomics Ltd Identifying antisense oligonucleotides
SE522077C2 (en) * 1997-09-05 2004-01-13 Lightup Technologies Ab Method of selecting sequence of probe for nucleic acid hybridization
GB2335035B (en) * 1998-03-03 2003-05-28 Brax Genomics Ltd Screening for functional antisense agents
US6361942B1 (en) * 1998-03-24 2002-03-26 Boston Probes, Inc. Method, kits and compositions pertaining to detection complexes
US6312906B1 (en) * 1999-01-15 2001-11-06 Imperial College Innovations, Ltd. Immobilized nucleic acid hybridization reagent and method

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Title
See references of WO02072885A1 *

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