WO2007054682A1 - Biodetecteurs commutables - Google Patents

Biodetecteurs commutables Download PDF

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Publication number
WO2007054682A1
WO2007054682A1 PCT/GB2006/004161 GB2006004161W WO2007054682A1 WO 2007054682 A1 WO2007054682 A1 WO 2007054682A1 GB 2006004161 W GB2006004161 W GB 2006004161W WO 2007054682 A1 WO2007054682 A1 WO 2007054682A1
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pro
switch
macromolecular structure
target
macromolecular
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PCT/GB2006/004161
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English (en)
Inventor
Peter Ghazal
Colin James Campbell
Amy Buck
Paul Dickinson
Jonathan Gordon Terry
Anthony John Walton
Elena Eduardovna Ferapontova Tereshkova
Christopher Mountford
Jason Crain
John Samuel Beattie
Andrew Raymond Mount
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The University Court Of The University Of Edinburgh
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Priority to EP06808456A priority Critical patent/EP1951896A1/fr
Publication of WO2007054682A1 publication Critical patent/WO2007054682A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to the use of novel switchable macromolecular structures for specific analyte detection.
  • DNA hybridisation assays are fundamental to much of life sciences research and especially in the application of DNA microarrays.
  • the generation of such arrays allows the simultaneous measurement of the expression of tens of thousands of the corresponding messenger RNA molecules.
  • Measurement of RNA molecules is generally achieved in an indirect manner by transformation to DNA via the multistage enzymatic process of reverse transcription. During this process, a certain proportion of fluorescently labelled nucleotide is introduced into the DNA molecule.
  • binding of target to probe molecules can be detected using confocal fluorescent scanning.
  • a macromolecular switch suitable for use in a data acquisition and/or processing device, comprising: a macromolecular structure having at least one ion binding site, and flippable between a plurality of discretely different conformations corresponding to different ion binding conditions at said at least one ion binding site, at least in the absence of any other binding to said macromolecular structure, said macromolecular structure being provided with at least one electrochemical input device formed and arranged for applying such different ion binding conditions to said ion binding site in response to corresponding external input signals.
  • a particularly preferred form of macromolecular structure switch is based on the Holliday Junction.
  • the DNA Holliday Junction structure is a junction of four double helices first reported in 1964 Holliday, R. Genet. Res. 1964, 5, 282.
  • the unique topological element of the junction is a branch point discontinuity formed at the intersection of the component strands.
  • the overall structure of the open junction is determined primarily by the strong electrostatic repulsion between the phosphate groups. In low ionic strength solutions, this repulsive Coulombic interaction favors an extended structure determined by maximum charge separation, an open branch point region and approximate fourfold symmetry.
  • HJ Holliday Junction
  • pro-HJ pro- Holliday Junction
  • the pro-HJ adopts an informal folded structure but on hybridization with a target oligonucleotide analyte forms a complete HJ (see Figure 1 which shows schematically a target analyte about to hybridize with a pro-HJ so as to form a corresponding HJ).
  • Hybridization of the pro-HJ with a target oligonucleotide forms a switchable HJ, whose conformation can be probed using various methods without the need for labels on the target oligonucleotide.
  • the present invention provides a pro-macromolecular structure switch suitable for use in detecting an analyte, wherein said pro-switch has an analyte binding site formed and arranged for binding said analyte with high specificity so as to form together therewith, a macromolecular structure switch having at least one ion binding site, and flippable between a plurality of discretely different conformations corresponding to different ion binding conditions at said at least one ion binding site, at least in the absence of any other binding to said macromolecular structure, wherein said discretely different conformations are capable of providing, in use, with an output signal reading system, characteristic output signals and wherein said pro-macromolecular structure switch is substantially incapable of being flippable between said plurality of discretely different conformations and providing, in use, with said output signal reading system, said characteristic output signals.
  • the present invention also provides a pro-macromolecular structure switch suitable for use in detecting an analyte, wherein said pro-switch has an analyte binding site formed and arranged for binding said analyte with high specificity so as to form together therewith, a macromolecular switch, comprising: an oligomeric macromolecular structure having at least one selective ion coordination site, and arms flippable between a plurality of narrowly defined discretely different conformations of said oligomeric macromolecular structure corresponding to different ion binding conditions at said at least one selective ion coordination site, at least in the absence of any other binding to said oligomeric macromolecular structure which would interfere with flipping between said different conformations in response to a change between said different ion binding conditions, wherein said discretely different conformations are capable of providing, in use, with an output signal reading system, characteristic output signals, and wherein said pro- macromolecular structure switch has a substantially different switching functionality from that of said oligo
  • the macromolecular structures used in accordance with the present invention are flippable between more or less well defined discretely different conformations or structural forms of a bound set, i.e. a finite limited predetermined number, of different conformations.
  • This is quite different from previously known systems in which it has been proposed to use macromolecules which are described as being switchable between different "states", which typically correspond to one state in which the macromolecule is bound to some other moiety, and another state in which it is not bound to said moiety, and/or involve one or more conformational states which is (are) not well defined to a greater or lesser extent and/or are part of a large spectrum or continuum of different conformations, and/or actually involve dissociation into multiple separate macromolecular structure entities.
  • the qualifier "pro-” is used herein in accordance with common practice in the field of biotechnology, to indicate a precursor which is converted, in use of the precursor, to the entity being qualified. Thus in the present case this term indicates a precursor with a “switching" functionality that differs significantly from that of the oligomeric macromolecular structure switch.
  • detecting indicates qualitative and/or quantitative detection, including inter alia simply detecting the presence or absence of a target analyte, and simply detecting a change in the concentration of a target analyte.
  • oligomeric macromolecular structure indicates a macromolecular entity of intermediate relative mass, hi general this corresponds to a structure consisting essentially of a switch device, including any associated components such as anchoring and/or switch state signaling component(s), but substantially free of any other non-functional components.
  • the oligomeric macromolecular structures of the invention will comprise not more than 500, preferably not more than 250, most preferably not more than 150, repeating units such as nucleotides, amino acids, saccharides.
  • selective ion coordination site indicates a site which can selectively bind certain ions.
  • said site may comprise an "ion coordination pocket".
  • ion coordination pocket indicates a structural feature which contributes energetically and/or spatially to the selectivity of the binding of ions thereto.
  • ion binding conditions includes physico-chemical conditions such as concentration, ionic strength, temperature, electric field strength etc, the effect of differences in the structure of the macromolecular structure switch such as those due to the specific sequence at the HJ branch point and also features such as the nature, identity, ion size, etc of the particular ion species involved in binding to the ion binding site(s).
  • the present invention provides a method for detecting a target analyte in a sample, which method includes the steps of: providing a pro-macromolecular structure switch, wherein said pro-switch has an analyte binding site formed and arranged for binding said analyte with high specificity so as to form together therewith, a macromolecular structure switch having at least one ion binding site, and flippable between a plurality of discretely different conformations corresponding to different ion binding conditions at said at least one ion binding site, at least in the absence of any other binding to said macromolecular structure, wherein said discretely different conformations are capable of providing, in use, with an output signal reading system, different characteristic output signals and wherein said pro-macromolecular structure switch is substantially incapable of being flippable between said plurality of discretely different conformations and providing, in use, with said output signal reading system, said different characteristic output signals; contacting said pro-switch with a said sample under binding conditions, so
  • the present invention also provides a method for detecting a target analyte in a sample, which method includes the steps of: providing a pro-macromolecular structure switch wherein said pro-switch has an analyte binding site formed and arranged for binding said analyte with high specificity so as to form together therewith, a macromolecular switch, comprising: an oligomeric macromolecular structure having at least one selective ion coordination site, and arms flippable between a plurality of narrowly defined discretely different conformations of said oligomeric macromolecular structure corresponding to different ion binding conditions at said at least one selective ion coordination site, at least in the absence of any other binding to said oligomeric macromolecular structure which would interfere with flipping between said different conformations in response to a change between said different ion binding conditions, wherein said discretely different conformations are capable of providing, in use, with an output signal reading system, characteristic output signals, and wherein said pro-macromolecular structure
  • biomolecule is used herein to encompass not only molecules found in living organisms, but any molecule having a character substantially similar to such molecules, howsoever, obtained.
  • biomolecule includes oligonucleotides or polypeptides whether made by processes having a biological character or purely organic chemistry synthetic processes, and oligonucleotides or polypeptides which are entirely artificial, i.e. are not found in nature.
  • references to "macromolecular structure” herein include both discrete individual molecules and macromolecular structure entities comprising more or less closely bound combinations of a plurality of discrete individual molecules, the integrity of which combinations is maintained across said discretely different conformations .
  • a Holliday Junction (HJ) (discussed in more detail elsewhere herein) is generally a combination of four nucleic acid strand sequences (or like entities) bound together into a single macromolecule entity with four arms, each said arm comprising respective portions of two strand sequences bound to each other.
  • these four sequences may all be part of separate strands, or two or more of said sequences may be part of a single strand with a molecular linkage extending between them, said strand being suitably folded to allow said sequences to hybridize with other sequence portions so as to form the HJ (or pro-HJ) structure.
  • One type of molecular linkage will use co-valent cross-linking between strands.
  • molecular linkages comprise strand sections which link the ends of strand sections which are hybridized together to form an arm of the pro-HJ.
  • Such linkages generally comprise any neutral sequence of nucleic acid bases which would not interfere with binding between complementary strand sequences to form the arms of the desired HJ structure, such as e.g.
  • a pro-HJ macromolecular structure switch comprises a single polynucleotide strand including six said strand sequence portions, wherein four said strand sequence portions are hybridized together so as to form two HJ arms (double- stranded), and the other two said strand sequence portions form two HJ pro-arms (single- stranded), which together provide said target analyte binding site, and when having target analyte bound thereto, constitute the other two arms of the HJ macromolecular structure switch.
  • the total output signal obtainable from a switch device of the present invention will depend on the number or population size of individual pro- macromolecule structures used in each device.
  • the device of the present invention has a plurality of said macromolecule structures. Desirably there is used at least 5, more desirably at least 10, preferably at least 100, advantageously at least 1000, most conveniently at least 10,000, pro-macromolecule structures, in each switch device.
  • the pro-macromolecular structure should generally be formed and arranged to facilitate applying the different ion binding conditions to said ion binding site of the macromolecular structure formed upon binding of the pro-macromolecular structure with target analyte. In general this involves retaining the pro-macromolecular structure in proximity to said input device, in a "fluid-interfaceable condition".
  • fluid — interfaceable condition means that the pro-macromolecular structure is supported in any convenient manner which would permit interaction between the macromolecular structure and a fluid medium.
  • the pro-macromolecular structure may be anchored in a suitable manner to a substrate having a suitable form, so that said structure can interact with components present in a fluid medium with which the device is contacted in use of the device.
  • the structure would generally be mounted on the surface of a solid substrate, it could also be embedded inside a more or less permeable substrate, which may moreover be more or less flexible, such as, for example a gel.
  • the gel should be chosen as to have a pore size sufficiently large to enable the target components in the fluid medium to be able to penetrate the gel and come into contact with the structure. It is also important that the gel pore size should be sufficient to allow adequate freedom of movement of the structure between its first and second conformations.
  • the pro-macromolecular structure could also in principle simply be suspended in solution within a cell or other vessel provided with said input device.
  • a cell may moreover be defined by fluid impermeable walls, or in some cases, at least partly, by fluid permeable walls such as dialysis membrane.
  • fluid permeable walls such as dialysis membrane.
  • pro-macromolecular structures may be susceptible to denaturation or other damage to a greater or lesser degree under certain conditions e.g. high and/or low pH, and it is therefore desirable that this should be taken into account in relation to the properties of any medium through which the pro-macromolecular structure is supported and/or interfaced through with biomolecule analytes.
  • pro-macromolecular structures may be modified so as to stabilize them to a greater or lesser extent against denaturation or disintegration, for example by means of co-valent and non-covalent cross-linking of the hybridized polynucleotide sequences within one or more arms of an HJ.
  • Examples of covalent cross linking includes psoralen, (Saffran et al. Proc. Natl. Acad. Sci. 1982, 79, 4594-8) and Iodo dT / phosphotioate modification (Roche, CJ. and Tse-Dinh, Y.C. Int. J. Biol. Macromol. 2001, 29, 175-180 ).
  • Examples of non-covalent cross linking include triple helix structures (Welch et al., Nucleic Acids Res. 1993, 21, 4548-4555) and hairpin polyamides (Herman, D.M.; Baird, E.E.; Dervan, P.B. J. Am. Chem. Soc. 1998, 120, 1382-139.
  • An example pro- HJ using cross linking is illustrated in Figure 2.
  • macromolecular structures may be stabilized by increasing the number of bonds. For example it is known that short armed HJ structures disassemble in low salt conditions (Shida, T. et al. J. Biochem. 1986, 119, 653-658 )) but longer-armed structures are more stable.
  • bridging sequences e.g. TTTT at the end of the HJ arms to form uni-, bi- or tri- HJs or pro-HJs, as described above, is that this would also increase the stability of the HJ structures against strand disassociation.
  • HJ structures are also obtainable by means of using synthetic polynucleotide analogues based on the use of size-expanded analogues of the natural bases (for example, using a three ring analogue of a bicyclic purine and a bicyclic analogue of a monocyclic pyrimidine as discussed by Liu et al in Science, VoI 302 Issue 5646, 868-871 (2003).
  • HJ is intended to encompass modified HJs.
  • Increased stability may also be achieved to a greater or lesser degree by the use of pro-HJs wherein the number of separate strands (as discussed hereinbefore) is minimized.
  • Modifications to the oligonucleotide(s) of oligomeric macromolecular structures of the present invention may also be used to modify the sensitivity of target analyte detection.
  • LNA locked nucleic acid
  • modifications wherein the ribose moiety of the LNA nucleotide is modified with an extra bridge connecting 2' and 4' carbons which "locks" the ribose in a 3'-endo structural conformation
  • LNA locked nucleic acid
  • the effect is dependent on the position within the pro- HJ with modifications remote from the branch point being preferred.
  • Nucleic acid modifications may also be used to enhance the switching performance of oligonucleotides macromolecular switches. Possible modifications which may be mentioned in this connection include LNA, PNA, phorphorthioates, non-natural bases such as size- expanded analogs of nucleotides, and other like chemical alterations to the polynucleotide backbone or base structure.
  • the electrode can be chosen from any of an assortment of conductors or semiconductors. These range from metals such as silver, gold, platinum, titanium, tantalum, tungsten, aluminium, nickel, zinc, copper and alloys of these or other metals to semiconducting or conducting oxides, nitrides or mixed oxynitrides of metals such as titanium, nickel, iron, tin, indium, tantalum, strontium, iron, tungsten, niobium, iridium, molybdenum, hafnium, zinc, aluminium and zirconium and doped versions thereof.
  • metals such as silver, gold, platinum, titanium, tantalum, tungsten, aluminium, nickel, zinc, copper and alloys of these or other metals to semiconducting or conducting oxides, nitrides or mixed oxynitrides of metals such as titanium, nickel, iron, tin, indium, tantalum, strontium, iron, tungsten, niobium, iridium
  • Electrodes may also be chosen from materials such as carbon, conducting and semiconducting molecular organic systems, inorganic and organic charge transfer salts (e.g. tetrathiafulvalene-tetracyanoquinodimethane), and semiconducting materials such as silicon, gallium arsenide or conducting polymers such as polypyrrole, polythiophene or polyaniline.
  • a biomolecule may generally be anchored to an electrode or other suitable substrate using one or more of three principal approaches: Physisorption, chemisorption or covalent attachment.
  • Chemisorption is a more specific form of orientation since it relies on the favoured interaction between a surface and a particular moiety on a molecule, examples of such are the interaction between thiols and gold (Whitesides et al., Chem. Rev. 2005 105, 1103-1169) and the interaction between phosphonic acids and metal oxides ( Long B., Nikitin K., Fitzmaurice D., J. Am. Chem. Soc. 2003, 125, 5152-5160).
  • Covalent attachment of biomolecules relies on the modification of an electrode surface so that it is intrinsically active to or can be activated to attach to specific, functional groups on the biomolecule.
  • Suitable treatment might be the oxidation of carbon electrodes to form carboxylic groups on the surface which can be activated to bind with amine groups Campbell et al., Langmuir 2001 17, 3667.
  • Another example would be the use of a functional silane compound (for example an epoxide terminated silane) to form a self-assembled, covalently attached layer that can be used to covalently attach a polymer on, for example, a metal oxide Cass, Anal. Chem 2001 73, 2676-2683.
  • epoxysilane can be polymerised onto a metal oxide such as titanium dioxide to form a surface with active epoxy groups that can react with amine functions on the surface of a biomolecule.
  • the surface coating can be made sufficiently thin so as to not hinder electron transfer across the interface.
  • an amino group on the 5 '-terminal of one arm of the HJ structure or an internal NH 2 dT linkage may itself be used for covalent bonding directly to a substrate.
  • a linking moiety such as a biotin molecule may be attached to the 5'-terminal of one arm of the HJ structure.
  • a polyion such as protonated poly-1-lysine can be physisorbed onto a charged electrode surface on the basis of charge pairing.
  • the poly-1-lysine is a polypeptide with amino side chains, the amino groups can be attached to activated carbonyls on a biomolecule or cross linked to a biomolecule in order to give covalent attachment.
  • Chemisorption of a bifunctional alkyl thiol can convert a gold surface into a surface reactive to biomolecular binding.
  • mercaptopropionic acid can be chemisorbed onto gold to give a self-assembled monolayer with carboxylic acid moieties reaching into the bulk solution.
  • carboxylic groups can be activated by carbodiimide chemistry to react with amine groups on a biomolecule.
  • the pro-switches can also be anchored by means of physical entrapment. This involves the formation of a polymer network or sol-gel or hydrogel substrate that has a pore size dimensioned so as to enable it to entrap a biomolecule during its formation. Although this can give a stable environment for the biomolecule, the surrounding matrix limits its interaction with other biomolecules to a greater or lesser degree. Also, since entrapment is not a covalent immobilisation it may be prone to leaching to a greater or lesser degree.
  • Suitable gels include agarose, polyacrylamide gels, polysaccharide hydrogels, for example carboxy-methyl dextran. It will be appreciated that when gels are used, the pro-macromolecule structures can also be covalently bonded to the gel matrix in order to prevent escape thereof over a period of time. Furthermore, pro- macromolecule structures can also be "anchored" by means of relatively strong non-covalent bonding e.g. biotin binding to streptavidin.
  • the pro-macromolecular structure switches of the invention may moreover be incorporated in a wide variety of different kinds of apparatus adapted for use in various types of in vitro assay system.
  • the pro-switches maybe incorporated in eppendorf tubes, multi-well plates, or in gels, or single channel analysis devices in conventional or micro- scale systems.
  • Example configurations are illustrated in Figures 7-12.
  • the switchable biosensors could be used in a industry standard multiwell plate format, containing typically 96, 384 or 1536 wells, or alternatively in a bespoke plate of multiple wells of the same or different sizes, with a population of pro-macromolecular switches deposited in each well, either in solution or with the pro-macromolecular switches immobilized to the inside of the well.
  • Wells would each typically contain distinct homogeneous populations of pro-macromolecular switches, with solutions of test sample added, but could include replicate assays or assays based on heterogeneous pro- macromolecular switch populations.
  • an optical separation-sensitive output device such as FRET could be read using a standard fluorescence plate reader, typically based on a cooled CCD camera, reading fluorescence intensity or fluorescence lifetime measurements.
  • FRET optical separation-sensitive output device
  • Figure 7 optical signals could be detected in optical fibres positioned into each microwell, or electrochemical signals could be detected using electrodes configured within the wells.
  • Ionic switching of the switchable biosensors could be achieved by introduction of ionic solutions into each well using fluid-handling robotics, or electrochemically generated using electrodes .configured within each well, hi a further variation, pro macromolecular structures could be attached onto microbeads, desirably of 1-70 ⁇ m, which are arranged in micro-scale wells of different or similar size as illustrated in Figure 9.
  • the switchable biosensors could be used in a microarray format, where a population of pro-macromolecular switches would be immobilized into micro-scale features or spots, with pro-macromolecular switches immobilized on a solid substrate or within a gel matrix as decribed hereinbefore. Spots would each typically contain distinct homogeneous populations of pro-macromolecular switches, but could include replicate assays or assays based on heterogeneous pro-macromolecular switch populations.
  • This arrangement is illustrated in Figure 8.
  • the test samples could be introduced onto the microarray using fluid-handling robotics, either across the whole array, across segmented array sections or individually to each spot using, for example, ink jet printing.
  • an optical separation-sensitive output device such as FRET could be read using a standard microarray reader, typically based on a cooled CCD camera, reading fluorescence intensity or fluorescence lifetime measurements.
  • optical signals could be detected in optical fibres positioned into each microarray, or electrochemical signals could be detected using electrodes configured on the microarray.
  • Ionic switching of the switchable biosensors could be achieved by introduction of ionic solutions onto the microarray using fluid-handling robotics, either across the whole array, across segmented array sections or individually to each spot using, for example, ink jet printing.
  • ionic switching would be electrochemically generated using electrodes configured on the microarray.
  • an array of electrodes could be constructed on the microarray which could be used to move fluid droplets around the array surface using electrostatics.
  • droplets of test solution and high/low salt buffer could be moved in turn onto an location upon which pro-macromolecular switches have been located, either in droplet form themselves or immobilized to the array surface. This configuration is illustrated in Figure 12.
  • the switchable biosensors could be used in a electrophoretic separation device, including conventional gels, microfluidic biochips or polymer tape-based configurations.
  • a population of pro-macromolecular switches which has previously been mixed with solutions of test sample would be located at one end of a channel of gel matrix as decribed hereinbefore.
  • Channels would each typically contain distinct homogeneous populations of pro-macromolecular switches, but could include replicate assays or assays based on heterogeneous pro-macromolecular switch populations.
  • Figure 10 standard electrophoresis methods can be used whereby an electrical potential is applied to induce molecular movement along the channel through the gel matrix, due to the inherent charge of the molecules.
  • the differing sizes and/or charges of the molecules will determine the distance moved by the macromolecular switches in each the gel channel. In this way it is possible to discriminate between pro macromolecular switches with no bound target and macromolecular switches with a bound target and this is illustrated by a standard gel assay in Figure 4.
  • ionic switching of the switchable biosensors could be achieved by direct introduction of ionic solutions into the gel channels or by in-situ ion-flux generation using electrochemistry.
  • the switching characteristics of a functional, macromolecular switch can then be detected using separation-sensitive output devices, as illustrated in Figure 4 using FRET.
  • An optical separation-sensitive output device such as FRET could be read using standard gel scanner technology, reading fluorescence intensity or fluorescence lifetime measurements.
  • optical signals could be detected in optical fibres positioned into each channel, or electrochemical signals could be detected using electrodes configured in the channel.
  • ionic switching would be electrochemically generated using electrodes configured in the gel channel.
  • the switchable biosensors could be used as an inline detection format where a population of pro-macromolecular switches would be moved in solution or immobilized into micro-scale beads through an in-line fluidic system incorporating detectors.
  • an optical separation-sensitive output device such as FRET could be read using an in-line fluorescence reader, typically reading fluorescence intensity or fluorescence lifetime measurements or electrochemical signals could be detected using electrodes configured into the fluidic apparatus.
  • Ionic switching of the switchable biosensors could be achieved by introduction of ionic solutions into the flow using valved fluid inlets or would be electrochemically generated using electrodes configured in the fluidic apparatus. This arrangement is illustrated in Figure 11.
  • the different conformational states of the macromolecular structure of the macromolecular structure switches formed in use of the present invention will correspond to different separation distances of particular (first and second) parts of the macromolecular structure, whereby changes in conformation state may be conveniently monitored through the change in output signal from a separation sensitive output device (described in more detail hereinafter).
  • Such devices will generally include respective output device components mounted (directly or indirectly) on relatively displaceable first and second parts of the pro- macromolecular structure switch.
  • pro-HJ switches such components are conveniently mounted at end portions of the HJ arms, preferably remote from the analyte binding site.
  • Example 4 shows how increasing the distance along arm length away from the branch point, at which the fluorophores are attached, increases the separation of the fluorophores in the 'open' state, reducing any background signal due to the closeness of these fluorophores when attached close to the branch point.
  • Position of the separation-sensitive output device components is a tuneable factor in selecting optimum macromolecular switch performance. The optimum will depend on the application for which the switch is used, will vary for different macromolecular structures and may be readily determined by trial and error. For example, deliberately placing the separation-sensitive output device components close enough that there is always a background signal, even in the open configuration, may be desirable as it forms a in-built control signal which if not present can detect malfunction of the bioswitch device.
  • separation sensitive signal output device may be used in accordance with the present invention.
  • such devices are formed and arranged so as to provide discretely different output signals in said discretely different conformations of said macromolecular structure.
  • a FRET system comprising a first separation sensitive output device component in the form of a donor fluorophore mounted on the first part and a second separation sensitive output device component in the form of an acceptor fluorophore mounted on the second part of the macromolecule structure.
  • an optical signal is "input" into the donor to excite the donor which then transfers energy (non-radiatively) to the acceptor which then provides an output optical signal.
  • the energy transfer from the donor to the acceptor is sensitive to the separation therebetween, thereby affecting the intensity of the output signal.
  • the donor and acceptor fluorophores must be in relatively close proximity- generally not more than 10 nm, advantageously from 0.2 to 10 nm, preferably in the range from 2 to 8 ran.
  • FRET does take place, as well as being indicated by the presence of an output signal from the acceptor fluorophore which is distinguishable from any donor fluorophore fluorescent radiation by having a longer wavelength, it can also be detected by a reduction in the donor fluorophore fluorescent radiation signal level.
  • acceptor fluorophore output signal is quantized and binary in nature in that it provides a 0 output signal in the absence of FRET and a 1 output signal in the presence of FRET.
  • acceptor fluorophore output signal is quantized and binary in nature in that it provides a 0 output signal in the absence of FRET and a 1 output signal in the presence of FRET.
  • non-separation sensitive fluorescent output signaling devices which can provide quantized output signalling.
  • a pro-HJ in which one of the Adenine nucleotides is replaced by a fluorophore such as 2- aminopurine (2AP) whose fluorescence is substantially quenched in the closed conformation of the corresponding HJ (but not in the open conformation), due to the influence of base stacking.
  • 2AP 2- aminopurine
  • one embodiment of the input and output signals of the separation sensitive signal separation sensitive output device reading system is optical signals which can readily be transmitted through an aqueous medium in which the macromolecule structure is supported in use of the device, and which can be more or less readily integrated with suitable electronic, opto-electronic, or optical signal processing elements.
  • the input signals to the macromolecular structure switches formed in use of the invention, for reading out analyte detection, may be produced in any convenient manner depending on the form and nature of the separation sensitive output device.
  • an electro-optical transducer such as LEDs or lasers to produce optical radiation at a frequency suitable for use with the separation sensitive separation sensitive output device reading system (as further discussed hereinbelow).
  • This optical radiation may be delivered from the electro-optical transducer substantially directly into the solution in which the "input connection" component of the separation sensitive output device of the macromolecular structure switch is supported, or via a suitable waveguide.
  • the separation sensitive output device reading system signal outputs from the switch devices in use of the invention may correspondingly be received at said another part of the output signal reading system using any suitable form of opto-electronic transducer which can collect the optical data carrier signal outputs and convert them into electrical signals for further processing.
  • non-separation sensitive systems for reading of the conformation state, including for example, systems based on changes in physical properties associated with the changes in conformation with switching of the macromolecule structure, e.g. electrochemical impedance changes generally based on changes in capacitance and/or resistance and/or inductance of a macromolecule structure population.
  • electrochemical impedance changes generally based on changes in capacitance and/or resistance and/or inductance of a macromolecule structure population.
  • other physical parameter monitoring systems based on structural physical changes such as a change in thickness of a macromolecule structure population layer (e.g. by dual polarization inferometry or other techniques), or as another example detection of microscale cantilever stress or resonant frequency changes.
  • the transformation or flipping of the macromolecule structure formed in use of the invention, between its different conformations is generally effected by applying suitable different ion binding conditions to the macromolecule structure ion binding site, depending on the general and/or particular nature of said macromolecule structure.
  • the macromolecule structure comprises a Holliday- Junction structure (conveniently referred to herein as an HJ) having two pairs of arms extending out from a "branch point".
  • HJ Holliday- Junction structure
  • HJs there are generally four possible “closed” conformers (two parallel, two anti-parallel) with different combinations of arms within the pairs and unconstrained HJs typically adopt the two anti-parallel conformations (Lilley, QIy Rev Biophysics 33,2 (2000) plO9-159).
  • the branch point migrates within the HJ structure.
  • the equilibrium position adopted between each of the two closed conformations may vary based on the specific HJ branch point sequence and/or the ion used and/or the chemical composition (e.g. DNA/RNA) (Ref: Grainger et al., Biochemistry 37, 23-32 1998).
  • DNA/RNA DNA/RNA
  • the position-sensitive output device such that an output signal were given whichever closed conformation each particular molecule of the population is in.
  • an acceptor dye to two opposite arms of the HJ with the donor dye attached to one of the other arms so that a FRET signal would be generated whichever of the two conformations is adopted by the HJ population.
  • multiple donors or acceptors to detect and distinguish between both conformers simultaneously.
  • ionic switching is sensitive to the efficiency and completeness of ion removal i.e. it is important to ensure that substantially all switching ion is removed to ensure a clean 'open' signal. This may be achieved for example by use of multiple spin columns (typically 99% removal) and/or use of an ion chelator such as ethylenediaminetetra(acetate) (EDTA).
  • EDTA ethylenediaminetetra(acetate)
  • the flipping of the HJ between open and closed conformations is affected by the ion binding conditions at an ion binding site in the region of the branch point, in at least two different ways including shielding of the negatively charged phosphate moieties of the sugar phosphate backbones within the branch point by positive ions so as to reduce repulsion therebetween, and specific binding of positive ions to the sugar phosphate backbones in the vicinity of the branch point. Hydroxyls of target RNAs in the branch point region could also participate in metal binding. Furthermore these effects can be additive.
  • metal cations including monovalent ones such as sodium and potassium, and divalent ones such as magnesium and zinc, which can be used to effectively control switching, with di- or tri- valent cations being effective at significantly lower concentrations than monovalent ones.
  • Advantageously switching ions may be selected to optimize one or more of various practically beneficial operating features such as minimal quenching, minimal absorbance, induction of a sharp transition between open and closed conformers of a macromolecular switch, and electrochemical activity in order to enable switching using electrodes.
  • the choice of ion to switch any particular macromolecular switch will also be dependent on the nature of the macromolecular switch used, and may readily be determined by trial and error. This may also include the use of combinations of two or more different ions.
  • particularly preferred tnetal cations are Mg 2+ , Zn + and Ca 2+ .
  • ions including buffer ions
  • ions which are electrochemically inactive and do not strongly switch the macromolecular devices at a known concentration
  • ions can be used to provide a sufficiently small screening length to act as background electrolyte for electrochemical studies and to help screen charges to optimise target hybridisation.
  • a particularly preferred cation is tetramethylammonium (or other tetralkylammonium ions) and a particularly preferred buffer cation is TrisH+, present in Tris/HCl buffer.
  • Also useful may be relatively highly charged cationic coordination complexes which are stabilised in solution by having the inner coordination shell of the metal occupied by strongly bound ligands. These systems are often electroactive, and their charge can be more or less readily varied electrochemically by changes in redox state , thereby readily facilitating electrochemical control of ionic binding conditions. This makes use of the fact that we have found there to be significant differences in ion concentration required for conformation flipping between ions of different charge.
  • One such suitable ion system which may be mentioned is Fe(bipy) 3 2+ ⁇ - -> Fe(bipy) 3 3+ + e " .
  • a further method for the electrochemical production of significant concentrations of redox inactive switching ions such as Mg 2+ is the production of redox active films (such as conducting polymers) containing covalently attached or insoluble cation chelating groups such as EDTA. If the reduced redox form of the film is neutral, Mg 2+ will be chelated in the film and the solution concentration near the film will fall. Upon electrochemical oxidation of the redox centres, an overall positive charge will be produced in the film which should lead to Mg + expulsion and a rise in the local concentration of Mg 2+ near the film.
  • the amount of cation(s) required will depend not only on the particular macromolecular structure used, and to some extent physical conditions (e.g. temperature), but also on the amount of macromolecular structure present. Suitable quantities in any given case may be readily determined by trial and error. Typically, though, for a macromolecular structure switch of the present invention containing 1 Micromolar of a suitable HJ, there would be required 100 micromolar OfMg 2+ (Duckett et al EMBO Journal 9 (1990) 583-590). It will be appreciated that various different polynucleotide sequences may be used in the HJs in accordance with the present invention.
  • Certain types of sequence are however preferred for various reasons including one or more of: dimensional stability of the HJ structure, resistance to damage e.g. by free radicals, functioning of the data carrier signal transmission system, etc.
  • dimensional stability of the HJ structure e.g. by free radicals
  • the donor and acceptor moieties are attached to the ends of the HJ arms to minimize background FRET signals, in so far as this will also reduce the level of the FRET signal generated.
  • a convenient positioning may readily be determined for each molecular structure design by trial and error.
  • the length of the polynucleotide sequence between the branching point and the donor and acceptor fluorophores should moreover be sufficiently long to fully avoid steric hindrance to opening and closing of the HJ, to ensure HJ stability and to ensure that data carrier signal transmission is substantially switched off in the open configuration of the HJ, without however being so long as to prevent data carrier signal transmission in the closed configuration.
  • the donor and acceptor fluorophores should preferably be mounted so as to mount these at from 4 to 20 bases from the branch point, advantageously from 6 to 12 bases therefrom.
  • the positioning of the output signaling system components within the pro-HJ can also be used to control the sensitivity of analyte detection. More particularly we have found that if one component (e.g. the Acceptor) is positioned at an end of a pro-arm (i.e. single stranded analyte binding arm) rather than internally (within an end portion of a non-pro-arm), then higher sensitivity to target analyte mutations at positions which bind the Acceptor-labelled pro-arm remote from the HJ branch point, can be obtained.
  • one component e.g. the Acceptor
  • the anchored arm of the HJ is preferably of a length sufficient to provide at least one complete double-helix turn in order to provide a more stable support for the other arms.
  • Dimensional stability may also be enhanced by using a so-called tripod mounting anchoring moiety which supports the anchored arm of the HJ in a substantially upright manner projecting out away from the substrate surface thereby reducing interference with the desired operation of the HJ structure in relation to one or more of interactions with target molecules, conformational switching etc (see for example Long B., Nikitin K., Fitzmaurice D., J. Am. Chem. Soc. 2003, 125, 5152-5160). Stability of the HJ structure may also be enhanced against separation of the polynucleotide strands of the arms, by binding them together with suitable bridging moieties.
  • the binding specificity of the analyte binding site will generally be dependent on the extent thereof. In the case of a pro-HJ switch, this will depend on the lengths of the polynucleotide strand sequence portions of the HJ single-stranded pro-arms. Desirably these arm portions should each have a length of at least 8 bases, preferably from 10 to 60, most preferably from 12 to 30. With covalent cross-linkage in the double- stranded arm, shorter double-stranded arm lengths less than 8 bases may be used, desirably from 4 bases, as the resultant macromolecular structure is more tightly bound in its assembled form
  • the various arms and pro-arms may have similar lengths or significantly different lengths, and hence that (where the pro-HJ is comprised by more than one polynucleotide strand), the strands may have various different lengths (bearing in mind also that in some cases an arm may have a non-hybridized strand sequence portion extending therefrom).
  • HJ-like structures which have first and second, discretely different, conformations flippable in substantially similar manner to conventional HJs (consisting essentially of polynucleotide sequences), may also be constituted by molecular species in which a greater or lesser portion of the four HJ arm structures is comprised by non-polynucleotide material, and may be of any other organic and/or inorganic material which is not incompatible with the operation and application of the macromolecular structure, provided that the branch point of the HJ-like macromolecular structure has at least the four interacting polynucleotide dimer sequence elements of an HJ.
  • HJ-like macromolecular structure may also be comprised by the corresponding polynucleoside components of polynucleotides, phosphorothioates or methyl phosphonates. Choice of these may be used to design the HJ to give the desired switching effect Ref: Liu et al, 2004 JMB 2004.) Accordingly any references to HJ structures herein are also intended to encompass such HJ-like structures unless the context specifically requires otherwise.
  • the fluorophore receives energy from an exciting wavelength radiation source - corresponding to an input signal, it fluoresces sending out radiation at another wavelength - corresponding to an output signal which can be detected.
  • the intensity of the output signal obtained is sensitive to any quenching agents which may be present in proximity to the fluorophore. Accordingly when the macromolecule structure is transformed from its first conformation to its second conformation, the quenching of the fluorescent radiation is changed thereby changing the output signal.
  • quenching modulation can also be made use of in conventional FRET systems (based on energy transfer from a donor fluorophore to an acceptor fluorophore) where energy transfer to the acceptor is quenched by close interactions between the donor and another material such as, for example, DNA.
  • hybridisation of a target molecule with part of the HJ can be used to reduce such interactions thereby reducing the quenching effect and increasing the output signal from the acceptor.
  • suitable precautions should be taken.
  • the pro-HJ structure may be maintained interfaced with an aqueous medium of HJ - compatible ionic strength.
  • the medium should be a buffer solution which contains one or more large monovalent cations at a concentration well below the concentration at which the ion switches the macromolecular structure.
  • 20 mM Tris/HCl buffer at pH 7.5 is a particularly convenient buffer, but other buffers may also be used.
  • the buffer pH is from 7-8. Nevertheless pro-HJ integrity may also be maintained in other ways, for example, by means of cross-linking together of the double stranded polynucleotide strands (as further discussed hereinbelow) - even under less favourable ionic strength or other conditions.
  • the ion-binding conditions so as to transform the structure between first and second conformations in various ways including physical and/or chemical perturbations such as change of ionic strength and/or composition in the aqueous medium, changing a voltage applied to the aqueous medium (including changing from no applied voltage), changing the temperature or pressure of the aqueous medium, and changing the concentration of particular organic or inorganic molecules in the aqueous medium which have specific interactions (for example multiple hydrogen bonding)- as distinct from general physical interactions such as those due to ionic strength.
  • it is also possible to change the ion binding conditions by discretely or progressively, replacing the fluid medium in which the macromolecular structure is supported.
  • magnesium ions have a specific effect in switching individual HJ structures from an "open" configuration to a “closed” configuration, and are effective at significantly lower concentrations than monovalent cations such as Na + and K + .
  • monovalent cations such as Na + and K + .
  • a Mg 2+ concentration of at least l ⁇ M, conveniently from lO ⁇ M to 10 mM is required to switch the HJ from open to closed configurations.
  • Various other divalent and multi-valent cations may also be used in this way.
  • Other switching cations which may be mentioned include Al(III), which can be electrochemically generated from insoluble Al, and Hexamminecobalt (III) [Co(NH 3 ) 6 3+ ], which allows electrochemical switching as it has a lower switching concentration than the soluble hexamminecobalt (II) [Co(NH 3 ) 6 2+ ] produced by electrochemical reduction.
  • an HJ structure can also be transformed from its open to its closed conformation in the presence of protonated polyamine species such as the protonated spermidine cation.
  • Suitable Polypeptide macromolecular structure switches according to the present invention also include inter alia metallo-enzymes that have discretely different conformations between which it is fiippable upon binding to an ion.
  • Particularly preferred macromolecular structures according to the present invention are those comprising at least one polynucleotide, and most preferably those comprising a Holliday Junction.
  • Particularly suitable other (Non-HJ based) pro-switch macromolecular structures which could be used in accordance with the present invention include 3 way nucleic acid junctions (Ref: Welch et al., NARS 1993, 21, 4548-4555, 1993), and zinc fingers (further described below).
  • Another type of macromolecular structure which may be used in accordance with the present invention is an ion-induced polysaccharide conformational switch such as E.coli Kl capsular polysaccharide (Koenig and McLean, Biometals VoI 12, Number I/March, 1999) or other synthetic polysaccharides e.g. Henry et al, Carbohydrate Research 315 (1999) 48-62.
  • biomolecule analyte binding sites in the case of HJ macromolecular structures, these can conveniently be in the form of so-called "aptamer" polynucleotide sequences which function as receptors for specific target biomolecule analytes from a wide range of biomolecule types including not only nucleotide sequences, but also polypeptide sequences, and which aptamers are obtainable by use of the well known SELEX in vitro selection technique in which very large numbers of random polynucleotide sequences are iteratively screened and amplified for binding to a particular target biomolecule analyte.
  • suitable polypeptide analogue biomolecule binding aptamers are obtainable by use of well known in vitro selection techniques such as Phage Display.
  • the so-called "zinc finger” which comprises a polypeptide which has a conformationally transformable portion which switches from a generally straight extended configuration into a loop which can bind DNA (see for example Wolfe et al Biochemistry 42 (2003) 13401-9).
  • the first and second, portions of the separation sensitive data carrier signal transmission portion e.g. donor and acceptor fluorophores of a FRET system
  • the separation sensitive data carrier signal transmission portion e.g. donor and acceptor fluorophores of a FRET system
  • a zinc finger involves the coordination of a metal ion with 4 amino acid residues at a metal coordination site - the amino acid residues for metal coordination being cysteine (C) and histidine (H).
  • C cysteine
  • H histidine
  • the zinc coordination site is split between two separate polypeptide arms. Each arm is comprised by two functional modules. In a given arm, one module is comprised of either one half of the metal coordination site (ie the C2 or H2 moiety) and the other module of the protein interaction domain. Labels for donor and acceptor are positioned on the polypeptide arms such that FRET will only be detected when a functional Zn finger coordination occurs and which prefers/requires intramolecular association. This will occur only if correct and specific association between binding modules occurs. Note that a restriction of such interactions is that they must be compatible with the formation of the zinc coordination centre (ie cannot be too bulky as to cause steric hindrance).
  • a protein interaction domain that would be feasible in the current invention is a leucine zipper (typically 6 - 24 amino acids long). There are over 300 members of this family and the association of a specific member is determined by its partner interaction.
  • a pro-switch could be formed using a "bait and prey" configuration for detecting protein- protein interaction (Fields, S. and Song, O.. Nature. 340 (1989) 245-6 ). Therefore one arm could include a 'bait' moiety - this a fixed probe sequence, and the other arm the 'prey' moiety(the target).
  • a 'bait' epitope would be made synthetically and assembled together with an attached polypeptide tail of 10-20 amino acids with cysteine (C) residues at either end, which is labeled with a donor fluorophore (D).
  • a sample containing the 'prey' epitope or target which could be a naturally occurring protein or a synthetic moiety depending on application, could be labeled with a polypeptide tail of 2-4 amino acids with histidine (H) residues at either end, and with an acceptor fluorophore (A) attached.
  • H histidine
  • A acceptor fluorophore
  • this acceptor labelled moiety could be attached post target binding, forming a label-free sandwich-assay system). This system is illustrated in Figure 3.
  • the bait and prey epitopes would selectively bind together upon specific leucine zipper partner association, the combined arms together forming a metal ion induced switch in conformation mediated by the Zn finger moieties.
  • this structure would form a zinc finger due to the specific chelation of the polypeptide tails, and a FRET signal could be generated.
  • the macromolecular structure In low salt conditions, the macromolecular structure would adopt a more open conformation and there would be a significantly lower FRET signal.
  • said first (open) and second (closed) conformations correspond to more or less narrowly defined stable states, whereby said transformation therebetween operates in an essentially quantized, binary (or ternary etc in some cases) or digital manner thereby yielding binary or digital output signals (0 or 1 etc) from said signal transmission portion.
  • the conformational flipping of the macromolecular structures can provide a substantially quantized output (with discretely different output signal values), it is also possible to use them to obtain analogue-form output signals.
  • analogue-type operation can be obtained in situations where the number of target molecules is significantly smaller than the number of macromolecule structures so that a positive output signal is obtained only from some of the macromolecule structures whereby the intensity of the output signal obtained depends on the concentration of the target molecule.
  • the macromolecular structure switches formed by binding of the pro-switches of the present invention with target analytes may, in principle, signal out the detection of the target analyte, in a variety of different ways.
  • transformation or flipping between different conformations may be obtainable by two or more different control signal inputs affecting ion binding conditions, for example, voltage and ionic strength, so that various combinations of different control signal inputs may be used which may have additive and/or subtractive effects (which may moreover be multi-dimensional), on the conformation transformations.
  • control signal inputs may have additive and/or subtractive effects (which may moreover be multi-dimensional), on the conformation transformations.
  • additive and/or subtractive effects which may moreover be multi-dimensional
  • the macromolecular structure switch is preferably configured so that the ionic binding conditions immediately before macromolecular structure switching, are close to the switching threshold for the ionic binding condition variable(s) being used to effect switching, in order to minimize the change in ionic binding conditions required for switching, hi another preferred form of the invention discussed elsewhere herein, the macromolecular structure may be pre-biased towards a switched conformation, against the restraint of a conformation "locking" device.
  • FRET fluorescence resonance energy transfer
  • Suitable donor fluorophores which may be mentioned include Cyanine 3 (Cy3) , FAM (carboxyfluoescein) and Fluorescein which are readily available commercially.
  • Suitable acceptor fluorophores include Cyanine 5 (Cy5), TAMRA (carboxytetramethylrhodamine) and Rhodamine (Tetramethylrhodamine) which are readily available commercially.
  • Cy3 has an excitation wavelength of 552 nm and Cy5 provides a maximum output radiation at 673 nm.
  • a suitable light source is used to supply radiation at a first wavelength to the donor fluorophore which transfers energy non-radiatively. If the acceptor fluorophore is within a predetermined range of the donor fluorophore (typically from 10 to 100 Angstrom), then the acceptor fluorophore will be able to convert energy received from the donor, into an output signal at a second wavelength which can be detected using suitable imaging spectroscopy apparatus.
  • separation sensitive output device designed to give quantized output
  • electrochemical where for example the environment around the redox-active active site of an enzyme tethered to one of the arms of the biomolecule can be altered by the change in conformation of the biomolecule, designed to induce a relative movement of an enzyme inhibitory substance tethered to the other arm. This would affect the enzyme reactivity, and hence the catalytic current due to the enzyme reaction, which could produce an electrochemical signal output. Such a catalytic system would also provide signal output amplification.
  • separation sensitive photoinduced charge transfer between, for example, a fluorophore and a quencher could be detected electrochemically.
  • fluorescent dyes should not be positioned next to a guanine residue to avoid quenching.
  • pro-HJs are not capable of flipping between discretely different conformations, in some cases they can nevertheless produce low level signals with separation sensitive detection systems (such as, for example, FRET) upon changing of ion binding conditions Typically though such signals are less than 30 % of the signal obtained with the corresponding HJ, and thus can readily be distinguished. Furthermore, it is possible to minimize this by positioning the fluorophores at increasing distances from the branch point, along the respective pro-HJ arms.
  • separation sensitive detection systems such as, for example, FRET
  • the liquid medium mediated input signal may take various different forms.
  • the input signal could be a mediator (e.g. NAD+/NADH, [Fe(CN)6] 3 -/4- or Fc/Fc+ where Fc is a soluble substituted ferrocene such as ferrocene carboxylic acid.
  • mediator e.g. NAD+/NADH, [Fe(CN)6] 3 -/4- or Fc/Fc+ where Fc is a soluble substituted ferrocene such as ferrocene carboxylic acid.
  • signalling in/out may be effected via two electrodes separated spatially either side of the HJs, one electrochemically generating mediator and the other ' detecting or collecting mediator which had undergone redox reaction (both electrodes could be held at the same potential).
  • Fc and GOD glucose oxidase
  • the rate of activity of the enzyme may be measured by the amount of Fc produced from electro generated Fc+, which is given by the current due to Fc — > Fc+ at the collector electrode.
  • the devices of the present invention will normally be employed with said pro- switches supported in an aqueous medium, it may be more convenient for storage and/or shipping purposes to present the devices in a lyophilised form, and accordingly the present invention also encompasses such forms thereof.
  • the invention may be used in a number of applications where the specific detection of target molecules, in particular nucleic acids, is a key feature. It is a particular advantage of the invention that the switchable macromolecular structure must both form AND it must display ion-controlled switching, in order to give a positive detection signal of the target molecule, as demonstrated in the following Examples. Detecting transition between different states depending on these two inputs allows a greater degree of interpretation about the target/pro-switch binding to be gained, and a greater degree of certainty about the interpreted result. This gives the specific benefit of a more definitive signal of target analyte detection, and less chance of false positive signals due to nonspecific binding.
  • transcript detection the available interpretations might comprise:
  • switchable biosensors will switch reversibly, so that the full characteristics of the macromolecular switch formed can be repeatedly tested, adding further robustness to the measurement Switchable biosensors are therefore more specific than the equivalent single- stranded oligonucleotide hybridization probes.
  • This feature leads to a specific advantage of the invention, namely the ability to detect target molecules with reduced false positive signals due to non-specific binding.
  • traditional single-stranded nucleic acid probe design involves a compromise between two factors, Short oligo probes (typically 20-3 Omer) are specific but less sensitive as they have fewer hydrogen bonds per molecule at a certain stringency, and long oligo probes (typically 50-70mer ) which have increased sensitivity but suffer from non-specific binding due to target loops and partial hybridization.
  • Short oligo probes typically 20-3 Omer
  • long oligo probes typically 50-70mer
  • the resulting trade-off results in either using large numbers of short oligo probes to detect a single target, which increases sensitivity while retaining specificity, or by careful design of a long oligo probe to reduce non-specific binding.
  • probes of shorter length than the equivalent single-stranded oligonucleotide hybridization probes could be used, simplifying oligo design and manufacture. In cases where multiple probes are used to screen out non-specific binding, the number of probes used can be reduced.
  • a pro-HJ is provided with a separation sensitive output device comprising a FRET dye pair ( Figure 1). Specific detection of an unlabelled target molecule results in the formation of the HJ switch, which can then be detected by operating the switch and sensing the FRET signal.
  • this approach offers benefits over other label-free techniques such as impedance, in that it retains the sensitivity benefits of a labelled detection system. This is further illustrated in Example 1.
  • hybrid macromolecular structure switches for example using a pro-HJ synthesized from DNA to specifically detect target RNA sequences, with high specificity and high sensitivity, without the need for analyte labeling.
  • arrays of switchable biosensors which will detect both DNA and RNA sequences by means of the use of a pro-macromolecular structure switch populations which binds together with a different components of the target analyte mixture, so as to form a functional macromolecular structure switch such as those described in our earlier WO2004/099767.
  • Branch point sequence is critical for the switching characteristics of the HJ switch, and in particular determines the ion binding affinity, and the thermodynamic equilibrium of the HJ conformers (Carlstrom & Chazin, 1996, Biochemistry 35, 3534 and Miick et al, 1997 PNAS 94, 9080). This provides the ability to recognize nucleotide mismatches at a specific position in the specifically bound target sequence which results in the combined benefits of specific sequence location together with specific base mutation detection. Switching characteristics are also influenced by the chemical composition at the branch point, and it is therefore possible to distinguish between DNA and RNA target sequences based on the different switching characteristics of the macromolecular switches formed.
  • switchable biosensors will have specific benefits in biological research, drug development and screening, diagnostic testing and as components of in- vivo medical devices.
  • the invention has particular application in the field of transcription profiling, where multiple analytes are detected in a single sample. It has advantages over existing techniques in the field, such as microarrays, in that it is free of sample labeling, it is less prone to false positives due to non-specific binding, can be used to monitor biodetection over time and can detect analytes of different types e.g. DNA, RNA, proteins.
  • the invention also has particular applications in the detection of single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • the switching characteristics of an HJ formed from a pro-HJ is particularly sensitive to the specific sequences at the HJ branch point, and so a single base pair mismatch can be detected, again in label free conditions.
  • the invention is also particularly advantageous in detecting a particular SNP in a region of DNA with other closely clustered SNPs.
  • There is also the potential of using single or small populations of pro-HJs which may allow detection of SNPs without the amplification required for most current SNP detection techniques.
  • the invention also shows particular benefits for the detection of RNA splicing.
  • an analyte binding site which corresponds to a target sequence that spans the junction between two exons - as illustrated in Figure 5, preferably with the branch point aligned with the boundary between the two exons.
  • Switchable bioswitch detection enables the use of a single probe to detect this splice junction, as in this case a successful positive result will only arise on the formation of a HJ with four double stranded arms.
  • the measurement of the switching characteristics described above would result in fewer false positives due to non-specific binding .
  • such a construction can be used to detect DNA insertions or deletion break points in which the target binding site spans the junction between the translocated DNA molecules - as illustrated in Figure 6, with the branch point is aligned with the break point.
  • the binding specificity of the analyte binding site - as indicated by hybridisation stringency in relation to HJ or HJ-like macromolecular structures will generally be dependent on a number of variables.
  • this will include inter alia the lengths of the polynucleotide strand sequence portions of the HJ pro- arms, and the base composition (ratio of G/C to A/T bases) within them, and their chemical composition e.g. DNA/RNA, as well as the presence of organic solvents and the extent of base mismatching, the combination of the various parameters being more important than the absolute measure of any one.
  • “Low” or “reduced stringency” refers to hybridisation and wash conditions of 6xSSC (standard sodium citrate) at 65 0 C.
  • “Normal stringency” refers to hybridisation and wash conditions of 2xSSC at 65°C while “high stringency” refers to hybridisation and wash conditions of 0. IxSSC at 65°C.
  • hybridisation may occur in the presence of 50% formamide at 42°C.
  • stringent conditions are selected to be approximately 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe.
  • stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH7 and the temperature is at least about 60°C.
  • pro-HJ target binding will require high stringency target binding, affording a high level of specificity.
  • further specificity can also be provided in lower stringency conditions due to the detection based on ionic switching characteristics.
  • the invention provides significant advantages over existing single- stranded oligo probes in which a compromise is made between sensitivity and specificity which results in lower fidelity signals. Switchable biosensors would allow greater sensitivity of detection over existing technology platforms.
  • Hybridisation kinetics may also be a factor.
  • existing methods of nucleic acid detection typically require hybridization at elevated temperatures over several tens of minutes.
  • Pro-HJ devices according to the present invention can be expected to bind target analytes and switch at room temperature within minutes, an improvement over current methods which require annealing at elevated temperature.
  • Branch point sequence is critical for the switching characteristics of the HJ switch, and in particular determines the ion binding affinity, and the thermodynamic equilibrium of the HJ conformers (Miick et al., 1997 PNAS 94, 9080).
  • the invention is less dependent on arm length for sequence specifity, because detection is more reliant on the switching characteristics of the particular branch point formed than complementary base pair binding of the target/single stranded pro-HJ arms.
  • these HJ arms should have a length of at least 8 base pairs, preferably from 10 to 60, most preferably from 12 to 30. It will be appreciated that the various arms and pro-arms may have similar lengths or significantly different lengths, and hence that (where the pro-HJ is comprised by more than one polynucleotide strand), the strands may have various different lengths (bearing in mind also that in some cases an arm may have a non-hybridized strand sequence portion extending therefrom).
  • HJ-like structures which have first and second, discretely different, conformations flippable in substantially similar manner to conventional HJs (consisting essentially of polynucleotide sequences), may also be constituted by molecular species in which a greater or lesser portion of the four HJ arm structures is comprised by non-polynucleotide material, and may be of any other organic and/or inorganic material which is not incompatible with the operation and application of the macromolecular structure, provided that the branch point of the HJ-like macromolecular structure has at least the four interacting polynucleotide dimer sequence elements of an HJ.
  • HJ-like macromolecular structure may also be comprised by the corresponding polynucleoside components of polynucleotides. Accordingly any references to HJ structures herein are also intended to encompass such HJ-like structures unless the context specifically requires otherwise.
  • HJ macromolecular structure switch As discussed hereinbefore various different HJ macromolecular structures are known with differing ratios of the two possible closed conformers (see for example Hays et al Biochemistry 42 2003) 9586-97). In this connection, the nature of the dimer sequence portions at the branch point of the HJ macromolecular structure switch, is especially influential. As noted above, the present invention is applicable in relation to various different kinds of macromolecular structure switch.
  • One well known form of macromolecular structure switch which may be mentioned here is the so-called "zinc finger" which comprises a polypeptide which has a conformational ⁇ transformable portion which switches from a generally straight extended configuration into a loop which can bind DNA (see for example Wolfe et al Biochemistry 42 (2003) 13401-9).
  • first and second, portions of the separation sensitive data carrier signal transmission portion are mounted on first and second parts of the zinc finger structure at opposite end portions of said conformationally transformable portion so that they are moved towards and away from each other as the zinc finger switches conformation.
  • a polynucleotide designed for traditional hybridising with a particular target DNA can also hybridise with a variant of that DNA in which one or two bases are no longer matched properly resulting in a lesser degree of hybridisation and hence weaker binding.
  • the variant of the target DNA might still form a functional macromolecular structure switch, and thereby provide a positive output signal.
  • the target analyte-binding portions of the pro-switch should be formed and arranged so that, when the target analyte is bound to the pro-switch, the SNP is located at the junction portion of the HJ type macromolecular structure, as the switching characteristics of the HJ switch are especially sensitive to the specific base sequence in this region.
  • the present invention provides a pro-HJ switch suitable for use in detecting a target analyte comprising an oligonucleotide substantially characteristic of said analyte, wherein said pro-HJ switch has an oligonucleotide-binding site formed and arranged for binding said oligonucleotide with high specificity so as to form together therewith, an HJ switch having an ion binding site, and flippable between a plurality of discretely different conformations corresponding to different ion binding conditions at said ion binding site, at least in the absence of any other binding to said HJ, wherein said discretely different conformations are capable of providing, in use, with an output signal reading system, characteristic output signals and wherein said pro-macromolecular structure switch is substantially incapable of being flippable between said plurality of discretely different conformations and providing, in use, with said output signal reading system, said characteristic output signals.
  • the present invention provides: a pro-HJ macromolecular structure switch suitable for use in detecting an analyte containing an SNP, wherein said pro-switch has an analyte binding site formed and arranged for binding said analyte with high specificity so as to form together therewith, a macromolecular structure having at least one ion binding site, and flippable between a plurality of discretely different conformations corresponding to different ion binding conditions at said at least one ion binding site, at least in the absence of any other binding to said macromolecular structure, wherein said discretely different conformations are capable of providing, in use, with an output signal reading system, characteristic output signals and wherein said pro-macromolecular structure switch is substantially incapable of being flippable between said plurality of discretely different conformations and providing, in use, with said output signal reading system, said characteristic output signals; and wherein said analyte binding site is formed and arranged for binding said analyte so that
  • said pro-switch is provided with at least one separation sensitive output device formed and arranged for providing different output signals in said discretely different conformations,
  • the present invention provides: a pro-HJ macromolecular structure switch suitable for use in detecting gene splicing, wherein said pro-switch has an analyte binding site which corresponds to a target sequence that spans the junction between two exons, preferably with the branch point aligned with the boimdary between the two exons.
  • the present invention provides a method for detecting a target analyte comprising an oligonucleotide substantially characteristic of said analyte, in a sample, which method includes the steps of: providing a pro-HJ switch, wherein said pro-HJ switch has an oligonucleotide binding site formed and arranged for binding said oligonucleotide with high specificity so as to form together therewith, an HJ switch having an ion binding site, and flippable between a plurality of discretely different conformations corresponding to different ion binding conditions at said ion binding site, at least in the absence of any other binding to said macromolecular structure, wherein said discretely different conformations are capable of providing, in use, with an output signal reading system, different characteristic output signals, and wherein said pro-HJ switch is substantially incapable of being flippable between said plurality of discretely different conformations and providing, in use, with said output signal reading system, said different characteristic output signals; contacting said pro-HJ switch
  • a pro-HJ which has an analyte binding site which corresponds to a target sequence that spans the junction between two exons, preferably with the branch point of the corresponding HJ, aligned with the boundary between the two exons.
  • a pro-HJ which has an analyte binding site which corresponds to a target sequence that includes the SNP, wherein the branch point of the corresponding HJ is aligned with the SNP.
  • the present invention provides: a pro-HJ oligonucleotide suitable for use as a pro-HJ structure for binding with a target oligonucleotide, said pro-HJ oligonucleotide comprising: first and second, target binding, portions, each having a base sequence such that together they are complementary to the base sequence of said target oligonucleotide, whilst being substantially non-self-complementary; third and fourth portions separated by a fifth, linkage, portion, said third and fourth portions being complementary to each other when folded back across each other about said fifth linkage portion; and sixth and seventh portions separated by a eighth, linkage, portion, said sixth and seventh portions being complementary to each other when folded back across each other about said eighth linkage portion, said third to eighth portions extending serially between said first and second portions.
  • said first and second portions are respective terminal portions of the pro-HJ oligonucleotide.
  • Figure 3 Peptide Zinc Finger Macromolecular switch
  • Figure 4 GeI-FRET assay showing independent processes of ion induced switching and target binding
  • Figure 11 Inline detection apparatus
  • Figure 12 Droplet Electrode Array Apparatus
  • FIG. 13 Switching characteristics shown as FRET ratio as a function OfMgCl 2 concentration in the presence (solid) and absence (hollow) of complementary DNA strand.
  • Figure 15 Alternative dye positions Figure 16 - Single nucleotide polymorphism (SNP) detection
  • Figure 20-Detection of specific alleles using HJ probes for SNP A-2131935 Pro-HJ's designed with sequences of A-2131935 C or A alleles have been analysed by gelFRET in the absence or presence of perfect match and mismatch alleles (- (no target), G (G target oligo) or T (T target oligo)). Analysis was performed in the presence of 5mM MgCl 2 . FRET ratios are shown below the band.
  • Pro HJ structures comprise a single polynucleotide (as shown in Figure 1) or 3 separate polynucleotides (as shown in Figure 2) with suitably chosen sequences such that respective inverted repeat or end portions bind together, and in the case of separate polynucleotides may be covalently or non covalently crosslinked, so as to produce a Pro HJ structure with two double stranded arms and two single stranded arms radiating out from a branching point.
  • polynucleotide sequences are based on similar known sequences (Kallenbach N.R., Ma R.I., Seeman N.C. (1983) Nature 305 829-830), and are selected so as to provide an immobile junction which substantially prevents migration of the branching point.
  • the polynucleotides have additional fluorescent dye moieties, Carboxyfluorescein (FAM) and Tetramethylrhodamine (TAMRA), attached either internally to the polynucleotide sequence or externally at the 5' end (Eurogentec, Belgium), to act as fluorescence resonance energy transfer (FRET) donor and acceptor moieties.
  • FAM Carboxyfluorescein
  • TAMRA Tetramethylrhodamine
  • the sequences present in the single stranded arms are designed to bind to a specific target polynucleotide sequence and have minimal self complementarity.
  • this Pro HJ molecule is hybridised to the complementary target sequence an immobile four- way junction is formed which is capable of undergoing a conformational transition by binding cations such as Mg 2+ .
  • a process of heat induced denaturation followed by a slowly ramped cooling to allow annealing of complementary sequences present in the Pro HJ double stranded arms is used to assemble the Pro HJ structures as described below.
  • the ion-induced switch characteristic of this DNA construct can be observed using Fluoresence Resonance Energy Transfer FRET between a donor (FAM) and an acceptor (TAMRA) only when the probe and target molecule are annealed to form a complete HJ.
  • FAM Fluoresence Resonance Energy Transfer FRET between a donor (FAM) and an acceptor (TAMRA) only when the probe and target molecule are annealed to form a complete HJ.
  • the buffer for assembly i.e. binding of pro-HJ to target so as to form HJ
  • the buffer for assembly was 20 HiM Tris/HCl ( ⁇ H7.5), with 5 mM MgCl 2 , 5OmM NaCl.
  • pro HJ oligonucleotides were mixed with or without target oligonucleotides and the assembly buffer detailed above and the mixture was heated to 8O 0 C for 30 minutes in a water bath and then allowed to cool to room temperature, while still jacketed by the water bath inside a polystyrene box to ensure a slow temperature ramp.
  • junctions were initially suspended in 2OmM Tris/HCl (pH7.5), 5mM MgCl 2 , 5OmM NaCl and the final step in the sample preparation was to remove the Mg 2+ and Na + ions by sequential application to two gel filtration columns to buffer exchange the sample into 2OmM Tris/HCl (pH7.5) in order to ensure HJ' s are in an open conformation.
  • this molecule When this molecule is hybridised to the complementary (target analyte) strand (GCATAGTGGATTGCA) necessary to form an immobile four- way junction it forms a functional macromolecular switch which undergoes a conformational transition by binding magnesium ions.
  • a l ⁇ M solution of pro-HJ was titrated with MgCl 2 over the range 0- 2OmM, with and without a 1 ⁇ M solution of the complementary oligo.
  • the FRET donor moieties are excited by a light source providing 476.5 nm radiation directed towards them and any output radiation from the FRET acceptor moieties is detected by a photomultiplier, using a Fluoromax Spectrofluorometer (Horiba Jobin Yvon Ltd., UK).
  • the HJ structure adopted a "closed" configuration in which FRET energy transfer to the FRET acceptor takes place resulting in an output radiation which can be detected.
  • a significant conformational change is seen due to switching of the HJ population (background corrected maximum FRET ratio, where the FRET ratio is the ratio of the peak acceptor to peak donor emission intensities, of 0.7) compared with the control case without the complementary strand (background corrected maximum FRET ratio of 0.1).
  • the switching transition is illustrated in Figure 13.
  • Example 2 The procedure of Example 2 was repeated using the proHJ (a) and the RNA target oligo UGGGAUUCGGACUAUGCA in place of the DNA target oligo TGGGATTCGGACTATGCA.
  • Example 5 The procedure of Example 5 was repeated using 1OnM of pro-HJ structure (a) from example 2 with RNA target oligo UGGGAUUCGGACUAUGCA at a very low concentration (10 nM). With addition of complementary target RNA a significant change in switching characteristic is seen. Ionic switching using 1OmM MgCl 2 shows FRET ratios of 0.53 compared with 0.18 with no target molecule present.
  • Example 7 Label-free detection of RNA from complex mixture at low target concentration
  • the procedure of Example 6 was repeated using the RNA target oligo UGGGAUUCGGACUAUGCA in the presence of varying background concentrations of non-complementary murine renal total RNA.
  • complementary target RNA With addition of complementary target RNA a significant change in switching characteristic is seen. Ionic switching using 1OmM MgCl 2 shows FRET ratios of 0.41 compared with 0.22 with no target molecule present.
  • Target oligonucleotide with the following sequences were made by solid phase synthesis: (a) no mismatch TGGGATTCGGACT ATGCA, (b) single base mismatch at the 11 th base position TGGGATTCGGGCTATGCA. .
  • Fig 16A illustrates the alignment of these targets with the pro-HJ probe.
  • a gelFRET assay (Ramirez-Carrozzi & Kerpolla, 2001, Methods 25, 31-43) was carried out. Samples were annealed overnight at a ratio of 10 ⁇ M pro-HJ probe: 50 ⁇ M target. The annealed samples were then diluted ten fold in 5.0 mM MgCl 2 and 45 mM Tris/Borate pH 8.3 prior to loading on the gel. Gels were run at 4 0 C for 90 min at 75 Volts.
  • Both targets bind to the pro-HJ, as shown by the gel shift when compared with the no target lane - Figure 16 B 1.
  • the resultant HJ structures switch to differing degrees in 5mM MgCl 2 .
  • the single nucleotide mismatch (b) has significantly reduced switching (RED) when compared with (a) the no mismatch structure (GREEN) i.e. the detection of single nucleotide differences in DNA targets, is possible
  • Figure 16 B 2 shows that these structures switch to the open conformation if the Mg 2+ is removed by soaking in 0.1 mM ethylenediaminetetra(acetate) EDTA (RED).
  • Figure 16 B 3 shows that the no mismatch structure switches back to the closed conformation (GREEN) if the MgCl 2 is reintroduced i.e. illustrates that the switchable biosensor is reversible.
  • the pro-HJ shown in Figure 4 was prepared as described in Example 1 and annealed overnight with/without the DNA target.
  • a gelFRET assay (Ramirez-Carrozzi & Kerpolla, 2001, Methods 25, 31-43) is then used. In this procedure the HJ samples are loaded onto polyacrylamide gels (90 mM Tris/Borate, pH 8.3, 10 % acrylamide and either 0 or 5.0 mM MgCl 2 ) at 2.5 uM (-/+ 5.0 uM DNA or RNA target). Gels were run at 4 °C for 90 min at 75 Volts.
  • Gels were then scanned using a laser to excite at 488 nm and emission filters to detect at 526 nm (the range is 500 — 540 nm) and 580 nm (the range is 565 - 595 nm); the bandpass is 560 nm.
  • the intensity of the signal obtained at 526 nm (represented by the intensity of the color red in the original figure) was overlaid with the intensity of the signal obtained at 580 nm (represented as the intensity of the color green in the original figure) to visualize the relative difference in FRET for each gel band.
  • the overall color of each gel band is indicated above the lane in the Figure, with GREEN meaning high levels of FRET and RED meaning low levels of FRET.
  • the DNA target binds to the pro-HJ (shown by the relative band positions of pro-HJ with and without targets in Figure 4). Switching of the HJ is induced by changing from 0 mM MgCl 2 (shown as RED in Figure 4) to 0.5mM MgCl 2 (shown as GREEN in Figure 4). This illustrates that target binding and ion-induced switching are independent processes.
  • HJ probes were designed to detect specific sequence of the mouse Ccl5 gene (Mm.284248) and specific sequence of the mouse Cxcl9 gene (Mm.766), in this case based on pro-HJ structures described in Example 1.
  • the selected probe designs are shown in Table 2.
  • T FAM deoxythymidine
  • T TAMRA deoxythymidine
  • the DNA target binds to the pro-HJ (shown by the relative band positions of pro-HJ with and without targets in Figure 18). Switching of the HJ is induced in the presence of 5mM MgCl 2 (shown as RED in Figure 18).
  • HJ probes can be designed to detect naturally-occurring nucleic acid sequences.
  • T FAM deoxythymidine
  • T TAMRA deoxythmidine
  • the DNA target binds to the pro-HJ (shown by the relative band positions of pro-HJ with and without targets in Figure 20). Switching of the HJ is induced in the presence of 5mM MgCl 2 (shown as RED in Figure 20) is only observed with binding of a perfectly matched allele. This illustrates that HJs can be used to discriminate between single nucleotides in naturally occurring SNPs.
  • Detection of naturally occurring SNPs in a clinical sample could be achieved after PCR amplification of the region encompassing the SNP and the use of a ProHJ to detect the amplified sample.
  • the region encompassing the SNP described in Example 11 could be amplified using the PCR primers described in Table 4 from clinical samples of genomic DNA.
  • a HJ has been constructed which has no donor or acceptor, but in which the adenine at the branch point has been exchanged for an analogue — 2-aminopurine (2AP) see figure 21 for details.
  • 2AP should fluoresce and on addition of Mg, when the HJ closes, the fluorescence should be quenched (due to base stacking) [Biochemistry 2001, 40, 946- 956, Probing Structure and Dynamics of DNA with 2-Aminopurine: Effects of Local
  • Figure 25 shows the change in FRET ratio with ion buffer exchange - the FRET ratio was calculated as the ratio between emission intensities: I 58 o/I 53 o.
  • This example illustrates the ability to form proHJ + target complexes with the probe immobilized on a surface which can switch based on ionic conditions.
  • Electrochemical Control of HJ Experiments were performed to induce HJ switching by the addition and removal of Zn 2+ switching ions into solution through electrochemical oxidation (stripping) and rereduction of zinc, to establish the principle of electrochemical control. It was found that Zn reversible plating and stripping at a zinc wire electrode was inhibited by the formation of an insulating zinc oxide surface layer in aqueous solution, and reversible quantitative electrochemical stripping and plating could not be achieved in aerated solution due to chemical oxidation of zinc by oxygen in aerated solution (corrosion) [Ferapontova et al, Electrochem. Cotnm., 9 (2007) 303-9].
  • F is the donor Carboxyfluorescein (known commercially as FAMTM) on oligonucleotide 1 and R is the acceptor Carboxytetramethylrhodamine (known as TAMRATM) on oligonucleotide 4.
  • TAMRATM Carboxytetramethylrhodamine
  • internal dyes were used to maintain dye position relative to the branch point.
  • the HJ was assembled in a solution of 20 mM Tris/TrisH + Cr (pH 7.5) buffer solution containing NaCl (50 mM) and MgCl 2 (5 mM), by heating to 8O 0 C for 30 minutes and then allowing the solution to cool slowly in a water bath to room temperature.
  • a ratio of 1:2:2:2 was chosen to ensure full incorporation of donor strand into the fully assembled 4-way junctions.
  • the final step in the sample preparation was to buffer exchange twice (Microspin G25 ion exchange columns (Amersham Biosciences)) to removing the Mg 2+ ions and produce HJ solutions (concentration 1 ⁇ M after dilution) in 2OmM Tris/TrisH + Cl " buffer (pH 7.5) as required for ion titration studies. 100 ⁇ M tetrasodium ethylenediamine(tetraacetate) Na 4 EDTA was then added to the solution to titrate any residual Mg 2+ ions and produce HJ exclusively in the open form.
  • the pro-HJ with the acceptor at the 3 'end (PD03) is sensitive to mutations in the target that are in proximitiy to the acceptor (even though they are not close to the branch point). This is not an issue for the probe with the internal acceptor (PD09).
  • the design PD09 may be preferred in applications where a single area of precise specific sequence detection is required such as SNP detection.
  • the design PD03 may be preferred if general sequence specification is required such as in transcript detection applications.
  • Fluorescence lifetime measurements have general advantages over intensity based assays in that the fluorescence lifetime of molecules is independent of local fluorophore concentration and local excitation light intensity.
  • FRET fluorescence lifetime measurement
  • the decay of the excited state population is rapid and time domain data can be used to determine the quantitative separation between fluorescent species using the nonlinear dependence of the FRET rate constant on separation.
  • TGCATAGTGGATTGCATTTTTGCAATCCTGAGCACATTTTTGTGCTCACCGAATC CCA-3' were synthesised (Eurogentec) with Tetramethylrhodamine (TAMRA) attached at the 5' end and Carboxyfluorescein (FAM) attached at a thymidine (19 nucleotides from the 5' end).
  • Probe variants were made with and without the FAM acceptor fluorophores.
  • Target variants were made with the base at point 11 modified with a C base (11C) and a G base (HG) (see Figure 28).
  • the probes (10 ⁇ M) were assembled in the presence of the target DNA oligonucleotide (purchased from Eurogentec) in 20 mM Tris/HCl (pH 7.5), 10 mM MgCl2 at five fold excess of target. Samples were incubated in a water bath at 8OC for 30 min, followed by a slow temperature decrease to room temperature. Time-resolved fluorescence data were collected by donor excitation from a mode locked, frequency doubled Coherent MIRA 900-F to produce vertically polarised, 200 fs pulses of wavelength 450 nm. Emission was collected in an Edinburgh Instruments FL-920 spectrometer with the monoclioromator set at 517 nm, the peak of the donor emission. The emission polariser was set at 54.7 to the vertical to avoid anisotropy effects. AU data were collected to a peak of 10,000 counts with a delay window of 20 ns and 4096 channels.
  • Figure 29 shows time-resolved FRET decays for a donor only molecule, plotted alongside data for a switch incorporating target HC and HG. This clearly shows that sufficient resolution is achieved to allow single base changes around the branch point to be resolved.
  • This example effectively illustrates the ability to form a 3 -state switch (closed, open I and open II) which adopts different states depending on the target molecule bound to the pro-HJ.
  • the match and mismatch case can be discriminated either by a change in one acceptor dye signal, or the other or both. This gives the opportunity to more precisely discriminate the specific match/mismatch cases from each other and from nonspecific binding.
  • the pro-HJ probe will give a signal output in the closed position based on the combined dye signal, regardless of the conformer.
  • probe design constraints i.e. designing a probe HJ to both form a switch and to achieve specific complementary binding
  • the data are normalized in the figure to maximal signal (matched target) compared to minimal signal (no target).
  • the unnormalized FRET ratios for probe in the absence of target and in the presence of matched target were 0.46 and 1.23 (5.0 mM MgCl 2 ) and 0.42 and 0.91 (0.1 mM MgCl 2 ), respectively.

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Abstract

L'invention porte sur un procédé de détection d'un analyte cible dans un échantillon à l'aide d'un commutateur à structure pro-macromoléculaire présentant un site de fixation de l'analyte où se fixe l'analyte pour former ensemble un commutateur macromoléculaire présentant un site de coordination d'ions et des bras pivotant entre des conformations discrètes différentes correspondant à différentes conditions de fixation des ions. Lesdites conformations fournissent avec un système de lecture des signaux sortants des signaux de sortie caractéristiques. Le pro-commutateur est mis en contact avec l'échantillon et on applique un signal extérieur au dispositif d'entrée pour changer les conditions de fixation des ions en conformations de commutateurs pivotant, les changements étant lus dans le signal de sortie. L'invention porte également sur un commutateur à structure pro-macromoléculaire.
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WO2021173767A1 (fr) * 2020-02-25 2021-09-02 Virginia Commonwealth University Capteur à molécule unique à base de frette dynamique pour la détection ultrasensible d'acides nucléiques

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