WO2013136088A1

WO2013136088A1 - Method and apparatus

Info

Publication number: WO2013136088A1
Application number: PCT/GB2013/050662
Authority: WO
Inventors: Cameron Alexander FRAYLING; Boris Breiner
Original assignee: Base4 Innovation Ltd
Priority date: 2012-03-16
Filing date: 2013-03-15
Publication date: 2013-09-19
Also published as: GB201204728D0

Abstract

A method for mapping the number and location of G4 Quadruplex constructs in a target nucleic acid comprises the steps of (1) translocating a target nucleic acid having detectable elements characteristic of the presence of G4 Quadraplex constructs therein through an analysing device comprising a nanopore and a detection window and (2) causing the detectable elements to be detected as they pass though the detection window. Typically the detectable elements are formed by selectively attaching one or more marker molecules to the G4 Quadruplex constructs e.g. by ligation. The data or signal obtained from the detection is suitably in the form of a distribution profile of the detectable elements, and therefore the G4 Quadruplex constructs along the length of the target nucleic acid and can be used to create a reference set of like distribution profiles against which new distributions can be compared. When the target nucleic acid is human DNA or RNA such comparisons enable valuable insights to be drawn about an individual's susceptibility to certain health conditions or the biochemical origins thereof.

Description

METHOD AND APPARATUS

The present invention relates to a method and apparatus for characterising a nucleic acid in terms of the number and location of G4 Quadruplex constructs present therein.

G4-Quadruplexes are nucleotide constructs formed in guanine-rich sections of nucleic acids such as DNA, RNA, LNA and PNA. They typically comprise square planar arrangements of guanine bases stabilized by Hoogsteen hydrogen-bonding, and an alkali-metal cation, typically potassium, located at their centre. Not only do they perform important functions in regulating the production of the enzyme telomerase, and the transcription and processing regulation of related RNA but they have also been linked to a variety of health conditions, including cancer, fragile X syndrome, Bloom syndrome, Werner syndrome and Fanconi anemia J. For these reasons, there has been widespread interest in finding suitable ways to detect the number and location of these constructs in human DNA with a view to understanding, for example, how they relate to an individual's increased susceptibility to these conditions or how these conditions might be pre-empted or ameliorated.

A number of small molecules have been shown to bond selectively to G4 Quadruplexes as part of research aimed at seeking to impair or eliminate the production of telomerase in cancer cells. For example, Mergny et al. (Proc. Natl. Acad, of Sci. USA, 98(6), pp.3062-3067, (2001)) have demonstrated that certain pentacyclic dibenzophenathroline compounds can be readily attached to G4 Quadruplexes whilst Kerwin (Current Pharmaceutical Design, 6(4), pp.441-471, (2000)) discloses a range of alternative ligands including porphyrins such as TMPyP4, perylene diimides and diimidoanthraquinones. Finally, Lemarteleur et al (Biochem. and Biophys. Res. Comm., 323, pp.802-808, (2004)) discloses an even wider range of candidate ligands including telomestatin which has shown especial usefulness and selectivity (see also Monchaud et al. J. Nucleic Acids 2010, Article ID 525862). We have now found that, when such molecules, which either fluoresce themselves or are modified to include an additional fluorophore, are employed they can be used as selective markers for the number and locations of G4 Quadmplex constructs in a target nucleic acid sample. Thereafter, using sequencing methods, including, inter alia, those we have disclosed previously in our patent application WO 2009/030953, it is therefore possible to map a nucleic acid sample in terms of these constructs thereby allowing for example correlations to be drawn between an individual's susceptibility to one of the conditions mentioned above and the precise disposition of these constructs in his or her genes.

EP-2343382-A1 relates to plasmonic force manipulation in nanostructures. Disclosed are a system and method for characterizing and/or manipulating particles such as DNA, wherein particles are translocated through a nanostructure at a speed that is influenced by a plasmonic force field generated by providing radiation to a metal surface of a substrate comprising the nanostructure.

WO 2005/045392 (EP- 1682673 -A2) discloses an apparatus and method for sensing a nanoscale moiety. The apparatus includes a substrate having a nanopore, an excitable molecule attached to the substrate adjacent to the nanopore and a light source for exciting the excitable molecule. The excitable molecule is quenched by a quencher molecule on the nanoscale moiety as it passes through the nanopore and past the excitable molecule. The modulation of the signal emitted by the excitable molecule as the quencher molecule moves through the nanopore is then detected.

According to the present invention, there is therefore provided a method for mapping the number and location of G4 Quadmplex constructs in a target nucleic acid which comprises the steps of (1) translocating a target nucleic acid having detectable elements characteristic of the presence of the G4 Quadruplex constructs therein through an analysing device comprising a nanopore and a detection window and (2) causing the detectable elements to be detected as they pass though the detection window. In this method, the detectable elements are suitably detected so as to output data or a signal in the form of a distribution profile of the detectable elements along the length of the target nucleic acid. The distribution profiles so obtained can be used as is or added to a database of like profiles so that over time an extensive reference set is built up which constitutes a valuable research tool enabling genetic, biochemical and therapeutic conclusions and insights to be drawn therefrom.

The term "nucleic acid" as used herein means a polymer of nucleotides. Nucleotides themselves are sometimes referred to as bases (in single stranded nucleic acid molecules) or as base pairs (in double stranded nucleic acid molecules) in an interchangeable fashion. Nucleic acids suitable for use in the method of the present invention are typically the naturally- occurring nucleic acids DNA or RNA or synthetic versions thereof. However the method can also be applied if desired to analogues such as PNA (peptide nucleic acid), LNA (locked nucleic acid), UNA (unlocked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid). The nucleic acids themselves in turn suitably comprise a sequence of at least some of the following nucleotides: adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) 4- acetylcytidine, 5- (carboxyhydroxylmethyl)uridine, 2-O-methylcytidine, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino-methyluridine, dihydrouridine, 2-O-methylpseudouridine, 2-O-methylguanosine, inosine, N6- isopentyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3- methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5- methy laminomethy luridine, 5 -methoxyaminomethy 1-2-thiouridine, 5 -methoxyuridine, 5 -methoxy carbonylmethyl-2-thiouridine, 5 -methoxycarbonylmethy luridine, 2- methylthio-N6-isopentenyladenosine, uridine-5-oxyacetic acid-methylester, uridine-5- oxyacetic acid, wybutoxosine, wybutosine, pseudouridine, queuosine, 2-thiocytidine, 5 -methy 1-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, 2-0-methyl-5- methyluridine and 2-O-methyluridine. Especially suitable nucleic acids are naturally occurring mammalian DNA or RNA most suitably of all being human DNA or RNA. Typically, the length of the target nucleic acid sequence is expressed in terms of the number of nucleotides it contains. For example, the term "kilobase" (kb) means 1000 nucleotides whilst "megabase" (Mb) means 1,000,000 nucleotides. The target nucleic acid used in the method of the present invention can in principle contain any number of nucleotides up to an including the number typically found in a human or other mammalian gene. However the method of the present invention is also applicable to smaller oligonucleotide fragments (e.g. fragments of a human gene) which are at least 10 bases (for single stranded nucleic acids) or base pairs (for double stranded nucleic acids) long, more typically at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 500 or more bases/base pairs long or lkb, 2kb, 5kb, lOkb, 20kb, 50kb, lOOkb, 250kb, 500kb or up to 1Mb or more long. The target nucleic acid itself may be derived directly or indirectly from any available biological sample including but not limited to materials such as blood, sputum or urine.

It is a feature of the method of the present invention that the target nucleic acid is further comprised of detectable elements characteristic of the former's G4 Quadruplex constructs. In principle, these detectable elements can be any element within or attached to the target nucleic acid at a site of a G4 Quadruplex construct which exhibits a detectable characteristic as the latter passes through the detection window of the analysing device. Typically the detectable elements will all be of the same type for a given nucleic acid sample, although a given target nucleic acid may be analysed multiple times using different detectable elements if so desired. In fact it may be beneficial in certain circumstances to map the same target nucleic acid in this way to ensure that all the G4 Quadruplex constructs are identified. Suitably, the detectable elements are such that they are able to generate, either directly or indirectly, corresponding characteristic data and/or a signal when caused to pass through the detection window. In a preferred embodiment of the invention, this characteristic data and/or signal is generated by the emission of photons characteristic of the detectable element fluorescing or Raman scattering incident light within the detection window. In the case of Raman scattering the detectable element may form part of the molecular structure of the G4 Quadruplex construct itself or may subsist in an element attached thereto which is made identifiable using Raman spectroscopy by the addition of the marker molecule referred to below.

Suitably, the detectable elements are generated by attaching a molecule capable of acting as a marker to the site of the G4 Quadruplex construct. Such marker molecules can be attached by either physical or chemical means and, in the case of the latter, by covalent, ionic, dative bonding or ligation. One preferred class of such marker molecules are ligands such as those mentioned above together with one selected from the group consisting of 2,7-substituted difluoreneones, acridines, ethidium derivatives, disubstituted triazines, fluoroquinoanthroximines, indoloquinolines, and telomestatin. If the method of detection is based on the detectable element fluorescing these marker molecules can be used unmodified (if they naturally fluoresce) or they can be chemically modified to include conventional fluorophore moieties including but not limited to xanthene moieties e.g. fluorescein, rhodamine and their derivatives such as fluorescein isothiocyanate, rhodamine B and the like; coumarin moieties (e.g. hydroxy-, methyl- and aminocoumarin) and cyanine moieties such as Cy2, Cy3, Cy5 and Cy7. Preferred fluorophore moieties are those derived from the following commonly used dyes: Alexa dyes, cyanine dyes, Atto Tec dyes, and rhodamine dyes. Examples include: Atto 633 (ATTO-TEC GmbH), Atto 740 (ATTO-TEC GmbH), Rose Bengal, Alexa Fluor 750 C₅-maleimide (Invitrogen), Alexa Fluor 532 C₂- maleimide (Invitrogen) and Rhodamine Red C₂-maleimide and Rhodamine Green. These fluorophore moieties can each be attached to the marker molecule used using chemical techniques known in the art.

If the distribution profile characteristic of the target nucleic acid is to be compared against a reference set of pre- determined profiles characteristic of known nucleic acid samples, this can be done using either best fit statistical methods or visual inspection. Typically, however the comparison is performed computationally and can be based on a set of logic decision rules, or on a range of regression and classification methods (linear or not), or on pattern matching and machine learning methods (such as neural networks, kernel methods or graphical models). For example, the comparison can be performed by a computer that has a database or reference set of distribution profiles for known nucleic acids and a memory containing instructions which, when executed by the processor, compare the distribution profile of the target nucleic acid to the reference set. In the case where no matching is found the target nucleic acid can be added to the reference set for future reference if so desired.

In the method of the present invention, the target nucleic acid having the necessary detectable elements is analysed by translocating it through an analysing device comprising a nanopore having a detection window. In the method, the target nucleic acid is translocated through both the nanopore and the detection window. Preferably this detection window is defined by a localised electromagnetic field generated by plasmon resonance. In such an embodiment, the interaction between this electromagnetic field, the detectable elements and incident electromagnetic radiation impinging on the detection window is used to generate an increased level of fluorescence or Raman scattering which can be easily detected and analysed. One example of such an analysing device can be found in our WO 2009/030953 the contents of which are incorporated herein by reference. Briefly, this analysing device comprises a nano-perforated substrate separating sample providing and receiving chambers. The nano-perforated substrate may either be fabricated from an inorganic insulator or from organic or biological material. Preferably the nano-perforated substrate is an inorganic insulator such as a silicon carbide wafer. Typically, the nanopore is between Inm and lOOnm in diameter preferably Inm to 30nm, Inm to lOnm, Inm to 5nm or 2nm to 4nm. The target nucleic acid is suitably caused to translocate from the sample to the receiving chambers via the nanopore by electrophoresis. Passage through the nanopore ensures that the target nucleic acid translocates in a coherent, linear fashion so that it emerges from the outlet thereof in a nucleotide by nucleotide fashion enabling the detectable elements and therefore the G4 Quadruplexes to be detected in sequence.

The analysing device is suitably provided with a detection window juxtaposed either within the nanopore or adjacent its outlet. Typically this detection window is defined by one or more metallic moieties fabricated from gold or silver capable of undergoing plasmon resonance under the influence of incident electromagnetic radiation from a coherent source such as a laser. This plasmonic resonance generates the strong localised electromagnetic field through which the target nucleic acid passes. The exact geometry of these metallic moieties determines the geometry of the detection window and hence affects the nature of the interaction with the detectable elements. For example, the geometry of the detection window can be chosen so as to be optimised for increased photon emission, rather than for lateral localisation. This is achieved by producing detection windows with a greater z length (the dimension along which the nucleic acid translocates), and modifying their geometry appropriately in the x and y dimensions in order to ensure their peak plasmonic resonance frequency is maintained at a desired wavelength. Preferably, the detection window is sized so that the length in the z dimension is from 1 to 100 preferably from 10 to 50 nanometres.

The signal generated by the interaction of the detectable elements and the electromagnetic field can be detected by a detector such as a photocounter in the case of fluorescence or a spectrometer in the case of Raman scattering. The output of such a device will typically be an electrical signal characteristic of the target nucleic acid's distribution profile of G4 Quadruplex constructs.

The method of the present invention may suitably employ multiple detectors and multiple analysing devices. For example, an array of pairs of detectors and analysing devices may be used with each detector being arranged to detect photons generated using its paired analysing device. Other detectors including other detectors for detecting fluorescence such as a photomultiplier or single photon avalanche diode may be used.

In another preferred analysing method, the characteristic data stream and/or signal is generated by fluctuations in an electrical property of the detection window and/or its contents (e.g. changes in voltage, resistance or current flow occasioned by the detectable element blocking or enabling the flow of ions in the nucleic acid's associated translocation medium between electrodes). In this latter case, it may be possible through careful choice of the marker molecule to avoid having to label the same with a fluorophore. This embodiment of the invention is therefore typically carried out as an alternative to optical detection using, for example, fluorescence or Raman scattering, and not in addition to such optical detection. Preferred translocation media used here are aqueous alkali metal electrolytes such as an aqueous potassium or sodium halide, nitrate or sulphate solution.

In a further aspect of the present invention there is provided an apparatus for identifying a target nucleic acid comprising detectable elements characteristic of G4 Quadruplex constructs the apparatus comprising: an analysing device comprising a nanopore having a detection window, wherein the analysing device is capable of plasmon resonance to produce a localised electromagnetic field which defines the detection window; a detector for detecting detectable elements of the target nucleic acid as they pass through the detection window to produce a distribution profile of the detectable elements along the target nucleic acid; and optionally a computer system for comparing the distribution profile to a reference set of distribution profiles for known nucleic acids. The computer system typically comprises a memory and a processor. Computer executable instructions can be provided which when executed by the processor compare the distribution profile of the target nucleic acids to a reference set of distribution profiles to identify the target nucleic acid or other relationships between it and the data in the database. The present invention will now be exemplified by the following figures in which:

Figure 1 is a flow diagram showing a method in accordance with an aspect of the present disclosure;

Figure 2 schematically illustrates an apparatus for the method of Figure 1 and

Figure 3a-b illustrates the evolution of a schematic distribution profile for the target

DNA analysed in the apparatus of Figure 2.

Figure 1 represents a flow diagram showing a method in accordance with the present invention. In one example, the method comprises, at step S10, translocating a target nucleic acid of human origin having detectable elements through a nanopore having a detection window. The nanopore is part of an analysing device which has a gold plasmonic structure that is capable of plasmon resonance under incident laser light to produce a localised electromagnetic field which defines the detection window. At step S12, the detectable elements are caused to fluoresce and are detected as they pass through the detection window to produce a distribution profile characteristic of the number and location of the G4 Quadruplex constructs in the target nucleic acid. At step S14, the distribution profile of the target nucleic acid is used to identify the number and location of the G4 Quadruplex constructs or compared against a reference set of distribution profiles.

Figure 2 schematically illustrates an apparatus for performing the method of Figure 1 comprising an analysing device 24, a photodetector 30, a data acquisition card 32 and a computer 34. 24 comprises a non-electrically conducting silicon carbide wafer perforated with a plurality of 4nm diameter nanopores 28 and associated gold plasmonic structures 26 (doughnut shaped) juxtaposed over the outlet of 28 to define detection windows 40. In use, the G4 Quadruplex constructs of a human patient's DNA 20 (isolated from a blood sample) are labelled with N,N- diethylthiocarbocyanine iodide in accordance with the method taught in Bioinorganic and Medicinal Chemistry Letters 11(18), pp.2411-2414, (2001) to create detectable elements 22 and the DNA itself caused to translocate though 28 and 40 by electrophoresis. 26 generate a localised electromagnetic field around the outlets of 28 which interacts with each 22 in turn causing them to fluoresce and emit photons 38 which are captured by 30. A laser (not shown) of frequency 750nm and power 12uW is used to induce the plasmon resonance in 26.

In an alternative detection method, 26 comprise one or more pairs of electrodes connected to each other via a battery and an ammeter (not shown) and the detectable elements created at the G4 Quadruplex constructs are sized so as to interfere with the flow of ions between these electrodes arising from the sample's associated translocation medium (in this case aqueous potassium chloride). Specifically, in this embodiment, a potential difference is continuously applied across the electrodes and the resulting fluctuations in the current flowing between the electrodes (or any equivalent voltage fluctuations or changes in electrical resistance) are continuously monitored as a function of time and/or the progress of the translocation event to generate a data stream analogous to that described in the previous paragraph.

32 is used to receive the output of 30 and transfer it to 34 for analysis. The output is an electrical signal representing the distribution profile of the G4 Quadruplex constructs in the DNA in the form of a profile of fluorescence over the length of at least a portion of its length. 34 comprises a processor and memory connected to a central bus structure which is in turn connected to a display via a display adapter and one or more input devices (such as a mouse and/or keyboard). 34 further comprises a communications adapter which is also connected to the central bus. The communications adapter can receive communications, in particular communications containing new distribution profiles for new nucleic acid samples, which can be sent to the computer over a suitable communications link such as the internet.

The computer 34 has a database or reference set 36 of distribution profiles for the G4 Quadruplex constructs of known human DNA samples and the memory of the computer contains instructions which when executed by the processor compare the measured distribution profile of the target DNA sample (shown in Figure 3) to this reference set to characterise the target DNA sample using pattern matching software.

Claims

1. A method for mapping the number and location of G4 Quadruplex constructs in a target nucleic acid which comprises the steps of (1) translocating a target nucleic acid having detectable elements characteristic of the presence of the G4 Quadruplex constructs therein through an analysing device comprising a nanopore and a detection window and (2) causing the detectable elements to be detected as they pass though the detection window.

2. A method according to claim 1 characterised in that it comprises the additional step of (3) retrieving data and/or a signal characteristic of the distribution profile of the detectable elements, and therefore the G4 Quadruplex constructs, in the target nucleic acid.

3. A method according to claim 2, characterised in that it comprises the additional step of comparing the distribution profile obtained in step (3) with a reference set of known distribution profiles.

4. A method according to any one of the preceding claims characterised in that the analysing device is capable of plasmon resonance to produce a localised electromagnetic field which defines the detection window.

5. A method according to any one of the preceding claims, characterised in that the detectable elements are comprised of a marker molecule attached to at least some of the G4 Quadruplex constructs.

6. A method according to any one of the preceding claims characterised in that the marker molecule comprises a means for ligating to the G4 Quadruplex construct.

7. A method according to any one of claims 2 to 6 characterised in that distribution profile is generated by measuring fluctuations in an electrical property of the detection window and/or its contents.

8. A method according to either claim 5 or 6 characterised in that the marker molecule is a fluorophore selected from the group consisting of porphyrins, perylene diimides and diimidoanthraquinones, dibenzophenathrolines, 2,7-substituted difluoreneones, acridines, ethidium derivatives, disubstituted triazines, fluoroquinoanthroximines, indoloquinolines, and telomestatin or fluorophore substituted derivatives thereof.

9. A method according to any one of claims 4 to 6 and 8 when dependent upon claim 2 or 3, wherein the distribution profile is a profile of fluorescence over the length of at least a portion of the target nucleic acid and wherein the plasmon resonance enhances fluorescent properties of the detectable elements.

10. A method according to any one of claims 4 to 6 when dependent upon claim 2 or 3 characterised in that the marker molecule can cause Raman scattering of light in a way characteristic of it.

11. A method according to claim 10, wherein the distribution profile is a profile of Raman scattering over the length of at least a portion of the target nucleic acid and wherein the plasmon resonance increases the level of Raman scattering from the detectable elements.

12. A method according to any one of the preceding claims, wherein the nanopore located in a nano-perforated substrate which is an inorganic insulator.

13. A method according to any one of the preceding claims, wherein the detection window is between 1 nanometre and 100 nanometres

14. A method according to any one of the preceding claims, wherein the detection window is between 10 and 50 nanometres.

15. An apparatus for mapping the number and location of G4 Quadruplex constructs in a target nucleic acid the apparatus comprising:

an analysing device comprising a nanopore having a detection window, wherein the analysing device is capable of plasmon resonance to produce a localised electromagnetic field which defines the detection window and

a detector for detecting detectable elements characteristic of the G4 Quadruplex constructs in the target nucleic acid as they pass through the detection window to produce a distribution profile of the detectable elements along the length of the target nucleic acid.

16. An apparatus as claimed in claim 15 characterised in that it additionally comprises a computer system for comparing the distribution profile to a reference set of distribution profiles for nucleic acids or a means for attaching such a computer system thereto.