WO2003076655A2 - Method for identifying a nucleic acid analyte - Google Patents

Abstract

The invention relates to a method for identifying a nucleic acid analyte by carrying out a hybridization with two or more hybridization probes.

Description

Method for determining a nucleic acid analyte

description

The invention relates to a method for determining a nucleic acid analyte by hybridization with two or more hybridization probes.

The use of molecular beacons as hybridization probes for the detection of nucleic acid target molecules in a sample is known (see US Pat. No. 5,607,834). Molecular beacons usually contain a so-called hairpin structure consisting of a star with two mutually complementary sequence sections and a loop, a fluorescent label group and a quench group. The stem sequence, which is not capable of binding to the target molecule, serves to keep the fluorescent labeling group and the quench group in proximity when the probe is not bound to the analyte. In this way, photons that are absorbed by the fluorescent label group are not emitted as fluorescent photons, but the energy is transferred to the quench group. The loop sequence is complementary to the target molecule to be detected. When the probe binds to the target molecule, the loop opens and hybridizes with the target molecule. In this way, the fluorescent label group is removed from the vicinity of the quench group, whereby fluorescence occurs and can be measured. Molecular beacons are used in particular for homogeneous detection methods in which it is not possible or desirable to isolate hybridization products of the target nucleic acid and the probe from an excess of the hybridization probes, such as in real-time monitoring of polymerase chain reactions. Furthermore, the use of a combination of hybridization probes in a detection method is known, one probe carrying a fluorescence donor group and the other probe carrying a fluorescence acceptor group. When hybridizing both probes to a target nucleic acid, a detectable energy transfer between the donor and acceptor groups can take place (see, for example, US Pat. No. 4,996, 143).

If target nucleic acid molecules are detected by simultaneously binding two or more different hybridization probes, undesired interactions (excitation with one wavelength, but detection in both channels) are a major problem, which leads to a deterioration in sensitivity and an increase in the measurement time. This problem exists in particular in fluorescence correlation spectroscopy, in which only a few molecules or even only single molecules are to be detected.

One object of the present invention is to develop a method for determining nucleic acid analytes in which the disadvantages mentioned above can be at least partially avoided.

This task is solved by using at least two co-res po ndies and h yb ri disie r ng sso ndengel. These corresponding hybridization probes are selected such that they have a hairpin structure, the loop sections of the hairpins comprising the probe sequences complementary to a nucleic acid analyte. The stars are formed by two mutually complementary arm sequences, which are arranged at the ends of the probe sequences. In addition, the sterns of both probe molecules are complementary to one another. The selection of star and loop sequences is such that a hybrid formed between the loop and a complementary sequence of the analyte is more stable than the hybrid of the complementary ones Sequences of the star is. This can be done by adjusting the length and / or the G / C portion of the respective hybridizing areas accordingly.

Both hybridization probes are furthermore selected such that they bind to the analytes adjacent to one another, preferably in the immediate vicinity without any space between, the adjacent stem sections of the probes additionally hybridizing with one another. A first label group is preferably attached to the 3 'end of the first probe and a second label group is preferably attached to the 5' end of the second probe. After hybridization to the analyte and the additional hybridization between the two probes, the star keeps the two labeling groups, preferably luminescence or fluorescence labeling groups, at a small spatial distance from one another. In this way, an interaction between the two marker groups, for example an energy transfer between an energy donor and an energy acceptor group, is possible. Thus, a luminescence or fluorescence donor group can be excited with a laser, which transfers its energy to the acceptor group and excites the latter to fluoresce. The fluorescence of the acceptor group is then detected. Alternatively or additionally, the analysis of the measurement signal can include a time-resolved determination.

The invention thus relates to a method for determining a nucleic acid analyte in a sample, comprising the steps:

(a) providing a sample that may contain the analyte,

(b) Providing at least two hybridization probes capable of binding to the analyte, each carrying different labeling groups, and, if they are not bound to the analyte, in a hairpin structure comprising a star with two mutually complementary sections and a loop comprising those capable of binding to the analyte Sequence, a first hybridization probe being able to bind to the analyte adjacent to a second hybridization probe, the labeling groups of the two hybridization probes being arranged such that, when the probes are bound to the analyte, they can interact with one another, the sequences in the asteroids of the the first and second hybridization probes are complementary to one another, that if the probes are bound to the analyte, hybridization can take place between sequences from the asterisks of the first and the second probe,

(c) contacting the sample with the hybridization probes under conditions where the probes can hybridize with the analyte, and

(d) Detect the simultaneous hybridization of at least two probes with the analyte.

The nucleic acid analytes determined by the method, e.g. DNA or / and RNA can be obtained from biological samples, especially from mammals such as humans, but also from other organisms, e.g. Microorganisms. In addition, analytes can also be detected that have been generated in vitro from biological samples, cDNA molecules that have been produced from mRNA by reverse transcription. The sample used for the method is preferably from an organism, e.g. from tissue or body fluid of an organism, especially from a human.

The method can be used, for example, to analyze gene expression, e.g. B. to determine a gene expression profile, or to analyze mutations, such as single nucleotide polymorphisms (SNP). However, the method is also suitable for determining enzymatic reactions on nucleic acids, for example for determining Nucleic acid amplification reactions, especially in one or more thermocycling processes.

The hybridization probes used for the method according to the invention are preferably oligonucleotides, in particular DNA molecules. However, corresponding nucleic acid analogs, such as peptide nucleic acids (PNA), locked nucleic acids (LNA) or other nucleotide analogs, can also be used. The length of the hybridization probes can in principle be chosen as in known probes and is usually in the range from 20 to 200 nucleotides.

The hybridization probes contain marker groups, preferably fluorescent marker groups, which differ in at least one measurement parameter, in particular selected from the emission wavelength and duration of the fluorescence. Particularly preferably, one probe contains a donor labeling group and the other probe contains an acceptor labeling group, wherein an energy transfer can take place between the donor and acceptor labeling groups.

The analytes are detected by determining the binding of both probes to the analyte. The determination preferably comprises the detection of an energy transfer between the marker groups of the first and second probe. Suitable combinations of donor and acceptor labeling groups for such energy transfer (FRET) are known to those skilled in the art (see e.g. U.S. Patent 4,996,143).

However, the method according to the invention can also include a so-called cross-correlation determination, in which at least two different markings, in particular fluorescence markings, are used, the correlated signal of which is determined. The The basic implementation of such a cross-correlation determination is, for example, in Schwüle et al. (Biophys. J. 72 (1 997), 1 878-1 886) and Rigler et al. (J. Biotechnol. 63 (1 998), 97-1 09).

The method according to the invention is based on the fact that a combination of hybridization probes is used which hybridize to adjacent sequence sections of the analyte. A combination of probes is preferably used, the first probe binding to a region of the analyte which is immediately 3 ′ to the region to which the second probe binds. In this case, the first probe conveniently carries its label group directly at its 3 'end and the second probe carries its label group at the 5' end. In this way, when the two probes are bound to the analyte, there is a or a very small distance between the two labeling groups, by means of which a change, e.g. B. E n e rg i etra n s f e between two marker groups, is made easier.

The hybridization probes used in the method according to the invention are preferably so-called molecular beacons which, when bound to the analyte, have an increase in the signal strength of the labeling group. This can be achieved, for example, if the signal of the labeling group is at least partially deleted by the presence of a quench group on the probe, if the probe is not bound to the analyte. The signal strength is preferably increased by at least a factor of 2, particularly preferably at least by a factor of 5.

The method according to the invention is preferably carried out with low concentrations of the analyte or hybridization probes present in the sample and with a very small sample volume. The sample volume is preferably in the range of <1 0 ^"6 I and particularly preferably <10 ^{~ 8} 1. To determine such samples a microwave structure with several recesses for receiving sample liquid can be used as the carrier, the individual wells having a diameter of between 10 and 1000 μm, for example. Suitable microstructures are described, for example, in DE 1 00 23 421 .6 and DE 1 00 65 632.3. Furthermore, the carrier can contain temperature control elements, for example Peltier elements, which enable temperature regulation of the carrier and / or individual sample containers therein.

The carrier used for the method is expediently equipped in such a way that it enables optical detection of the sample. A carrier which is optically transparent at least in the region of the sample containers is therefore preferably used. The carrier can either be completely optically transparent or contain an optically transparent base and an optically opaque cover layer with cutouts in the sample containers. Suitable materials for supports are, for example, composite supports made of metal (e.g. silicon for the cover layer) and glass (for the base). Such supports can be produced, for example, by applying a metal layer to the glass with predetermined cutouts for the sample containers. Alternatively, plastic carriers, e.g. made of polystyrene or polymers based on acrylate or methacrylate. It is further preferred that the carrier has a cover for the sample containers in order to provide a closed system which is essentially isolated from the surroundings during the measurement.

Furthermore, electrical fields, in particular in the area of the sample containers, can be generated in the carrier in order to achieve a concentration of the analytes to be determined in the measurement volume. Examples of electrodes which are suitable for generating such electrical fields are described, for example, in DE 101 03 304.4. The analyte is particularly preferably detected by fluorescence correlation spectroscopy (FCS). The measurement of one or a few analyte molecules is preferably carried out in a measurement volume which is part of the sample volume, the concentration of the analyte molecules to be determined preferably being <10 ^"6 mol / l and the measurement volume preferably being <10 ^{" 14} I. Measurement signals originating from the marker groups of the hybridization probes are determined by luminescence measurement. For details of apparatus details of the method and devices suitable for carrying out the method, reference is made to EP-B-0679251.

Alternatively, the detection can also be carried out by time-resolved decay measurement, a so-called time / gating, the signal of the marker groups, e.g. the fluorescence within the measurement volume is excited and then the detection interval on the detector is preferably opened at a time interval of> 100 ps. This measurement method is described, for example, by Rigler et al. in "Ultrafast Phenomena" D.H. Auston, ed., Springer 1984.

In a particularly preferred embodiment of the method, a confocal single-molecule analysis is carried out such that the distance between the measurement volume in the sample liquid and the focusing optics of the light source used to excite fluorescence marking groups in the sample liquid is> 1 mm and the sample liquid is a light source, in particular from the Focusing optics is thermally insulated. Such a method and a device suitable therefor are described, for example, in DE 101 11 420.6. By thermally isolating the sample liquid from equipment components, the temperature of the sample can be set independently and varied during the process. This means that temperature-variable processes, e.g. the determination of nucleic acids Hybridization melting curves or the determination of nucleic acid amplification reactions can be carried out.

The carrier is preferably a microstructure with several, preferably at least 10 ² containers for holding a sample liquid, the sample liquid in the separate containers being able to come from one or more sources. The sample liquid can be introduced into the containers of the carrier, for example by means of a piezoelectric liquid dispensing device.

The containers of the carrier are designed in such a way that they enable the detection reagent to be bound with the analyte in solution. The containers are preferably indentations in the carrier surface, these indentations generally being able to have any shape, for example circular, square, diamond-shaped, etc. The carrier can also comprise 10 ³ or more separate containers.

The optical excitation device comprises a strongly focused light source, preferably a laser beam, which is focused on the measurement volume in the sample liquid by means of corresponding optical devices. The light source can also contain two or more laser beams, which are then focused on the measurement volume by different optics before entering the sample liquid. The detection device can contain, for example, a fiber-coupled avalanche photodiode detector or an electronic detector. However, excitation or / and detection matrices, consisting of a dot matrix of laser dots, generated by diffraction optics or a quantum well laser, and a detector matrix, generated by an avalanche photodiode matrix or electronic detector matrix, for example a CCD camera, can also be used , be used. In a preferred embodiment, the light source comprises one or more laser beams, which are split into multiple foci by guiding through one or more diffraction-optical elements, as described in DE 101 26083.0.

The carrier can already be provided in a prefabricated form, luminescence-labeled detection reagents, preferably luminescence-labeled hybridization probes or primers, being introduced into several separate containers of the carrier. The carrier containing the detection reagents is then advantageously dried.

In a preferred embodiment of the invention, a prefabricated carrier is provided which contains a large number of separate, e.g. 100 containers, into which different detection reagents, e.g. Reagents for the detection of nucleic acid hybridization, such as primers and / or probes, are present. This carrier can then be mixed with an organism to be examined, e.g. a human patient, sample can be filled so that different analytes are determined from a single sample in the respective containers. Such carriers can be used, for example, to create a gene expression profile, e.g. for the diagnosis of diseases or for the determination of nucleic acid polymorphisms, e.g. to detect a certain genetic predisposition.

Fluorescent labels, such as fluorescein, rhodamine, phycoerythrin, CY3, CY5 or derivatives thereof, are preferably used as labeling groups. The fluorescent labeling groups can be differentiated using the emission wavelength, the lifetime of the excited states or a combination thereof.

Furthermore, the invention also relates to a reagent kit for determining nucleic acid analytes, comprising at least two hybridization probes capable of binding to the analyte, each carrying different labeling groups, and, if they are not bound to the analyte, in a hairpin structure comprising a star with two mutually complementary sections and a loop comprising the sequence capable of binding to the analytes, one of which can bind the first hybridization probe adjacent to a second hybridization probe to the analyte, the labeling groups of the two hybridization probes being arranged such that, when the probes are bound to the analyte, they can interact with one another, the sequences being in the asterisks of the first and second hybridization probes are complementary to one another, that when the probes are bound to the analyte, hybridization between sequences from the asterisks from the first and the second probe can take place.

This reagent kit is preferably used for the method according to the invention.

Another way of reducing interference in cross-correlation is to carry out a time-resolved measurement by time gating. This method can optionally also be carried out independently of the probes described above for the determination of (any) analytes by cross-correlation or coincidence analysis by means of optical excitation of several luminescent molecules in one or more confocal detection volumes. Here, several spectrally distinguishable marker groups bound to the analyte are repeated with a high pulse frequency, which is in the order of the dead time of the detector, preferably in the range from 1 0 to 0, 1 MHz (corresponding to time intervals of 0, 1 -10 μs), for example of about 1 MHz (corresponding to 1 μs). You can do this by appropriate electronic background analysis, calculating a Correlation or / and coincidence curve "correctly" occurring correlation signals can be distinguished from background signals which are created by a newly desired interferer z (C rossal k) with the marker groups.

The invention preferably comprises the combination of three

Technologies, namely:

(i) multi-color detection, preferably using a

Excitation laser, (ii) combination of molecular beacon hybridization probes, such that energy transfer between the two marker groups of the hybridization probes can only take place when bound to the correct analyte and

(iii) specific detection of the target molecule by multiple laser excitation with generation of a coincidence function suitable for the specific detection of the analyte.

The method is particularly suitable for the detection of single molecule analytes by confocal detection, preferably by means of fluorescence correlation spectroscopy. Due to the low concentrations of analytes and hybridization probes prevailing in the context of fluorescence correlation spectroscopy, there is almost no unspecific energy transfer between the two labeling groups. A high level of sensitivity can be achieved by suitable statistical data evaluation. The method allows cross-correlation determination with only a single excitation light source, in particular a single laser. Undesired interference between the marker groups can be largely eliminated by using the molecular beacon probes. Overall, this leads to a significant increase in sensitivity compared to known fluorescence correlation spectroscopic detection methods. Figure 1 shows an embodiment of the method according to the invention. A sample containing a nucleic acid analyte (T) is brought into contact with a detection reagent, comprising two hybridization probes (S _{1 f} S ₂ ), which are present as hairpin structures, each with an asterisk and a loop. The probe S binds directly to the target nucleic acid T on the 3 'side of the probe S _2. The probe S has a first fluorescent labeling group (F,) at its 3' end and a quench group (Q ^ at its 5 'end The probe S ₂ has a second fluorescent labeling group (F ₂ ) at its 5 'end and a quench group (Q ₂₎ at its 3' end, if the probes are not bound to the analyte (T) fluorescent marker groups and the Quenchgruppen in close spatial proximity, so that an originating from the fluorescent groups signal is at least deleted teileise Upon binding to the analyte, the spatial proximity of the fluorescent labeling groups {FF ₂₎ suspended over the Quenchgruppen (Q _{1 #} Q _2). so that they have a higher fluorescence intensity.

When bound to the analyte, the two fluorescent labeling groups (F _{1 #} F ₂ ) are arranged in close spatial proximity to one another, since the 5'-sided stem section of S ₂ can hybridize with the 3'-sided stem section of S ,. Because of this spatial proximity, there is the possibility that the two fluorescent marker groups (F _{1 #} F ₂ ) act as fluorescent donor and fluorescent acceptor groups, so that when the first group is excited, energy is transferred to the second group and a fluorescent radiation emitted by the second group is detected can.

Another aspect of the invention relates to a method for determining an analyte by cross-correlation or coincidence analysis by means of optical excitation of several luminescent, for example fluorescent Molecules in at least one confocal detection volume, comprising the steps:

(a) Providing a sample, comprising different species of luminescent molecules, (b) irradiating the sample with an optical excitation and focusing device, comprising several separate light sources, each for exciting individual species of luminescent molecules in at least one confocal detection volume, the part of the sample is where. the irradiation of individual light sources takes place at different time intervals and repetitively and

(c) collecting emission radiation from the at least one measurement volume by one or more detectors and evaluating the collected radiation, signals which are collected during a time interval in which a first species is excited by luminescent molecules but which originate from a second species , are not taken into account in the evaluation or are filtered out.

Figure 2 shows an embodiment of this aspect of the invention. Two spectrally different luminescent labeling groups, e.g. a green (g) and a red (r) fluorescent marker group, sequentially excited at short clock intervals, which are of the order of the detector's dead time. Green (g) and red (r) laser pulses are used for excitation.

The photons striking the detector (s) are evaluated. A "correct" signal is when a green photon (g) is picked up with a green laser pulse and a red photon (r) with a red laser pulse. If, on the other hand, a red photon (r *) is caught with a green laser pulse, this is an undesirable interference (crosstalk). This - with conventional ones Methods that cannot be resolved - event can be filtered out of the overall signal by suitable electronic measures, whereby a new correlation or coincidence curve can be determined. In this way a significant reduction in the background is achieved.

Claims

Expectations A method for determining a nucleic acid analyte in a sample, comprising the steps: (a) providing a sample that may contain the analyte, (b) Providing at least two hybridization probes capable of binding to the analyte, each carrying different labeling groups, and, if they are not bound to the analyte, in a hairpin structure comprising a star with two mutually complementary sections and a loop comprising those capable of binding to the analyte Sequence are present, wherein a first hybridization probe can bind adjacent to a second hybridization probe to the analyte, the labeling groups of the two hybridization probes being arranged such that, when the probes are bound to the analyte, they can interact with one another, the sequences in the asteris of the first and second hybridization probes are complementary to one another, that if the probes are bound to the analyte, hybridization can take place between sequences from the asterisks of the first and the second probe, (c) contacting the sample with the hybridization probes under conditions where the probes can hybridize with the analyte, and (d) Detect the simultaneous hybridization of at least two probes with the analyte. 2. The method according to claim 1, characterized in that the hybridization probes carry fluorescent labeling groups which are in at least one measurement parameter, in particular selected from the emission wavelength and / or lifetime of the Fluorescence. 3. The method according to claim 1 or 2, characterized in that step (d) the detection of an energy transfer between the Includes labeling groups of the first and second probes. 4. The method according to any one of claims 1 to 3, characterized in that hybridization probes are used, the first probe binding to a region of the analyte which is immediately 3 ′ to the region to which the second probe binds. 5. The method according to claim 4, characterized in that the first probe contains its labeling group at the 3 'end and the second probe contains its labeling group at the 5' end. 6. The method according to any one of claims 1 to 5, characterized in that the labeling group of at least one probe has a lower signal strength when the probe is not bound to the analyte than when the probe is bound to the analyte. 7. The method according to claim 6, characterized in that the signal of the labeling group is at least partially deleted by a quench group when the probe is not bound to the analyte. 8. The method according to any one of claims 1 to 7, characterized in that step (d) comprises detection by confocal detection. 9. The method according to claim 8, characterized in that the detection comprises a single molecule detection. 1 0. Method according to one of claims 8 or 9, characterized in that step (d) comprises detection by fluorescence correlation spectroscopy or / and time gating. 1 1. Method according to one of claims 8 to 10, characterized in that a single light source is used to excite the marker groups. 1 2. The method according to any one of claims 8 to 10, characterized in that one uses several light sources to excite the marker groups. 13. The method according to any one of claims 1 to 12, characterized in that the light source (s) used to excite the marker groups is (are) irradiated into the sample in the form of repetitive pulses. 14. The method according to claim 13, characterized in that the frequency of the pulses is in the range of 10 to 0.1 MHz. 15. Reagent kit for the determination of nucleic acid analytes, comprising at least two hybridization probes capable of binding to the analyte, each carrying different labeling groups, and, if they are not bound to the analyte, in a hairpin structure comprising a stem with two mutually complementary sections and a loop comprising the there is a sequence capable of binding the analyte, a first hybridization probe being able to bind to the analyte adjacent to a second hybridization probe, the labeling groups of the two hybridization probes being arranged such that, when the probes are bound to the analyte, they can interact with one another, whereby the sequences in the asterisks of the first and second hybridization probes are complementary to one another, that when the probes are bound to the analyte, a Hybridization between sequences from the asterisks from the first and the second probe can take place. 16. Use of a reagent kit according to claim 15 in a method according to one of claims 1 to 14. 7. A method for determining an analyte by cross-correlation or coincidence analysis by means of optical excitation of a plurality of luminescent molecules in at least one confocal detection volume, comprising the steps: (a) providing a sample comprising different species of luminescent molecules, (b) irradiating the sample with an optical excitation and focusing device, comprising a plurality of separate light sources, each for excitation of individual species of luminescent molecules in at least one confocal Detection volume, which is part of the sample, the irradiation of individual light sources taking place at different time intervals and repetitively and (c) collecting emission radiation from the at least one measurement volume by one or more detectors and Evaluation of the collected radiation, signals which are collected during a time interval in which a first species is excited by luminescent molecules, but which originate from a second species, are not taken into account in the evaluation. 8. The method according to claim 1 7, characterized in that the time intervals are each in the range of 0.1-10 s.