EP1440313A2 - Arrays with hairpin structures - Google Patents

Abstract

The invention relates to arrays of nucleic acids immobilized on a carrier, which are present at least partly in the form of secondary structures such as hairpin structures. The invention also relates to methods for the production of said arrays and their applications.

Description

Arrays with hairpin structures

description

The invention relates to arrays of nucleic acids immobilized on a carrier, which are at least partially in the form of secondary structures, such as hairpin structures. Methods of making such arrays and uses thereof are also disclosed.

In recent years, the technology of receptor arrays immobilized on a carrier, e.g. DNA chips, a valuable tool that enables complex analyte determination procedures to be carried out quickly and in high parallel. The biophysical principle on which the receptor arrays are based is that of the interaction of a specific immobilized receptor with an analyte present in a liquid phase, for example by nucleic acid hybridization, with a large number of receptors, e.g. Hybridization probes are attached which are compatible with analytes present in the sample, e.g. specifically bind complementary nucleic acid analytes.

A binding event between immobilized receptor and analyte is usually detected by detection of a label group that is bound to the analyte. A carrier and a method for analyte determination which allow integrated synthesis of receptors and analysis are e.g. described in WO 00/1 301 8.

In order to be able to deal with complex biological questions (gene expression studies, target validation, sequencing by hybridization, re-sequencing) with receptor arrays, e.g. DNA chips, it is of fundamental importance that the hybridization between receptor and target is carried out as error-free as possible can. The The detection system must therefore be able to distinguish between a so-called "fill match", ie if the probe and target are completely complementary, and a "mismatch" if one or more incorrect base pairings are present. It is of course particularly difficult to distinguish between a "single mismatch" when only 1 base pairing is faulty and the "fill match". Furthermore, for thermodynamic reasons, particularly terminal (terminal) base mismatches are difficult or insufficient to detect. Mismatches in the middle of a sequence, on the other hand, are easier to detect for the same reasons.

Known DNA chips have nucleic acid receptors in single-stranded form if possible. When selecting the sequences for the receptors, care is therefore taken to avoid any formation of secondary structures. Base mismatches are now detected by not only applying the actual sequence to be queried to the DNA chip, but also, as a comparison, the corresponding sequence with a mismatch in the middle of the base sequence as a negative control. Whether it is a "fill match" or "mismatch" can be detected by the different signal intensities in each case, which result from hybridization of the sample (target) to the probe or its negative control sequence (mismatch sequence). However, since a clear decision cannot always be made here, not only a single sequence, but several (e.g. 20 sequences per gene) sequences (R. Lipschutz et al., Nature Genetics, 1 999, 21, 20ff), which each result as fragments of the sample to be detected, are used with the associated control sequences (mismatch sequences). As a result, not only a single nucleic acid sequence is required on the DNA chip for the detection of a single sample sequence, but there are usually 20 sequences plus the respective 20 negative sequences. Control sequences (mismatch sequences) required. This results in a considerable additional effort in the production of DNA chips and significantly reduces their information density.

There are currently no ways to detect terminal base mismatches on DNA chips.

It is therefore an object of the invention to provide a system which allows base mismatches to be detected very precisely. Furthermore, the system according to the invention should not only recognize base mismatches in the middle of a sequence, but also at the end (terminal) on an array in a highly parallel manner.

This object is achieved by providing receptor arrays which contain nucleic acid receptors which are at least partially in the form of hairpins.

Hairpins are a special form of secondary structures in nucleic acids, which are composed of two complementary sequence sections in the so-called stem and a further sequence section in the so-called loop (FIG. 1 a). Here there is a balance between the closed shape and the open shape (Figure 1 b). Hairpin structures have already been used in solution for the label-free detection of hybridization events (Tyagi et.al, Nature Biotechnology 1 995, 14, 303-308). These hairpin structures (FIG. 2 type A) are distinguished by the fact that the recognition sequence is in the loop of the hairpin (Marras et al, Genetic Analysis; Biomolecular Engineering, 1 999, 14, 1 51 -1 56). In a particular embodiment, a quencher and a fluorophore molecule are in close spatial proximity in the closed state, so that the fluorescence is quenched. If a hybridization event occurs with the recognition sequence in the loop, the hairpin opens, whereby Fluorophore and quencher are spatially separated. As a result, a fluorescence signal can be observed. In addition to known dyes, quenchers can also be poly-deoxyguanosine sequences (M. Sauer, BioTec, 2000, 1, 30ff). This has the advantage that the hairpin structure only has to be labeled with a fluorophore (M. Sauer et.al., Anal.Chem. 1999, 71 (14), 2850ff) that the incorporation of a quencher molecule is omitted.

Studies on the behavior of hairpin structures on a fixed phase - i.e. Arrays with hairpin structures - are known (US 5770772), but only use the presence of the double-stranded structure of a hairpin as a recognition site for e.g. Proteins. Evidence of analyte binding by dissolving the hairpin structure is not disclosed. In particular, no hairpin structures are known which use the sequence information in the stem of the hairpin as a recognition sequence for hybridization. Furthermore, no hairpin structures have yet been known whose connection to the solid phase is not via a terminal end.

The invention relates to a method for determining analytes, comprising the steps:

(a) Providing a carrier with a plurality of predetermined areas, on each of which different receptors selected from nucleic acids and nucleic acid analogs are immobilized, the receptors in one or more of the predetermined areas

The absence of an analyte specifically capable of binding is at least partially present as a secondary structure,

(b) bringing the carrier into contact with a sample containing analytes and (c) determining the analytes via their binding to the receptors immobilized on the carrier, the binding of an analyte to a receptor specifically capable of binding the detection of the Resolution of the secondary structure present in the absence of the analyte comprises.

Another object of the invention is a device for determining analytes, comprising

(i) a light source matrix,

(ii) a carrier with a plurality of predetermined positions, at each of which different receptors are immobilized on the carrier, (iii) means for supplying fluids to the carrier and for removing fluids from the carrier, and (iv) a detection matrix with a plurality of detectors, which are assigned to the predetermined positions on the carrier.

Surprisingly, the inventions according to the structure of the structure can be used for a very precise discrimination of base mismatches on a solid phase, in particular on an array. The hairpin structures according to the invention can be generated highly parallel both in situ on the solid phase, but can also, if prefabricated, be immobilized on this.

The receptors are selected from nucleic acid biopolymers, e.g. Nucleic acids such as DNA and RNA or nucleic acid analogues such as peptide nucleic acids (PNA) and locked nucleic acids (LNA) as well as combinations thereof. Nucleic acids are particularly preferably determined as analytes, the binding of the analytes comprising hybridization. However, the method also enables the detection of other receptor-analyte interactions, e.g. the detection of nucleic acid-protein exchange effects.

The method according to the invention preferably comprises a parallel determination of several analytes, ie a carrier is provided, that contains several different receptors that can react with different analytes in a single sample. The method according to the invention preferably determines at least 50, preferably at least 100 and particularly preferably at least 200 analytes in parallel.

The receptors can be immobilized on the carrier by covalent binding, non-covalent self-assembly, charge interaction or combinations thereof. The covalent bond preferably comprises the provision of a support surface with a chemically reactive group to which the starting components for receptor synthesis can be bound, preferably via a spacer or linker. The non-covalent self-assembly can, for example, on a noble metal surface, e.g. a gold surface, by means of thiol groups, preferably via a spacer or linker.

The present invention is preferably characterized in that the detection system for analyte determination combines a light source matrix, a microfluidic carrier and a detection matrix in an at least partially integrated structure. This detection system can be used for integrated synthesis and analysis, in particular for the construction of complex supports, e.g. Biochips, and for analysis of complex samples, e.g. for genome, gene expression or proteome analysis.

In a particularly preferred embodiment, the synthesis of the receptors takes place in situ on the carrier, for example by passing fluid with receptor synthesis building blocks over the carrier, the building blocks are immobilized in place-specific and / or time-specific manner on predetermined areas on the carrier and these steps are repeated, until the desired receptors have been synthesized on the respective predetermined areas on the support. This includes receptor synthesis preferably at least one fluid-chemical step, a photochemical step, an electrochemical step or a combination of such steps and online process monitoring, for example using the detection matrix.

The light source matrix is preferably a programmable light source matrix, e.g. selected from a light valve matrix, a mirror array, a UV laser array and a UV LED (diode) array.

The carrier is preferably a flow cell or a micro flow cell, i.e. a microfluidic carrier with channels, preferably with closed channels, in which the predetermined positions with the differently immobilized receptors are located. The channels preferably have diameters in the range from 10 to 10000 μm, particularly preferably from 50 to 250 μm, and can in principle be designed in any form, e.g. with a round, oval, square or rectangular cross-section.

As already stated, the secondary structures of the receptors preferably comprise a hairpin structure, which is composed of a stem and a loop. In a first embodiment of the method according to the invention, the sequence of the receptor which is bindable with the analyte can be located in the area of the loop of a hairpin. The hairpin structure is opened by binding the loop to the receptor. This hairpin opening can in turn be detected by suitable measures (eg see above). In a particularly preferred embodiment, however, the sequence of the receptor that is specifically bindable with the analyte is located in the stem of the hairpin structure. In this embodiment too, the binding of the analyte to the receptor brings about a detectable dissolution of the hairpin structure. According to a preferred embodiment, the hairpin structures according to the invention with a recognition sequence in the stem (FIGS. 2B, 3A and 3B) comprise complementary sequences A and A * in the stem and a linker unit L in the loop. There are building blocks in the loop of the hairpin that cannot make base pairings (e.g. polyethylene glycol, alkyl, polyethylene glycol phosphate or alkyl phosphate units) or building blocks that can only make weak base pairings (e.g. a T _n loop with n = 2- 8th). Both sequence sections A (FIG. 3B) or A * (FIG. 3A) in the stem can serve as recognition sequences. If a hybridization experiment is carried out, for example a sequence A in the sample to be examined competes with the reference sequence A in the stem of the hairpin for the sequence A * (FIG. 3A). This competitive situation is used to increase the specificity of the hybridization. If, for example, sequence A in the sample is not completely complementary to A * (ie mismatches occur), the pairing between the two sequences A and A * is more stable in the hairpin, which has the consequence that the hybridization balance on the left side is shifted to the closed form of the hairpin (FIG. 3A). If a labeled sample A is used for hybridization, this means that no or only a small signal can be detected, since the equilibrium lies on the side of the closed hairpin. Signals can only be detected when the hairpin structure is in the open state, ie a stable pairing between A in the sample to be examined and A * in the hairpin is possible, and the equilibrium is on the right, ie with an open hairpin.

In addition to common variables of stringency conditions, such as salt concentration, temperature, probe and target concentration, another variable is introduced with which the stringency of a hybridization experiment can be influenced. Furthermore, the hybridization balance (and thus the stringency) of the reference probe or the recognition sequence can be varied, inter alia, by the fact that these sequences contain building blocks of nucleic acid analogues which are distinguished by the fact that they bind more strongly with DNA than DNA with DNA. PNA or LNA building blocks or other building blocks known to the person skilled in the art having the described characteristics can be used for this purpose.

The procedure described ensures that, in contrast to the usual procedure (use of 1 perfect match + 1 single base mismatch probe), only one probe needs to be used for the discrimination between perfect match and single base mismatch, and thus parking spaces on the array can be saved or more information can be queried with a predetermined amount of parking spaces. Furthermore, terminal mismatches can also be queried, since the presence of the reference sequence in the same molecule allows higher stringency conditions to be set than if two separate probes were used to discriminate perfect match and single base mismatch.

In a further preferred embodiment, the hairpin structures comprise two complementary sequences (A, A *) and two non-complementary units (Z, X) in the stem and a linker unit (L) in the loop

(Figure 4). The recognition sequences can both be close to the solid phase

Sequence sequence A-Z (Figure 4A) as well as that of the solid phase

Sequence sequence A * -Z (Figure 4B) serve. Crucial is the fact that X and Z don't pair up. For this purpose it is proposed according to the invention that X is one or more nucleic acid building blocks capable of pairing and Z is one or more non-pairing building blocks. Z can be, for example, an “abasic site” (DNA or RNA building block without a heterobase) or a building block known to the person skilled in the art which does not enter into base pairing, but does

DNA structure does not interfere. L is to be understood as a linker which preferably consists of nucleic acid building blocks which cannot be paired, for example Polyethylene glycol phosphate units (R. Micura, Angew. Chemie, 2000, 39 (5), 922ff) or building blocks that can only form weak base pairs (eg a T _n loop with n = 2-8). This ensures that terminal mismatches in particular can be better detected. This is done in that in this embodiment (FIG. 4A) the target A-X ^{* has} further bases available for pairing, but not the reference AZ. This has the effect that the balance between the closed form of the hairpin (left) is advantageously shifted to the right side (open hairpin) if an additional base pairing from the target AX ^* with the sequence region X in the hairpin can take place. If mismatches occur between Target AX ^* and the sequence area X in the hairpin, this is not the case and the balance is not increasingly shifted to the right (open) side.

Due to the presence of additional mating sections X in the hairpin structure of the receptor, which can be further increased with the analyte to be detected, the co-p l e m e n t a r s i n d, k a n n s o m i t d i e st r i n g e n s of the hybridization experiment (FIGS. 4A and 4B). Here too, as described above, nucleic acid analogs can be used which bind more strongly with DNA than DNA with DNA.

In a further embodiment, Z can also be the mixture of the 4 bases adenosine, guanosine, cytidine and thymidine or uracil.

In yet another embodiment, the hairpin structure contains a marker group which is at least partially quenched in the closed state, for example a fluorophore. By dissolving the hairpin structure, the signal originating from the marker group increases and this signal increase is demonstrated. A hairpin structure (FIG. 5) according to the invention thus contains, for example, a quencher (Q) and a fluorophore (F), which are located at opposite ends of the hairpin nucleic acid sequence. According to the invention Fluorescence in the closed hairpin is quenched by the spatial proximity of Q and F. Fluorescence can be detected in the open state. Molecule combinations Q and F are well known to those skilled in the art.

In yet another embodiment, hybridization can also be carried out using double-stranded targets (FIG. 6). If both strands are marked, the light intensity detectable for a parking space can be increased.

In a further embodiment, the hairpin structures of type A (recognition sequence in the loop) and type B (recognition sequence in the stem) can be connected to the carrier not only terminally but also internally (FIG. 7). It also discloses type A hairpin structures internally bonded to the carrier. Combinations of terminally and internally immobilized receptors on a carrier are also possible.

An improvement over the prior art is achieved in particular in that the hairpin structure according to the invention contains the recognition sequence in the stem. The strand complementary to the recognition sequence is used as a reference sequence for mismatch discrimination. In a hybridization experiment, a target sequence in the sample solution competes with the reference sequence (A *) for the probe sequence (A). If desired, the stringency can be further increased by incorporating special nucleic acid building blocks (PNA, LNA) in the reference strand. This means that only hybridization will take place - ie change the hairpin to the open form - if the target sequence in the sample solution is exactly complementary to the probe sequence. If this is not the case, the reference sequence integrated in the hairpin ensures that the hairpin does not change to the open form and therefore no hybridization with a target sequence can take place. Furthermore, the hairpin structure according to the invention allows the discrimination of terminal base mismatches. These are made possible by hybridizing the target sequence to position X. Base pairings complementary to terminal position X decide whether the hairpin changes to the open form and whether a hybridization event can be detected as a result.

As a result, in contrast to conventional DNA arrays, significantly fewer sequences have to be applied for deciding whether it is a “fill match” or “mismatch”. The result of this is that with the a priori given maximum parking space density of an array, more different target sequences can be processed. This significantly increases the information density of the array.

By incorporating fluorescence (F) and / or quencher (Q) building blocks into the hairpin structure according to the invention, it is also possible to achieve a label-free detection of DNA arrays (FIG. 5).

In a preferred embodiment of the invention, an integrated system for determining analytes is provided, which allows highly parallel in situ production of complex populations of hairpin receptors immobilized in microstructures for the detection of analytes.

A device comprising:

(i) a light source matrix,

(ii) a microfluidic carrier having a plurality of predetermined ones

Positions at which in each case different receptors selected from nucleic acids and nucleic acid analogs are immobilized on the support, in one or more of the predetermined ones

Specific areas of the receptors in the absence of one bindable analytes are at least partially present as a secondary structure, (iii) means for supplying fluids to the carrier and for deriving fluids from the carrier and (iv) a detection matrix comprising a plurality of detectors which are assigned to the predetermined areas on the carrier.

In the device according to the invention, preferably at least components (ii), (iii) and (iv) are present in an integrated form. The carrier is particularly preferably arranged between the light source matrix and the detection matrix. The detectors of the detection matrix are preferably made of photodetectors and / or electronic detectors, e.g. Electrodes selected.

The device according to the invention can be used for the controlled in situ synthesis of nucleic acids, e.g. DNA / RNA oligomers are used, and photochemical, fluid-chemical and / or electronically removable protective groups can be used as temporary protective groups. The location- and / or time-resolved receptor synthesis can be carried out by targeted activation of electrodes in the detection matrix, targeted fluid supply in defined areas or area groups on the support or / and targeted exposure via the light source matrix.

The present invention is further to be illustrated by the following figures:

FIG. 1A schematically shows a hairpin structure and FIG. 1B shows the balance between the closed and open form of the hairpin.

Figure 2 shows two types of surface-bound hairpin structures. According to FIG. 2A, the recognition sequence of the receptor that is bindable with the analyte is in the loop. According to Figure 2B there is the recognition sequence in the stem which is bindable with the analyte. There are two options for arranging the recognition sequence, ie near the surface (A) or away from the surface (A ^* ). The arrangement of the recognition sequence shown in FIG. 2B represents a preferred embodiment of the invention.

FIG. 3 shows an embodiment of the hairpin structure according to the invention and its mode of action, where S is the solid phase and L is a linker sequence. In FIG. 3A, A ^{* is} the recognition sequence of the receptor which is capable of binding to the analyte and A is a sequence (reference sequence) of the receptor which is complementary thereto. In FIG. 3B, A is the recognition sequence of the receptor which is capable of binding to the analyte and A ^{* is} a sequence (reference sequence) of the receptor which is complementary thereto.

FIGS. 4A and 4B show a further embodiment of the hairpin structures according to the invention with an additional recognition sequence X and a sequence Z which is not complementary to it within the star or its mode of action.

FIGS. 5A and 5B show an embodiment of the hairpin structures according to the invention, which enables label-free detection, i.e. for the detection of unmarked analytes and their mode of action. F represents a fluorescent labeling group, Q a quencher.

Figure 6 shows the mode of action of hairpin structures according to the invention in double-strand hybridization, i.e. the analyte to be determined is in the form of a double-stranded target.

FIG. 7 shows hairpin structures according to the invention which are not bound to the solid phase via the ends, but inside the sequence. According to FIG. 7A the recognition sequence is in the loop and according to FIG. 7B the recognition sequence is in the stem. FIG. 8 shows an example of a hybridization on an array with hairpin structures in which the recognition sequence is in the loop.

FIG. 9 shows an example for a hybridization on an array which contains hairpin structures with a recognition sequence in the stem.

Claims

Expectations 1 . Method for determining analytes, comprising the steps: (a) providing a carrier with a plurality of predetermined ones Areas on which different receptors selected from nucleic acids and nucleic acid analogues are immobilized, the receptors being at least partially present as a secondary structure in one or more of the predetermined areas in the absence of an analyte that can specifically bind to them, (b) bringing the carrier into contact with a sample containing an analyte and (c) determining the analytes via their binding to the receptors immobilized on the carrier, the binding of a Analytes on a receptor that can specifically bind with it includes the detection of a dissolution of the secondary structure present in the absence of the analyte. 2. The method according to claim 1, characterized in that a microfluidic carrier with channels, in particular with closed channels, is used, in which the predetermined areas with the receptors immobilized there are located. 3. The method according to claim 1 or 2, characterized in that DNA molecules are used as receptors. 4. The method according to any one of claims 1 to 3, characterized in that the binding of an analyte to the receptor comprises hybridization. 5. Method according to one of claims 1 to 4, characterized in that several analytes are determined in parallel in the sample. 6. The method according to claim 5, characterized in that at least 50, preferably at least 100 analytes are determined in parallel. 7. The method according to any one of claims 1 to 6, characterized in that the receptors are immobilized on the carrier by covalent binding, non-covalent self-assembly, charge interaction or combinations thereof. 8. The method according to any one of claims 1 to 7, characterized in that the synthesis of the receptors takes place in situ on the carrier. 9. The method according to claim 8, characterized in that the synthesis of the receptors comprises: passing fluid with receptor synthesis building blocks over the carrier, the location and/or time-specific immobilization of the building blocks at predetermined positions on the carrier and repeating this Steps until the desired receptors have been synthesized at the respective predetermined positions. 0. The method according to claim 8 or 9, characterized in that the synthesis of the receptors comprises at least one fluid chemical step, a photochemical step, an electrochemical step or a combination of two or more such steps. 1 . Method according to one of claims 8 to 10, characterized in that the synthesis of the receptors includes on-line process monitoring. 2. Method according to one of claims 1 to 1 1, characterized in that the analyte determination takes place in a device comprising: (i) a light source matrix, (ii) a microfluidic carrier, (iii) means for supplying fluids to the carrier and for discharging fluids from the carrier; and (iv) a detection matrix comprising a plurality of detectors associated with predetermined areas on the carrier. 3. The method according to claim 1 2, characterized in that a programmable light source matrix selected from a light valve matrix, a mirror array and a UV laser array is used. 4. The method according to claim 1 2 or 1 3, characterized in that a device is used in which at least the components (ii), (iii) and (iv) are present in integrated form. 1 5. The method according to any one of claims 1 to 14, characterized in that the secondary structures of the receptors have a hairpin structure comprising a stem and a loop. 1 6. The method according to claim 1 5, characterized in that the sequence of the receptor that can specifically bind to the analyte is located in the loop of the hairpin structure. 17. The method according to claim 1 5, characterized in that the sequence of the receptor that can specifically bind to the analyte is located in the stem of the hairpin structure. 1 8. The method according to claim 17, characterized in that the loop of the hairpin structure does not contain any building blocks that can enter into strong hybridization reactions with the analyte. 1 9. The method according to claim 17 or 18, characterized in that the stem consists of sequence regions that are complementary to one another and sequence regions that are not complementary to one another. 20. The method according to claim 1 9, characterized in that the non-complementary sequence regions of the stem comprise sections capable of base pairing and/or sections not or only weakly capable of base pairing. 21. Method according to one of claims 1 to 20, characterized in that the receptors are immobilized terminally and/or internally on the predetermined areas of the carrier. 22. The method according to any one of claims 1 to 21, characterized in that a hairpin structure is used which contains a marking group that is at least partially quenched in the closed state, and that an increase in a signal originating from the marking group caused by dissolution of the hairpin structure is detected. 23. Device for determining analytes, comprising (i) a light source matrix, (ii) a microfluidic carrier with several predetermined ones Positions at which different receptors selected from nucleic acids and nucleic acid analogs are immobilized on the carrier, the receptors being at least partially present as a secondary structure in one or more of the predetermined areas in the absence of an analyte that can specifically bind to them, (iii) means for supplying fluids to the carrier and for draining fluids from the carrier and (iv) a detection matrix comprising a plurality of detectors assigned to the predetermined positions on the carrier. 24. The device according to claim 23, in which at least components (ii), (iii) and (iv) are in integrated form. 25. Device according to claim 23 or 24, characterized in that the carrier is arranged between the light source matrix and the detection matrix. 26. Use of a device according to one of claims 23 to 25 in a method for the parallel determination of a large number of analytes.