JP2002529715A - How to replicate molecular arrays - Google Patents

Abstract

(57) Abstract: A method for producing a molecule array immobilized on a substrate, comprising an array of a first molecule group (3) and a second molecule group immobilized on a first substrate (1). (4) forming a hybrid array with the (ie, the group of molecules to be immobilized), thus defining a spatial array of the second group of molecules, wherein the first substrate (1) and the second substrate (6) in close proximity, wherein the second substrate (6) and the second group of molecules (4) can be interconnected, linking them while retaining the spatial array The method comprising the steps of printing the second group of molecules (4) on the second substrate (6), and separating the respective substrates.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

 The present invention relates to a method for replicating or cloning a molecular array.

[0002]

[Prior art]

Much of cell and molecular biology is based on specific, non-covalent interactions between molecules, often referred to as molecular recognition. These interactions are not permanent and are mainly based on hydrophobic interactions and hydrogen bonding, so that the bond joining the two molecules is reversible. A molecule that specifically recognizes another molecule can be defined as a cognate molecule or a complementary molecule. For a DNA strand, the cognito molecule can be a strand of complementary sequence. The molecular cognite can be a protein, such as a DNA sequence, or a transcriptional regulatory protein that binds a zinc finger. In addition, the cognite molecule may be an antibody that recognizes an antigen, an enzyme that binds to a specific substrate, or a receptor that binds to a ligand, or vice versa.

[0003]

[Problems to be solved by the invention]

Many laboratory techniques have used molecular recognition. For example, in Western blots, separated biomolecules are transferred from one substrate to another for subsequent interrogation with cognite (transfer). However, while this transfer may preserve the spatial arrangement from the first substrate, it does not allow, for example, repeated transfer of a tightly packed, addressable array of biomolecules.

[0004] WO-A-93 / 17126 discloses a two-component oligonucleotide array and the transfer of hybridized oligonucleotides by the blotting technique. The transfer is non-specific. Non-specific transfer of colony material using a colony lift membrane is disclosed in US-A-5491068.

[0005] WO-A-95 / 12808 describes the transfer of DNA from solution to different binding sites by applying a relatively positive potential and then applying a relatively negative potential to one location. Selective transfer is disclosed. The first step in the process is to bind the target with the non-target DNA, and the second step is to release the non-target DNA.

[0006] US-A-5795714 discloses a method for transferring a DNA molecule complementary to a DNA molecule arrayed on a second surface. The transfer is performed by directly contacting the second surface with a solution containing the complementary DNA,
The solution is contained in a distinct vial present on the first substrate. In a preferred embodiment, an avidin-biotin interaction is required to assist in the transfer. The method relies on diffusion to transfer complementary DNA. Any lateral diffusion will limit the resolution achieved when performing a transfer from a high density array.

[0007]

[Means for Solving the Problems]

According to the present invention, a method for producing an array of molecules immobilized on a substrate comprises the following steps: an array of a first group of molecules immobilized on a first substrate and a second group of molecules (ie, ,
Forming a hybrid array with the group of molecules to be immobilized, thus defining a spatial array of the second group of molecules, bringing the first substrate and the second substrate into close proximity, where Second substrate and second
The groups of molecules can be interconnected, linking them, printing the second group of molecules on the second substrate while retaining the spatial array, separating each substrate .

In one preferred embodiment of the invention, a particular class of molecule (s) is attached to a surface using strong linkages (eg, covalent bonds) to form a spatially addressable array. be able to. If the surface is subsequently exposed to various cognites and allowed to reach near equilibrium, one cognite will bind to the molecule with the strongest affinity for it. Unbound molecules can be removed by washing. Because the cognite is attached only to the molecules it recognizes by non-covalent interactions, the application of an appropriate electric field (under conditions with a net positive or negative charge) will cause the second surface to be in close proximity. Can be transferred.

In another preferred embodiment of the invention, the cognite comprises a covalent or non-covalent coupling group at one end. After contacting the molecules on the surface, the cognite can be transferred to a second surface by contact printing. The second surface should first be treated to include complementary covalent or non-covalent coupling groups to react with those at the cognate end. No charge is required to perform the transfer.

Thus, if the first surface has specific regions and each region contains a different molecule,
Cognate molecules are printed on a second surface and a spatial array of cognites (sp
atial array). Preferably, the cognite binds to the second surface with high affinity, but this is not required. It is only necessary if it is desired to repeat the process on the arrayed cognite to form a molecular positive array (eg, make a copy of the original array). For example, a stronger bond can be formed by forming a covalent bond between the cognite and the second surface or through a strong non-covalent interaction (eg, avidin-biotin bond).

The novel method described above is applicable to both single molecule arrays and multi-molecule arrays, ie, arrays of different individual molecules and arrays of different regions, each region containing multiple copies of one individual molecule. is there. The advantages of the method are countless. In particular, only one
It is necessary to make two spatially addressable arrays, which means that multiple copies can be made for screening and diagnostics.

[0012] The molecular array can be characterized before printing. For example, the array can be spatially addressed by sequencing so that each molecule on the array is known.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION

The first or master molecule array used in the present invention comprises a protein, eg, an antibody or an enzyme, immobilized on a solid surface. The protein can interact with other molecular species (cognites) that are transferred to the second substrate, such as a protein, small molecule or polynucleotide. For example, the arrayed proteins are zinc finger proteins, which can bind to polynucleotides with sequence selectivity. See Choo et al., PNAS USA, 91: 11163 (1994).
In some situations, the cognito may be unknown and further characterization may be required to determine exactly what activity or function the cognite exerts.

[0014] Alternatively, the arrayed molecule (s) may be polynucleotides. "
The term "polynucleotide" is used herein to refer to DNA, RNA and DNA, RN
A synthetic derivative or mimic that can interact with A, such as a thioester (t
hioate), amidate, and PNA. Whatever the group of molecules in the first array, the term "cognite" refers herein to a molecule that has specific recognition for a molecule that differs in structure from itself. Each molecule will typically have a complementary portion.

In particular, according to the present invention, multiple copies of one molecule (eg, DNA or RNA) array are produced from a single molecule array (eg, a polynucleotide array), which may be spatially addressed (master copy). be able to. The method is
The complementary (eg) DNA on the array is converted from the complementary (eg) DNA on the array such that its complementary DNA hybridizes to the original DNA in the array, for example, using a DNA polymerase or by direct hybridization from a mixture of oligonucleotides.
Based on making NA and then printing the complementary DNA on a second surface. In this step, a second substrate is brought into proximity with the hybrid (hybrid) array. The complementary polynucleotide is then printed thereon, for example, by charging the second substrate.

In another embodiment of the invention, it is possible that the molecule to be transferred is not the cognite itself, but a product of the reaction between the cognite and the array molecule. For example, an array molecule may be an enzyme that reacts with its substrate (cognite) to form a product. Since this is a local result, the product can be transferred to the second surface. In this case, the product is understood as the molecule to be transferred.

In a related embodiment, the array molecule is able to “capture” the cognite, but the cognite reacts with another substrate, and the product of the reaction is transferred to a second surface. For example, the array molecule can capture a specific enzyme so as to retain its active site. Thereafter, the products of the enzyme-catalyzed reaction are transferred to a second substrate.

[0018] In yet another embodiment, the first and second groups of molecules do not hybridize directly. The indirect association may involve phage-bacteria-phage or antibody-cell-
Antibody interaction (an embodiment of the present invention where each array molecule can be the same or different).

By way of example only, the present invention is described herein with reference to DNA polynucleotides and spatially addressable arrays. The manufacture and use of such arrays is based on PC
T / GB99 / 02487, the contents of which are incorporated herein by reference. The density of such arrays is at least 10 ⁴ , for example, at least 10 ⁵ or 10 ⁶ / cm ² , up to or more than 10 ⁹ / cm ² , the same or different, optionally immobilized on beads. Consisting of molecules (typically about 1 μm beads packed at a density of 10 ⁸ per cm ² ). The fact that the molecules can be different gives the invention a wide applicability.

In one embodiment of the invention, one DN, for example on glass or silicon
The A-array is copied by hybridization of a library of single-stranded DNA under conditions such that members of the library hybridize to their complementary DNA strands on the array. Alternatively, an array complementary to the master copy is generated by enzymatic synthesis using DNA polymerase, appropriate primers and dNTP's.

[0021] Once the DNA array has been prepared, all unhybridized DNA is removed by washing. As a result, all or most of the DNA in the spatially addressable array will hybridize to its complementary DNA. The complementary array can now be transferred and attached to a second substrate.

Each means of attaching DNA to the first and second substrates should preferably be orthogonal to achieve clean transfer. Using the same means, no transfer may occur if complementary DNA can bind to the master. Assuming that this can be prevented, each mounting
Each attachment may be of equal or different intensity, but each attachment is greater than the hybridization intensity of the DNA-DNA duplex. Of course, for example, known procedures such as heating,
Alternatively, the strength of the latter can be reduced by altering the salt concentration to destabilize the duplex.

The DNA of the original array preferably has strong binding to the surface of the first substrate (eg,
(Covalent bond) or via avidin-biotin, which has an equivalent strength to the covalent bond. To achieve binding to a second substrate, the complementary DNA preferably has a chemically reactive or activatable end group;
The result is that it reacts with the second substrate and is thus attached to it. DN
When the A-hybrid is formed, this end group can be positioned furthest away from the surface of the first substrate. For example, the terminal group may be biotin, or avidin, in which case the second substrate surface is covered with a layer of avidin or biotin for attachment. An example of a group that can be activated is “caged” biotin, which can be photoactivated during the transfer step to achieve printing on a second substrate. As each substrate is separated, the relatively weak bonds between the hybridized molecules are broken, and each molecule is retained on its respective substrate.
Spatial decomposition is maintained.

As suggested above, transfer is accomplished in an electric field without contacting the second substrate with cognite in the hybrid array. In this case, both substrates should be conductive. The first substrate may be a metal or a doped semiconductor such as silicon. The master array is attached to its surface by covalent bonds (preferably) or by strong specific interactions. The transfer may take place in the electrolyte, since its specific interaction with cognite molecules often involves multiple hydrogen bonds. For example, DNA duplexes are stable in salt solutions, but are unstable in pure water.

When a sufficiently strong electric field is applied, cognite will be transferred from the hybrid array to another array and may be anchored to the copy surface by specific interactions. In addition, positive ions will migrate to the negative electrode, or vice versa, ions will migrate to the electrode. If this electrolysis lasts too long,
May damage the array. In order to keep the required potential low and to ensure good spatial transfer, the electrodes are made of non-conductive spacers such as Teflon®, for example 0.1-10 μm, often 5-10 μm. Separate (ie, as close as possible without shorting the electrodes). To transfer the cognite from the master to the copy, a potential of 1 mV to 1 V can be applied to the electrode for a short time, for example 1 ms to 1 s, without damaging the master. The potential and time depend on the distance between the electrodes and the ionic strength of the electrolyte and can be optimized. The polarity of the applied potential will depend on the charge on the cognite. The cognate can be attached by non-specific interaction to the copy electrode or by having the appropriate molecular layer on the electrode and the appropriate group on the cognate so that once transfer occurs, the specific binding Can be formed.

The second substrate is preferably a semiconductor (eg, a surface coated with silicon or gold). The transfer can be performed in the presence of a material that mediates the transfer of the complementary polynucleotide, such as a polymer gel, or water,
Other suitable liquid thin films. However, transfer in air or vacuum is also possible. Solution conditions during the printing process, or heating the array, may also help ensure good transfer.

[0027] Printing on the second substrate is facilitated by any suitable means. For example, the second substrate is charged or can be charged. Charging may be done by static electricity. Preferably, a positive potential is applied to the semiconductor surface by a suitable voltage source. The effect is to attract the negatively charged phosphate backbone of the hybridized DNA and pull it onto the semiconductor surface. As already demonstrated, hybridized DNA can be removed at a moderate potential of 300 Vm ^-1 . See PNAS USA 94: 119 (1994).

During the printing process, it is very important that the surfaces of the respective substrates are close to each other in order to obtain an exact copy and to ensure that adjacent elements at the switch positions of the array are not problematic. Desirable. Proximity greatly facilitates generating an electric field strong enough to attract the hybridized DNA to the second substrate surface.

If the hybridized DNA can be polarized sufficiently to attract it,
Any semiconductor or insulator surface is suitable. In order to apply a positive potential, the second substrate is provided with a cover electrode used directly with a metal electrode or other electrode directly behind it.
A thin film of glass such as a slip may be used. The second substrate is preferably not a metal. Because when the array is used with a fluorescent probe, it may quench the fluorescence.

In another aspect of the invention, the transfer may be performed simply by bringing the two surfaces together so that the second surface can be directly attached to the molecule to be transferred. There is no need to apply an electric field. In this method, it is desirable that the cognite have a coupling group that adheres to a complementary coupling group attached to the second surface. Thus, the transfer is mediated by the interaction between the respective coupling groups, providing a point of attachment deformation and preserving spatial integrity. Suitable coupling groups are as described above, for example, biotin / avidin, thiol linker and the like.

The surface of both substrates should be as flat as possible. Suitable silicon wafers are readily available.

[0032] One or each substrate may consist of beads to which DNA is attached. In this case, the beads are used to keep the two surfaces apart,
One surface may be located directly on top of the other surface, the spacing of which depends on the diameter of the beads.

Beads are particularly preferred when the process employs contact printing without any charge. Since the contact is between the top of the bead and the surface on which printing occurs, the presence of the bead between the two surfaces will facilitate transfer. It is also beneficial to have a non-rigid surface, for example, by reducing the thickness of the substrate or using a deformable material such as thin plastic.

Following the transfer, the original, ie, the master copy of the DNA, is attached with stronger binding to the surface of the first substrate than to the complementary DNA, but remains attached. The complementary DNA is printed on a second substrate and stripped away, leaving the original master array intact, ready for further printing. If necessary or desired, this step can be repeated with complementary DNA copies to obtain an exact copy of the original array.

In this sense, reference is made to FIG. A first substrate 1 carrying beads 2 with an array of DNA molecule (s) 3 thereon is shown. DNA molecules 3 are covalently attached to the beads. The complementary DNA molecule (s) 4 has a reactive functional group 5. The second substrate 6 is modified to carry a group 7 that reacts with said complementary DNA molecule, covalently attaching them.

It will be apparent to one skilled in the art that the method of the present invention can be applied to any molecular species involved in molecular recognition. For example, antibody-antigen recognition is compatible with the present invention, and DNA binding proteins can also be used, either as immobilized template arrays, or as cognate molecules. In addition, enzymes and their substrates can also be used.

It will also be apparent that the main advantage of the method is that only one master array needs to be made, from which multiple copies can be printed. This also greatly increases the speed of array production and can be widely used for diagnostics, genotyping and expression monitoring.

The following examples illustrate the invention.

[0039]

【Example】

(Preparation of glass slide) A glass slide to which DNA is to be transferred is soaked in 1: 1 concentrated HCl-MeOH for 1 hour, cleaned, rinsed with Milli-Q water, and soaked in concentrated H ₂ SO ₄ for 1 hour. And rinsed again with water. The cleaned slide was stored in mQ water.

The slide was amino-functionalized with a silane reagent, N- [3- (trimethoxysilyl)
[Propyl] ethylenediamine (DETA). Silanization of the cleaned glass substrate was performed in a 1% solution of DETA in 1 mM glacial acetic acid for 1 hour. Rinse slides with mQ water, 120 ° C. and dried at N _2, was baked for 5 minutes.

The silanized slide was then reacted with SMCC, a heterobifunctional linker capable of reacting with amine and thiol groups (see FIG. 2). 15 mg (4
(5 μmol) of SMCC was dissolved in 200 μl of DMSO. This was diluted to 120 ml with 80:20 MeOH-DMSO. Silanized slides immersed in 3 hours the solution at room temperature, then rinsed well with mQ water and dried with N _2. The maleimide-derivatized slide was stored in a vacuum desiccator.

(Control Experiment) In order to confirm that the maleimide surface was reactive with thiol, 1
One slide was tested. 5'-SH, 3'TMR 20-mer oligonucleotide (SEQ ID NO: 1
) Was used. DT from the sample by passing the sample through a NAP-5 gel filtration column
T was removed because it interfered with the reaction. 500 μl thiol oligo solution into SM
500 μl were placed on a CC-reacted glass slide and 500 μl on a control glass slide and placed in a humid environment at room temperature for 2 hours. The slide was then rinsed with mQ water to remove any DNA that did not covalently attach to the surface, and the SPSC
It was left in a buffer solution (50 mM NaPi, 1 M NaCl) for 12 hours. Slides were rinsed and visualized with FluorImager dried with N ₂ (488 nm excitation, 570 nm filter).

(Transfer) An ethanol solution of 1.0 μm silica beads having a 20-mer DNA sequence (SEQ ID NO: 2) was spotted on a clean glass slide, and the ethanol was evaporated to form a monolayer of beads. . Five such slides were prepared. 10 mM K
25 μl of a 10 μM solution of 5′-SH, 3′-TMR in pi, 100 mM NaCl, 1 mM DTT was added to the circular patch of the bead on each slide and hybridized for 1 hour at room temperature. The slide was rinsed well with buffer (10 mM Kpi, 100 mM NaCl) to remove DTT and unhybridized oligos (5 × 5 ml wash). Slides derivatized with reactive SMCC were carefully placed on slides with duplexes hybridized on beads. To increase the weight and the amount of contact with the beads, 0, 1, 2,
Three and four extra slides were placed on each SMCC slide. Place the slides in a humid environment for 2 hours at room temperature, then carefully remove the top slides of the reactivity and place in SPSC buffer for 12 hours to remove any oligos not covalently attached to the surface Was. Slides were rinsed, dried, and visualized using a FluorImager (488 nm excitation).

(Results) DNA transfer from the bead array to the glass surface was achieved. The observed circular image corresponds to the shape of the original bead patch on the surface. Based on control experiments, the observed fluorescence can represent only fluorescent oligonucleotides transferred from the bead array and covalently attached to the opposite surface.

[Sequence list]

────────────────────────────────────────────────── ─── Continuation of the front page (31) Priority claim number 9919605.7 (32) Priority date August 18, 1999 (August 18, 1999) (33) Priority claim country United Kingdom (GB) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Clenarman, David United Kingdom, Cambridge CB2 1 Idabrew, lens Field Road, Department of Chemistry, University of Cambridge F-term (reference) 4B024 AA20 BA80 CA01 CA11 HA14

Claims (19)

[Claims] 1. A method for producing a molecular array immobilized on a substrate, comprising: an array of a first molecule group immobilized on a first substrate and a second molecule group (ie,
Forming a hybrid array with the group of molecules to be immobilized, thus defining a spatial array of the second group of molecules, bringing the first substrate and the second substrate into close proximity, where Second substrate and second
The groups of molecules can be interconnected, linking them to print the second group of molecules on the second substrate while retaining the spatial array, and separating each substrate The method comprising the steps of: 2. The method according to claim 1, wherein the first and second groups of molecules are indirectly associated in the hybrid array. 3. The method of claim 1, wherein the first and second groups of molecules comprise complementary moieties. 4. The method according to claim 1, wherein the first group of molecules is a protein. 5. The method according to claim 1, wherein the second group of molecules is a protein, for example a zinc finger protein. 6. The method according to claim 3, wherein the first and second groups of molecules are polynucleotides. 7. The method of claim 6, wherein the second group of molecules comprises a library of single-stranded polynucleotides. 8. The method of claim 6, wherein the second group of molecules in the hybrid array is formed in situ using a polymerase and a nucleotide triphosphate. 9. The method according to claim 1, wherein the linkage comprises a covalent bond or an avidin-biotin bond. 10. The method according to claim 1, wherein the first group of molecules is immobilized on microbeads bound to a solid support. 11. The method according to any of the preceding claims, wherein the second substrate and the second group of molecules are contacted and linked and the respective substrates are subsequently separated. 12. The method according to claim 1, wherein the second group of molecules is printed on a second substrate by applying an electric field. 13. The method according to claim 13, further comprising the step of introducing a substance that mediates the transfer of the second group of molecules from the hybrid array to the second substrate between the first substrate and the second substrate. The method according to claim 12. 14. The method according to claim 12, wherein one or each substrate comprises a semiconductor surface. 15. The method of claim 14, wherein said semiconductor surface comprises silicon. 16. The method according to claim 1, wherein the facing surface of each substrate is flat. 17. An array wherein one or each array comprises a plurality of different molecules, at least 10 ^Five /cm^TwoThe method according to any of the preceding claims, characterized in that the method comprises: 18. A hybrid substrate with a third substrate that prints the first molecule group thereon by repeating the steps of any one of the preceding claims and using the first molecule group. Forming a copy of the array of the first group of molecules immobilized on the first substrate. 19. Any one of the preceding claims, wherein the method is repeated any desired number of times, thereby producing multiple copies of the immobilized molecule having a spatial array.
The method described in the section.