Direct synthesis of oligonucleotides on microtomed tissue slices.

Abstract

The present invention provides a method for synthesizing oligonucleotides on a surface of a biological sample, comprising the steps of: a. attaching a plurality of primer molecules to spatial locations on a surface of a biological sample having a stochastic surface distribution, thereby generating oligonucleotides bound to the biological sample; b. providing to the biological sample an A, T, C or G nucleotide having a protection unit at the 3' position; c. incorporating one of A, T, C or G nucleotides having a protection unit at the 3' position at the 3' end of at least one oligonucleotide bound to the biological sample by adding a terminal transferase, thereby extending the oligonucleotide; d. adding at least one photoactivated cleavage agent capable of removing a protecting unit from the incorporated protected nucleotide; e. removing protecting units from incorporated protected nucleotides by activating a photoactivated cleavage agent with light provided to at least one spatial location of the biological sample; f. repeating steps b) through e) to incorporate additional nucleotides into at least one oligonucleotide; The present invention relates to a method for producing a semiconductor device.

Description

[Technical field]

[0001] The present invention relates to the direct synthesis of oligonucleotides on tissue slices to add spatially known barcodes. [Background technology]

[0002] Methods using spatially known barcode oligonucleotides have been described, which associate presynthesized oligonucleotide sequences with specific locations.Typical DNA synthesis uses phosphoramidite methods in anhydrous conditions and organic solvents, and is therefore not compatible with biological materials such as tissue slides.

[0003] Since the oligonucleotides are pre-synthesized on a DNA synthesizer, they must somehow be "placed" in the correct position, but this method is inflexible and precise placement is difficult.

[0004] On the other hand, water-based oligonucleotide synthesis is known. It is carried out using the enzyme terminal transferase and uses 3'-protected nucleotide triphosphates. Several companies, such as DNA script and molecular assemblies, have commercialized kits and procedures in this field.

[0005] Thus, chemical methods for the synthesis and deprotection of 3'-protected nucleotides are known.

[0006] It was an object of the present invention to provide a method for the direct synthesis of oligonucleotides, preferably on a surface or tissue, optionally obtaining spatial information of the oligonucleotide relative to the surface or tissue. Summary of the Invention

[0007] The present invention relates to the use of terminal transferase to synthesize oligonucleotides with identical or different sequences on a surface, protein, antibody, or any biological sample, or to synthesize extended barcode sequences directly on existing primers, optionally. Terminal transferase is an enzyme, used in aqueous and biologically compatible conditions, and requires protected building blocks. The necessary deprotection step is also biologically compatible, as it is similar to the similar step in DNA sequencing.

[0008] An object of the present invention is therefore a method for synthesizing oligonucleotides on the surface of a biological sample, comprising the steps of: a. attaching a plurality of primer molecules to spatial locations on a surface of a biological sample having a stochastic surface distribution, thereby generating oligonucleotides bound to the biological sample; b. providing to the biological sample an A, T, C or G nucleotide having a protection unit at the 3' position; c. incorporating one of A, T, C or G nucleotides having a protection unit at the 3' position at the 3' end of at least one oligonucleotide bound to the biological sample by adding a terminal transferase, thereby extending the oligonucleotide; d. adding at least one photoactivated cleavage agent capable of removing a protecting unit from the incorporated protected nucleotide; e. removing protecting units from incorporated protected nucleotides by activating a photoactivated cleavage agent with light provided to at least one spatial location of the biological sample; f. repeating steps b) through e) to incorporate additional nucleotides into at least one oligonucleotide; The method includes:

[0009] Specific locations of the barcode can be created either by using a photoactivated cleavage agent, such as a protected phosphine, to locally cleave the 3' protecting group for further extension, or by physically separating the four nucleotides at various locations on the tissue. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 illustrates the general method of the present invention. [Diagram 2] FIG. 2 illustrates the general method of the present invention. [Diagram 3] FIG. 3 illustrates the general method of the present invention.

[0011] Detailed Description In the present invention, light is used to spatially activate the deprotection reaction for spatially controlled deprotection of nucleotides and, further, for spatially controlled extension of the resulting oligonucleotides. As a result, oligonucleotides with defined sequences can be added to defined locations on the surface of a sample.

[0012] Photodeprotection is a known subject, for example disclosed by Vaughan et al., JACS, 2013, 135(4) 1197-1200, and is used in different techniques to quench fluorescence. Any such photodeprotection technique can be used in the present invention. For example, TCEP, which is deactivated by reaction with a cyanine dye, or a molecule that reacts similarly with a phosphine, can be used as a photoactivated cleavage agent.

[0013] In the methods of the invention, preferably the A, T, C or G nucleotides having protection units at the 3' position are provided as a mixture.

[0014] Depending on the further use of the oligonucleotide, in addition to A, T, C or G nucleotides, nucleotides linking all nucleotides may be incorporated by further providing an inosine nucleotide (I) with a protection unit at the 3' position.

[0015] Additionally, the oligonucleotide may comprise multiple thymine nucleotides (T) in part, thereby creating an oligonucleotide having a poly-T sequence capable of binding m-RNA from a sample.

[0016] Figure 1 shows the first step of the method of the invention, in which tissue (biological sample) is placed on a surface and a primer molecule is provided. The primer molecule is an oligonucleotide starter unit and may be provided with a protection unit at the 3' position (indicated with an "*"). In an alternative variation, a terminal transferase is added to provide the primer with an A, T, C or G nucleotide with a protection unit at the 3' position, thereby extending the oligonucleotide. Again, the 3' position of the oligonucleotide is protected (indicated with an "*").

[0017] The sample is then provided with at least one photoactivated cleavage agent capable of removing the protection unit.

[0018] Preferably, A, T, C, G and optionally I nucleotides with a protection unit at the 3' position are subsequently provided, where after step c) unincorporated nucleotides are removed from the biological sample.

[0019] Nucleotides may be provided at spatial locations where a photoactivated cleavage agent is activated with light. This is shown in Figure 2, where a photoactivated cleavage agent is activated with light (shown as a grey triangle) provided to at least one spatial location of a biological sample. This process removes protective units at at least one spatial location of a biological sample, making the oligonucleotides at these locations ready for extension with additional nucleotides.

[0020] Figure 3 shows that the resulting unprotected oligonucleotide is extended in the presence of terminal transferase by providing an A, T, C or G nucleotide bearing a protecting unit at the 3' position, so that at the deprotected site a new nucleotide (cross in circle) is incorporated that is 3' protected (star).

[0021] After each nucleotide addition (eg, A) and optional subsequent washing of the excess, unactivated cleavage solution is applied over the entire surface.

[0022] The positions where the next nucleotide (e.g., G) needs to be attached are exposed to light, which photoreleases a cleavage agent that, in solution, deprotects the adjacent 3' protecting groups. At these positions, the 3' end of the primer is deprotected and can be extended with the next nucleotide by terminal transferase.

[0023] The other positions that were not exposed to light remain protected because the cleavage agent did not deprotect them: they are not extended with new nucleotides.

[0024] The number of oligonucleotides depends on the dimensions of the laser beam, its precision, and the method of controlling the diffusion of the deprotected phosphine in the space. Due to the spatially controlled method of the method, the oligonucleotides can serve as barcode information for further sequencing of the tissue, i.e., the barcode is "written" into the oligonucleotides in a spatially controlled manner.

[0025] This sequence may be repeated as necessary and at desired sample locations, thereby extending the oligonucleotides in a defined order and in a spatially controlled manner.

[0026] In theory, the sequence of steps may be repeated indefinitely, however, in practice, steps b) through e) may be repeated 1 to 100 times to incorporate additional nucleotides into at least one oligonucleotide.

[0027] In an embodiment of the invention, nucleotides are subsequently added to an oligonucleotide at a first spatial location by sequential addition, and then a photoactivated cleavage agent is activated with light provided to the first spatial location, where the photoactivated cleavage agent removes the 3' protecting unit from the 3' terminal nucleotide and the deprotected 3' terminal nucleotide is bonded to a new nucleotide.

[0028] A sequence common to the entire tissue can be added to the localisation sequence. This sequence can be used as the target sequence for primer "B". Primer "B" can have a sequence complementary to the sequence in the tissue that is extended by the sequence that targets the specific mRNA. The first primer can be further extended by a normal polymerase. Once the duplex is formed, primer "B" can be removed (if dU was used in its production and the USER enzyme is used) and primer A has the mRNA target sequence that captures the mRNA.

[0029] After capture of the mRNA and its reverse transcription (RT), the newly formed cDNA contains the location sequence and the mRNA sequence, both of which can be sequenced separately.

[0030] In a variation of this method, the oligonucleotide comprises a sequence of nucleotides that codes for the spatial location of the oligonucleotide on the sample.

[0031] In another variation of the method, the oligonucleotide comprises a nucleotide sequence encoding the sample.

[0032] Preferably, after providing a plurality of primer molecules on the surface of the biological sample, the biological sample is imaged to obtain spatial information of the location of the primer molecules.

[0033] Synthesis of oligonucleotides according to the methods of the invention may be controlled by providing at least four photoactivated cleavage agents that are activated by light having different wavelengths.

[0034] Oligonucleotides can be removed from a sample by providing a photocleavable primer molecule.

Claims

1. 1. A method for synthesizing oligonucleotides on the surface of a biological sample, comprising: a. attaching a plurality of primer molecules to spatial locations on the surface of said biological sample having a stochastic surface distribution, thereby creating oligonucleotides bound to said biological sample; b. providing said biological sample with an A, T, C, or G nucleotide having a protection unit at the 3' position; c. incorporating one of the A, T, C or G nucleotides having a blocking unit at the 3' position onto the 3' end of at least one oligonucleotide bound to the biological sample by adding a terminal transferase, thereby extending the oligonucleotide; d. adding at least one photoactivated cleavage agent capable of removing the protecting unit from the incorporated protected nucleotide; e. removing the protecting unit from the incorporated protected nucleotide by activating the photoactivated cleavage agent with light provided to at least one spatial location of the biological sample; f. Repeating steps b) through e) to incorporate additional nucleotides into at least one oligonucleotide; A method comprising:

2. 2. The method of claim 1, wherein the A, T, C, or G nucleotides having a protection unit at the 3' position are provided as a mixture.

3. 2. The method of claim 1, wherein the A, T, C or G nucleotide is provided with a protection unit at the 3' position, and subsequently, after step c), the unincorporated nucleotides are removed from the biological sample.

4. 2. The method of claim 1, wherein the nucleotide is provided at the spatial location where the photoactivated cleavage agent is activated with light.

5. 2. The method of claim 1, wherein the oligonucleotide comprises a sequence of nucleotides that encodes the spatial location of the oligonucleotide on the sample.

6. 2. The method of claim 1, wherein the oligonucleotide comprises a nucleotide sequence encoding the sample.

7. 10. The method of claim 1, wherein after providing a plurality of primer molecules on the surface of the biological sample, the biological sample is imaged to obtain spatial information about the location of the primer molecules.

8. 2. The method of claim 1, further comprising providing an inosine nucleotide with a protection unit at the 3' position.

9. 10. The method of claim 1, comprising providing at least four light-activated cleavage agents that are activated by light having different wavelengths.

10. 10. The method of claim 1, wherein the oligonucleotides are removed from the sample by providing a photocleavable primer molecule.

11. 2. The method of claim 1, wherein the oligonucleotide comprises at least in part a poly-T sequence capable of binding mRNA derived from a sample.

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