Photoresponsive oligonucleotide

Abstract

To provide a spatially controlled nucleic acid amplification reaction or sequencing reaction.SOLUTION: The present invention provides a method comprising hybridizing a photoresponsive oligonucleotide to a nucleic acid by supplying an oligonucleotide complementary to the nucleic acid, where the oligonucleotide serves as a starting point for a polymerase for nucleic acid synthesis, the photoresponsive oligonucleotide having at least two photoresponsive elements that change from a first conformation to a second conformation when irradiated with light, thereby disabling or enabling oligonucleotide hybridization. In addition, the present invention provides a method in which spatially controlled hybridization of an oligonucleotide to a specific site by the spatial illumination of a non-target region is performed, and thereby the conformation of an oligonucleotide is changed to an unbound state.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to the fields of nucleic acid analysis, spatial transcriptome analysis, and next-generation sequencing.

[0002] Background Nucleic acid analysis is a crucial tool in molecular biology. Typical applications include next-generation sequencing and spatial transcriptome analysis. All methods rely on the specific binding of oligonucleotides, which are used to detect specific sequences in a sample or as starting points for nucleic acid synthesis reactions for amplification or sequencing. Notable examples of these techniques are disclosed below: smFISH (AM Femino, FS Fay, K. Fogarty, RH Singer, Science 1998, 280, 585; A. Raj, P. Van Den Bogaard, S. Rifkin, Nat. Methods 2008, 5, 877), MERFISH (KH Chen, AN Boettiger, JR Moffitt, S. Wang, X. Zhuang, Science 2015, 348, aaa6090; G. Wang, JR Moffitt, X. Zhuang, Sci. Rep. 2018, 8, 4847; F. Chen, AT Wassie, AJ Cote, A. Sinha, S. Alon, S. Asano, ER Daugharthy, JB Chang, A. Marblestone, GM Church, A. Raj, ES Boyden, Nat. Methods 2016, 13, 679), smHCR(S. Shah, E. Lubeck, M. Schwarzkopf, TF He, A. Greenbaum, CH Sohn, A. Lignell, HM Choi, V. Gradinaru, NA Pierce, L. Cai, Development 2016, 143, 2862), seqFISH(E. Lubeck, AF Coskun, T. Zhiyentayev, M. Ahmad, L. Cai, Nat. Methods 2014, 11, 360; S. Shah, E. Lubeck, W. Zhou, L. Cai, Neuron 2016, 92, 342.), seqFISH+(C.-H.L. Eng, M. Lawson, Q. Zhu, R. Dries, N. Koulena, Y. Takei, J. Yun, C. Cronin, C. Karp, GC Yuan, L. Cai, Nature 2019, 568, 235), osmFISH(S. Codeluppi, LE Borm, A. Zeisel, G. La Manno, JA van Lunteren, CI Svensson, S. Linnarsson, Nat. Methods 2018, 15, 932), RNAscope(D. Clair, Bio-Techne Announces Commercial Release of RNAscope®HiPlex Assay: A Multiplex In Situ Hybridization Assay For Tissues 2016; D. Schulz, VRT Zanotelli, JR Fischer, D. Schapiro, S. Engler, XK Lun, HW Jackson, B. Bodenmiller, Cell Syst. 2018, 6, 25. e5; A. Mavropoulos, B. Allo, M. He, E. Park, D. Majonis, O. Ornatsky, Cytometry, Part A 2017, 91, 1200). .

[0003] The application of spatial transcriptome analysis focuses, for example, on nucleic acid analysis in tissue samples. Primers (oligonucleotides) are used to initiate nucleic acid synthesis and sequencing reactions. The added molecules bind to all complementary nucleic acids at all locations in the sample without the ability to control hybridization to the target region. In addition, it is difficult to use the sample for multiple analyses because existing primers must be removed under harsh conditions. This often results in sample damage or residual contamination that interferes with downstream reactions.

[0004] Oligonucleotide hybridization and removal are typically functions of the melting temperature (Tm) of the dimer. This temperature is a function of nucleic acid length and sequence; %GC; nucleic acid (RNA or DNA) type and modification (a small number among a myriad: phosphorothioate, 2'Fluoro, LNA; Nucleic Acids Research, 2007, Vol. 35, Web Server issue W43 - W46; Howley PM, Israel MF, Law MF, Martin MA. A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes. J. Biol. Chem. 1979;254:4876 - 4883; Breslauer KJ, Frank R, Bloecker H, Marky LA. Predicting DNA duplex stability from the base sequence. Proc. Natl Acad. Sci. USA. 1986;83:3746 - 3750; Sugimoto N, Nakano S, Yoneyama M, Honda K. Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res. 1996; 24:4501 - 4505).Based on this, oligonucleotide hybridization can be controlled by salt concentration (monovalent like Na+ or divalent like Mg2+), temperature, presence or absence of crowding agents (PEG, dextran); presence or absence of destabilizers (formamide; ethylene carbonate, urea) and pH-specific buffering conditions (Lee, Je Hyuk, et al. “Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.” Nature protocols 10.3 (2015): 442-458.; MERFISH (KH Chen, AN Boettiger, JR Moffitt, S. Wang, X. Zhuang, Science 2015, 348, aaa6090; G. Wang, JR Moffitt, X. Zhuang, Sci. Rep. 2018, 8, 4847; seqFISH+ (C.-HL Eng, M. Lawson, Q. Zhu, R. Dries, N. Koulena, Y. Takei, J. Yun, C. Cronin, C. Karp, GC Yuan, L. Cai, Nature 2019, 568, 235), osmFISH (S. Codeluppi, LE Borm, A. Zeisel, G. La Manno, JA van Lunteren, CI Svensson, S. Linnarsson, Nat. Methods 2018, 15, 932).

[0005] One major drawback of existing methods is that certain buffering components can introduce contaminants into the reaction system, which can lead to sample damage or potentially interfere with downstream reactions. Furthermore, current methods offer limited control over the binding or removal of localized oligonucleotides.

[0006] The inventors have found that this problem can be solved by using photoresponsive oligonucleotides. Controlling the reaction using light (ultraviolet or visible light) has many advantages over other external stimuli: (1) Light does not introduce contaminants into the reaction system; (2) the excitation wavelength can be controlled by designing the photoresponsive molecule; and (3) the irradiation time and / or local excitation can be controlled.

[0007] The application of DNA-mediated bioprocesses using photoresponsive DNA elements is discussed in H. Asanuma, et al. and other literature (Angew. Chem. Int. Ed. 2001, 40, 2671 - 2673; H. Asanuma, et al., Nat. Protoc. 2007, 2, 203 - 212; Y. Kamiya and H. Asanuma, Acc. Chem. Res. 2014, 47, 1663 - 1672; XG Liang, et al., J. Am. Chem. Soc. 2003, 125, 16408 - 16415; H. Ito, et al., Org. Biomol. Chem. 2010, 8, 5519 - 5524; XG Liang, et al., J. Am. Chem. Soc. 2002). This is disclosed in 124, 1877-1883; M. Zhou, et al., Angew. Chem., Int. Ed. 2010, 49, 2167-2170; M. Liu, et al., J. Am. Chem. Soc. 2003, 128, 1009-1015). These documents describe the modification of nucleic acids with azobenzene derivatives, as well as their applications to biotechnology and nanotechnology. In addition, European Patent Application Publication No. 3015555 describes the use of photoresponsive nucleic acid templates for isothermal DNA amplification. However, these documents do not describe the use of photoresponsive oligonucleotides for nucleic acid analysis, spatial transcriptome analysis, and next-generation sequencing.

[0008] The inventors have surprisingly discovered that photoresponsive oligonucleotides can control and gently remove the binding of these structures while maintaining sample integrity, without using harsh conditions. Furthermore, the inventors have found that photoresponsive oligonucleotides have great potential for spatially controlling their binding in a sample.

[0009] Summary of the Invention The object of the present invention is to provide a method for hybridizing a photoresponsive oligonucleotide to a nucleic acid by supplying an oligonucleotide complementary to the nucleic acid, wherein the oligonucleotide functions as a starting point for polymerase for nucleic acid synthesis, characterized in that the photoresponsive oligonucleotide has at least two photoresponsive elements that change from a first conformation to a second conformation when irradiated with light, thereby making the hybridization of the oligonucleotide impossible or possible.

[0010] In addition, the present invention provides a method for performing spatially controlled hybridization of oligonucleotides to a specific site by spatial illumination of a non-target region, thereby changing the conformation of the oligonucleotide to an unbound state.

[0011] Reversible hybridization of oligonucleotides can be used to control several reactions, such as rolling circle amplification and sequencing reactions.

[0012] Description of the drawing The drawings illustrate the method and embodiments of the present invention without limiting the scope of the claims.

[0013] Figure 1 shows an example of a photoresponsive oligonucleotide, which is part of a padlock system. A nucleic acid, such as mRNA, serves as a template for a nucleic acid synthesis (gap filling) reaction (Figure 1A). After the padlock has bound to the nucleic acid, it can be removed from the nucleic acid by illumination with light having a second wavelength disclosed herein. This induces a conformational change of the oligonucleotide to an unbound state, resulting in the unbinding of the padlock (Figure 1D). The gap of the padlock, which is not illuminated with the second wavelength and therefore still bound to the nucleic acid, is then filled and ligated with a complementary nucleotide (Figure 1B). In this way, a cyclic template containing the photoresponsive oligonucleotide is formed (Figure 1C).

[0014] Figures 2 and 3 illustrate a spatially controlled rolling circle nucleic acid synthesis reaction. A photoresponsive oligonucleotide is added to a sample containing a cyclic nucleic acid template supplied, for example, by a padlock system. Unasymmetric regions in the sample are illuminated with light having a second wavelength disclosed herein. In response, oligonucleotides localized in these regions change their conformation to an unbound state. Therefore, if the oligonucleotide is already bound to the nucleic acid, it neither binds nor dissociates, thus hindering the nucleic acid synthesis reaction (Figure 2B). Photoresponsive oligonucleotides localized in unilluminated regions or regions of the sample illuminated with light having a first wavelength disclosed herein maintain a first conformational state (bound state; Figure 2A). In the regions where the photoresponsive oligonucleotide is bound to the template, rolling circle amplification is initiated. Based on the rolling circle amplification, nucleic acids (rollony) containing several copies of the initial cyclic nucleic acid template are generated (Figure 3).

[0015] Figure 4 shows spatially controlled oligonucleotide binding for nucleic acid synthesis using a Loroney as a nucleic acid template. A photoresponsive oligonucleotide is added to a sample containing a nucleic acid template supplied by the Loroney. Subsequently, non-asymmetric regions in the sample are illuminated with light having a second wavelength disclosed herein. Oligonucleotides localized in these regions change their conformation to an unbound state. This causes dissociation or unbinding of the oligonucleotide from the nucleic acid, thus hindering the nucleic acid synthesis reaction (Figure 4B). Photoresponsive oligonucleotides localized in the unilluminated regions or regions of the sample illuminated with light having a first wavelength disclosed herein maintain a first conformation (bound state; Figure 4A). In these regions, the photoresponsive oligonucleotide binds to the template, and the nucleic acid synthesis reaction is initiated.

[0016] Figure 5 illustrates a spatially controlled sequencing reaction in which Rollonie serves as a template. Oligonucleotide bonding is possible in the unilluminated region of the sample or in the region illuminated with light having the first wavelength disclosed herein. This then initiates the sequencing reaction. In the region illuminated with light having the second wavelength, the reaction does not initiate because oligonucleotide bonding is not possible in these regions.

[0017] Figure 6 illustrates a spatially controlled nucleic acid amplification reaction. Oligonucleotide binding is possible in the unilluminated region of the sample or in the region illuminated with light having the first wavelength disclosed herein. This then initiates the nucleic acid amplification reaction. In the region illuminated with light having the second wavelength, oligonucleotide binding is not possible in these regions, and therefore the reaction does not begin. As a result of such amplification reactions, an amplified nucleic acid product (concatamer) is produced. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows an example of a photoresponsive oligonucleotide, which is part of the padlock system. [Figure 2]This figure shows a spatially controlled rolling circle nucleic acid synthesis reaction. [Figure 3] This figure shows a spatially controlled rolling circle nucleic acid synthesis reaction. [Figure 4] This figure shows spatially controlled oligonucleotide bonding for nucleic acid synthesis, using Rollonie as a nucleic acid template. [Figure 5] This figure exemplifies a spatially controlled sequencing reaction in which Rollonie functions as a template. [Figure 6] This diagram illustrates a spatially controlled nucleic acid amplification reaction.

[0019] Detailed explanation This invention provides a method for reversible and spatially controlled hybridization of photoresponsive oligonucleotides with nucleic acids in a sample using light. Therefore, the photoresponsive oligonucleotides can be removed under mild conditions, thereby improving the integrity of the sample and enabling multiple analyses.

[0020] In a first aspect, the present invention provides a method for hybridizing an oligonucleotide to a nucleic acid by supplying an oligonucleotide complementary to the nucleic acid. The oligonucleotide functions as a starting point for polymerase for nucleic acid synthesis and is characterized by comprising at least two photoresponsive elements. These photoresponsive elements can change from a first conformation to a second conformation when irradiated with light, thereby making the hybridization of the oligonucleotide impossible or possible.

[0021] Photoresponsive oligonucleotides Several photo-responsive structures are known in the art as molecular photo-switches. They are defined as chemical structures that convert into two or more isomers under light irradiation. Isomerization between chemical structures proceeds through two basic mechanisms: trans-to-cis isomerization and 6π electrocyclic reaction of triene systems. Some photo-responsive structures such as azobenzene and their heteroaromatic analogs, indigo, hemithioindigo, stilbene, hydrazone, iminothioindoxyl, diarylethene, spiropyran, Stenhouse adduct (DASA) 15 and fulgide are known in the art (Ilse M. Welleman, Mark W. H. Hoorens, Ben L. Feringa, Hendrikus H. Boersma and Wiktor Szymanski Chem. Sci., 2020, 11, 11672 - 11691).

[0022] Azobenzene derivatives are the most common photo-responsive molecules for versatile applications because they are readily available and chemically stable. Irradiation with light of one wavelength can yield a planar trans form, and irradiation with light of the other wavelength can yield a non-planar cis form. Therefore, azobenzene can be reversibly photo-isomerized between the first conformation (trans) and the second conformation (cis) when irradiated with light of an appropriate wavelength.

[0023] Phosphoramidite monomers having an azobenzene group by a covalent bond and linked by an amide bond to D-threoninol as a scaffold can be synthesized as described below: Enantioselective introduction of azobenzene into oligodeoxyribonucleotides for effective photo-regulation of double-strand formation (H. Asanuma, T. Takarada, T. Yoshida, D. Tamaru, X. Liang et M. Komiyama, Angewandte Chemie-International Edition 2001, 40, 2671 - 2673).

[0024] By using this monomer, an azobenzene cartridge can be easily introduced into an oligonucleotide as a photoreactive element (X), and thus the oligonucleotide can be rendered photoreactive. Therefore, according to the present invention, the terms "oligonucleotide" and "photoreactive oligonucleotide" can be used interchangeably.

[0025] Based on the properties of the azobenzene cartridge, the light regulation of the oligonucleotide is achieved. The photoreactive oligonucleotide changes from a first conformation to a second conformation when irradiated with light having a first wavelength, and changes from the second conformation to the first conformation when irradiated with light having a second wavelength. According to the present invention, the first (initial) conformation of the photoreactive oligonucleotide is trans. These terms can be used interchangeably. In this form, the oligonucleotide can hybridize to a nucleic acid. In contrast, the oligonucleotide in the second conformational state (cis form) cannot bind due to steric hindrance. The second conformational state is also referred to as the "non-binding state" or "cis". According to the present invention, the terms "hybridization", "binding" and their grammatically equivalent terms can be used interchangeably.

[0026] The conformational change of the oligonucleotide disclosed in the specification to the second conformation can be induced by illumination with light having a first wavelength. The first wavelength is in the range of 300 to 400 nm, more preferably 330 nm. It should be noted that the conformational change is reversible, that is, the conformation can be changed from the second conformation (cis) to the first conformation (trans) by illumination with light having a second wavelength. The second wavelength is in the range of 400 to 600 nm, more preferably 532 nm or 525 nm.

[0027] To impart the function of azobenzene to oligonucleotides, a photoresponsive element (X) is introduced between the base pairs (N) of the nucleic acid (e.g., NNN → NXNN). The modified photoresponsive oligonucleotide chain can further form a double helix with its complementary chain, and all base pairs are maintained in the double helix. To achieve efficient photocontrol, it is effective to introduce multiple azobenzene residues. It should be noted that replacing native nucleotides with X is not recommended as it causes double helix destabilization.

[0028] In one embodiment of the present invention, the structure of the photoresponsive oligonucleotide has an X:N ratio of 1:2 or 1:3, where N is a nucleotide and X is a photoresponsive element. In a more preferred embodiment of the present invention, the photoresponsive oligonucleotide has the formula N(XNN)nXN, where N is a nucleotide and X is a photoresponsive element, with n = 3 to 29, preferably 9, 14, or 23. By using this design strategy, the iterative and efficient regulation of hybridization and dissociation of nucleotide double strands can be facilitated by alternating illumination between a first wavelength and a second wavelength. The photoresponsive oligonucleotide has a nucleotide length of at least 15, 50, or 70, more preferably 20, 30, or 50.

[0029] Method / Application Photoresponsive oligonucleotides can hybridize to complementary regions of nucleic acids. This can then function as a starting point for polymerases involved in nucleic acid synthesis.

[0030] The nucleic acids described above can function as templates for polymerase to generate complementary nucleotide chains, thereby creating synthesized nucleic acid chains (nucleic acid synthesis). During nucleic acid synthesis, complementary nucleotides are added by polymerase to the 3' end of the bound oligonucleotide, thus producing complementary nucleic acids. Standard polymerases that can be used in these reactions include, for example, T7 DNA polymerase, Klenow fragments, and T9 DNA polymerase.

[0031] Nucleic acid synthesis reactions in which nucleic acids function as templates include, but are not limited to, cDNA synthesis, linear DNA amplification, sequencing, gap-filling reactions, or rolling circle amplification.

[0032] The reaction template is supplied by nucleic acids in a sample, such as a tissue sample, chip, or hydrogel. The nucleic acid may be a polynucleotide chain made of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may have a linear or cyclic conformation. In certain embodiments of the present invention, the nucleic acid is cyclic and serves as a template for rolling circle amplification.

[0033] The specimens to be analyzed by the disclosed methods may originate from any specimen, such as whole animals, organs, tissue sections, cell aggregates, or single cells, from invertebrates (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., zebrafish, Xenopus laevis), and mammals (e.g., mice, humans). Biological specimens may have the form of tissue sections, cell aggregates, suspension cells, or adherent cells.

[0034] In one embodiment of the present invention, a photoresponsive oligonucleotide may be used for the non-destructive removal of a synthesized nucleic acid chain. Following hybridization of the photoresponsive oligonucleotide to the nucleic acid, a nucleic acid synthesis reaction is initiated, thus producing a synthesized nucleic acid chain containing the photoresponsive oligonucleotide. The conformation of the photoresponsive oligonucleotide changes to an unbound state when irradiated with light, thus allowing the synthesized nucleic acid to be dissociated from the template.

[0035] In another embodiment of the present invention, the photoresponsive oligonucleotide is part of a padlock system, such as that disclosed in European Patent Application Publication No. 3936623 (Figure 1). The nucleic acid serves as a template for a gap-filling reaction (Figure 1A). After the padlock is bound to the nucleic acid, the gap in the padlock is filled with complementary nucleotides and ligated (Figure 1B). In doing so, a cyclic template is formed (Figure 1C), which includes the photoresponsive oligonucleotide. This template can be further used in nucleic acid synthesis reactions.

[0036] In one embodiment, the padlock can be removed from the nucleic acid by illumination with light having a second wavelength disclosed herein, thereby inducing a conformational change to an unbound state of the oligonucleotide. This can then result in the unbinding or partial unbinding of the padlock (Figure 1D).

[0037] The method of the present invention may be used for spatially controlled oligonucleotide hybridization. Oligonucleotide binding can be controlled by spatial illumination of an asymmetric region. This induces a conformational change of the oligonucleotide to an unbound state, thereby controlling the hybridization of the oligonucleotide to a specific site. More specifically, during illumination with a first wavelength, a conformational change to a second conformational state (cis) of the photoresponsive oligonucleotide is initiated in the illuminated region. This then leads to the selective removal of the oligonucleotide.

[0038] In another embodiment, non-target regions may be illuminated with light of a first wavelength before and / or during oligonucleotide binding, thus generating a light-based mask. In these regions, the oligonucleotides only have a second conformation (unbound), thus preventing their binding to the illuminated regions.

[0039] In one embodiment, spatially controlled oligonucleotide hybridization can be used for spatially controlled nucleic acid synthesis. Photoresponsive oligonucleotides are supplied to a sample containing nucleic acids. Subsequently, controlled spatial illumination of the non-asymmetric region is performed with light having a second wavelength as disclosed herein. This induces conformational changes to the unbound state of the oligonucleotides in the non-asymmetric region. These oligonucleotides cannot bind or dissociate if they are already bound to the nucleic acid in those regions. In contrast, oligonucleotides in the un-illuminated region or the region illuminated with light having a first wavelength as disclosed herein are in a bound state. Therefore, they can bind to the nucleic acid. After binding, nucleic acid synthesis can be initiated in these regions.

[0040] In specific embodiments of the present invention, the spatially controlled nucleic acid synthesis reaction described above may be rolling circle amplification, as illustrated in Figures 2 and 3. Herein, a photoresponsive oligonucleotide is supplied to a cyclic nucleic acid template in a sample supplied, for example, by a padlock system. Unasymmetric regions in the sample are illuminated with light having a second wavelength, as disclosed herein. Oligonucleotides localized in these regions change their conformation to an unbound state. This results in the unbinding (dissociation) of these oligonucleotides, thus hindering the nucleic acid synthesis reaction (Figure 2B). In contrast, conditions for photoresponsive oligonucleotide binding are provided in the unilluminated regions or regions of the sample illuminated with a first wavelength, as disclosed herein, and the oligonucleotides are maintained in a bound state, which is a first conformation (Figure 2A). In the regions where the photoresponsive oligonucleotide is bound to the template, rolling circle amplification can be initiated. Based on the rolling circle amplification, nucleic acids containing several copies of the initial cyclic nucleic acid template are produced (Figure 3B). This structure is defined as a Rollonie. In another embodiment of the present invention, the Rollony can function as a template for spatially controlled nucleic acid synthesis reactions, such as nucleic acid amplification or sequencing (Figure 4).

[0041] In certain embodiments, the present invention can be used in a spatially controlled sequencing reaction in which a Rollonie serves as a template. Oligonucleotide bonding is enabled in the unilluminated region of the sample or in the region illuminated with light having a first wavelength disclosed herein. The sequencing reaction can then be initiated. In the region illuminated with light having a second wavelength, the reaction cannot be initiated because oligonucleotide bonding is ineffective in these regions (Figure 5).

[0042] In another specific embodiment, the present invention may be used for a spatially controlled nucleic acid amplification reaction. Oligonucleotide binding is possible in an unilluminated region of the sample or in a region illuminated with light having a first wavelength disclosed herein. This then initiates the nucleic acid amplification reaction. In regions illuminated with light having a second wavelength, oligonucleotide binding is not possible in these regions, and therefore the reaction does not initiate. As a result of such an amplification reaction, an amplified nucleic acid product (concatamer) is produced (Figure 6).

Claims

1. A method for hybridizing at least one oligonucleotide to a nucleic acid by providing at least one complementary oligonucleotide to the nucleic acid, wherein the oligonucleotide serves as a starting point for a polymerase for nucleic acid synthesis, wherein the oligonucleotide has at least two photoresponsive elements that change from a first conformation to a second conformation when irradiated with light, thereby disabling or enabling hybridization of the oligonucleotide.

2. 2. The method of claim 1, wherein the nucleic acid serves as a template for the production of a complementary nucleotide strand by a polymerase, thereby creating a synthesized nucleic acid strand.

3. 2. The method of claim 1, wherein the nucleic acid is circular and serves as a template for rolling circle amplification.

4. 2. The method of claim 1, wherein the photoresponsive oligonucleotide is part of a padlock system and the nucleic acid serves as a template for a gap-filling reaction, in which the gap in the padlock is filled with complementary nucleotides and ligated to form a circular template.

5. The method of claim 2 or 4, wherein the synthesized nucleic acid strand comprises the photoresponsive oligonucleotide, wherein the conformation of the photoresponsive oligonucleotide changes to an unbound state when irradiated with light, thereby allowing the synthesized nucleic acid to dissociate from the template.

6. 2. The method of claim 1, wherein the provided nucleic acid serves as a template for a sequencing reaction.

7. The method of claim 1, characterized in that oligonucleotide binding is controlled by spatial illumination of non-target areas, thereby changing the conformation of the oligonucleotide to an unbound state and thereby controlling hybridization of the oligonucleotide to a specific site.

8. 2. The method of claim 1, wherein the photoresponsive oligonucleotide is at least 15, 50, or 75 nucleotides in length.

9. 2. The method of claim 1, wherein the structure of the photoresponsive oligonucleotide has an X:N ratio of 1:2 or 1:3, where N is a nucleotide and X is a photoresponsive element.

10. 2. The method of claim 1, wherein the photoresponsive oligonucleotide has the formula N(XNN)nXN, where N is a nucleotide, X is a photoresponsive element, and n=3-29.

11. 2. The method of claim 1, wherein the photoresponsive oligonucleotide changes from a first conformation to a second conformation when irradiated with light having a first wavelength, and changes from the second conformation to the first conformation when irradiated with light having a second wavelength.

12. 12. The method of claim 11, wherein the first wavelength is between 300 and 400 nm and the second wavelength is between 400 and 600 nm.

13. The method of claim 1, wherein the nucleic acid is provided by a tissue sample, a chip, or a hydrogel.

14. The method of claim 1, wherein the nucleic acid is linear or circular.

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