Methods and Products for Making Functionalized Single-Stranded Oligonucleotides

Abstract

The present invention relates to functionalized single-stranded oligonucleotides, and in particular to a method for making functionalized single-stranded oligonucleotides, comprising the steps of: (a) providing a circular DNA molecule comprising an oligonucleotide sequence flanked by cleavage domains; (b) performing a rolling circle amplification (RCA) reaction with the circular DNA molecule of (a) and one or more functionalized nucleotides (dNTPs) as a template; and (c) enzymatically cleaving the product of the RCA reaction at the cleavage domain to release a single-stranded functionalized oligonucleotide.

Description

[Technical field]

[0001] The present invention relates to a method for making functionalized single-stranded oligonucleotides. In particular, the present invention provides a method for generating a large number (e.g., a library) of functionalized oligonucleotides using functionalized nucleotides in an enzyme-mediated rolling circle amplification (RCA) reaction. Kits for use in the method and functionalized single-stranded oligonucleotides obtained by the method are also provided. In particular, the present invention provides a library comprising a plurality of functionalized single-stranded oligonucleotides obtained by the method. [Background technology]

[0002] Single-stranded nucleotides are useful for a wide range of applications due to their ability to hybridize, for example intermolecularly, to complementary sequences by Watson-Crick base pairing. In addition, single-stranded oligonucleotides can hybridize to themselves (i.e., intramolecular complementarity) to form complex topological geometries and molecular assemblies, including secondary structures known as aptamers. These aptamers can bind to biological targets with high affinity through non-covalent interactions to provide a range of different functions. The utility of oligonucleotides can be enhanced by the addition of functionality (i.e., the addition of reactive chemical groups) to nucleotides, particularly nucleobases, which functionality does not interfere with Watson-Crick base pairing. However, the synthesis of such functionalized single-stranded oligonucleotides can be problematic, which has hindered the growth of the above-mentioned applications.

[0003] Currently, single-stranded oligonucleotides are generally produced using solid-phase synthesis, in which nucleotides are added in a stepwise manner to a growing chain that is attached to a solid support. However, this method can be severely limited in terms of the length and precision of the oligonucleotides produced. The error rate in the production of oligonucleotides by solid-phase synthesis is significantly higher than that of polymerase enzymes observed in nature, and increases dramatically with the length of the oligonucleotide, with only 70% purity being common for commercially available oligonucleotides of approximately 50 residues in length. Furthermore, the availability of single-stranded oligonucleotides that contain functionalized nucleotides at high density has been limited due to the constraints on the length, quality and cost of solid-phase synthesis. In particular, the number of functionalized nucleotides that can be incorporated into single-stranded oligonucleotides produced by solid-phase synthesis is limited due to, for example, cross-reaction caused by functional groups.

[0004] The need for high levels of purity for many of the uses of synthetic oligonucleotides means that an additional step of purification by methods such as high performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) is often necessary to isolate the desired product of the solid-phase synthesis reaction. This makes current methods not only labor intensive but also expensive. Moreover, even after these additional purification steps, practical problems due to residual impurities can still be observed.

[0005] Given these problems with solid-phase methods, a need exists for alternative methods to generate functionalized oligonucleotides, particularly cost-effective methods that can generate longer oligonucleotides with greater precision. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 5,449,603 [Patent Document 2] U.S. Patent No. 5,534,407 [Patent Document 3] U.S. Patent No. 6,867,028

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Summary of the Invention

[0008] Modified (e.g. functionalized) nucleotides have been used in enzymatic reactions to generate oligonucleotides, but only to generate functionalized DNA by polymerase chain reaction (PCR) or primer extension (PE) reactions. These reactions require the use of specific primers that must then be removed afterwards, and require not only that the modified nucleotides are incorporated by the polymerase, but that the functionalized product is recognized as a template during PCR. This can be problematic for some modifications, thus limiting the usefulness of these methods. Furthermore, primers are synthesized by solid-phase methods, resulting in amplification of errors in the newly synthesized DNA rather than verified sequences. In addition, PCR-based methods result in a preponderance of double-stranded DNA products, and therefore require additional steps of elution and purification to obtain functionalized single-stranded oligonucleotides. [Means for solving the problem]

[0009] The present inventors have previously described a method for enzymatically generating "monoclonal stoichiometric" (MOSIC) single-stranded DNA oligonucleotides from sequence-verified templates (Ducani et al., 2013, Nature Methods, pp. 647-652). The present inventors have surprisingly determined that the MOSIC method can be successfully adapted to generate single-stranded functionalized oligonucleotides.

[0010] In a representative example, the method includes the design and preparation of a linear sequence comprising one or more oligonucleotide sequences, each flanked by a hairpin sequence containing a restriction enzyme site.The linear sequence is then circularized into a double-stranded rolling circle amplification (RCA) template, nicked, and amplified by RCA in the presence of one or more functionalized nucleotides to generate partially single-stranded linear concatemers containing functionalized nucleotides.The RCA product is then treated with a restriction enzyme that recognizes the restriction site in the hairpin region described above and cuts the concatemers, releasing a plurality of functionalized single-stranded oligonucleotides, i.e., generating a library of functionalized oligonucleotides.

[0011] As shown in the examples, the inventors have advantageously determined that, contrary to expectations, functionalized nucleotides were efficiently incorporated by DNA polymerases with strand displacement activity and did not prevent the formation of hairpin structures or interfere with the stability or effective cleavage of the hairpin structures. Furthermore, the inventors have discovered that functionalized nucleotides can be utilized in the MOSIC method while still maintaining the beneficial features associated with the method. For example, in contrast to the PCR-based methods described above, the method does not require primers and can directly generate functionalized single-stranded oligonucleotides. This does not require any additional steps to allow primer annealing or convert the double-stranded product into a single-stranded oligonucleotide. Furthermore, the method does not require the functionalized nucleotide to be recognized as a template for further amplification, which means that functionalized residues can be incorporated in large quantities.

[0012] Thus, in a broad sense, the present invention may be viewed as providing the use of a circular DNA molecule comprising an oligonucleotide sequence flanked by a cleavage domain in the production of a single-stranded functionalized oligonucleotide. In particular, the present invention may be viewed as the use of a circular DNA molecule comprising an oligonucleotide sequence flanked by a cleavage domain and one or more functionalized nucleotides in the production of a single-stranded functionalized oligonucleotide.

[0013] It becomes evident that when functionalized nucleotides are used in combination with equivalent conventional nucleotides, the functionalized nucleotides may be randomly incorporated into the concatemers produced by the RCA reaction, and thus cleavage of the concatemers results in a large number (e.g., a library) of single-stranded functionalized oligonucleotides, i.e., functionalized nucleotides are incorporated at different positions in the oligonucleotide. It becomes even more evident that the diversity of the functionalized oligonucleotides produced can be increased by using circular DNA molecules containing multiple oligonucleotide sequences, each flanked by a cleavage domain. The oligonucleotide sequences may differ in their sequence and / or length. Additionally or alternatively, the diversity of the functionalized oligonucleotides may be increased by using a combination of functionalized nucleotides in the RCA reaction. Still further diversity may be introduced into the library of functionalized oligonucleotides by modifying the oligonucleotides after synthesis, for example, by conjugating molecules or moieties to the functionalized nucleotides in the oligonucleotides. It becomes evident, therefore, that the present invention advantageously provides a new method for enzymatically producing highly functionalized single-stranded DNA oligonucleotides that may open new applications in DNA microengineering, nucleic acid imaging diagnostics, and biomedicine. Thus, in one particular embodiment, the present invention provides a method of making a single-stranded functionalized oligonucleotide, said method comprising: (a) providing a circular DNA molecule comprising an oligonucleotide sequence flanked by cleavage domains; (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule of (a) as a template and one or more functionalized nucleotides (dNTPs); (c) cleaving the product of the RCA reaction at the cleavage domain to release a single-stranded functionalized oligonucleotide.

[0014] As described in more detail below, the step of providing a circular DNA molecule may be accomplished by any suitable means and may depend on the structure of the circular DNA molecule. In this regard, the circular DNA molecule may be a single-stranded DNA molecule or a double-stranded DNA molecule.

[0015] In embodiments where the circular DNA molecule is a double-stranded molecule, it will become apparent that the circular DNA molecule must be processed to provide the RCA template, and thus in some embodiments the method includes the additional step of cleaving one strand of the circular DNA molecule to provide the RCA template before the RCA reaction is carried out.

[0016] The present invention can be advantageously used to create functionalized oligonucleotides that contain any oligonucleotide sequence. Thus, any suitable sequence can be used as the oligonucleotide sequence in the circular DNA molecule of the present invention. By suitable sequence, it is meant that the oligonucleotide sequence domain should not interfere with (i.e., inhibit or distort) the creation or cleavage of the RCA product. For example, in some embodiments, the oligonucleotide sequence may be designed to avoid the creation of secondary structures that may inhibit or displace the progression of the polymerase that performs the RCA reaction. Nevertheless, in some embodiments, the oligonucleotide sequence may be an aptamer or may code for it.

[0017] Additionally, the oligonucleotide sequence can be designed such that it does not specifically hybridize to the cleavage domain in the RCA product. Alternatively, the cleavage domain flanking the oligonucleotide sequence can be designed such that it does not specifically hybridize to the oligonucleotide sequence in the RCA product.

[0018] In the embodiment where the circular DNA molecule comprises multiple oligonucleotide sequences, each sequence can be designed not to specifically hybridize to other oligonucleotide sequences in the RCA product.However, in some embodiments, it may be desirable to create functionalized oligonucleotides that contain regions of complementarity such that the oligonucleotides can interact, especially after being released from the RCA product.Thus, in some embodiments, oligonucleotide sequences can be designed to promote the interaction (e.g., hybridization) of the functionalized oligonucleotides created by the method, as long as the interaction does not interfere with the creation or cleavage of the RCA product, i.e., the creation of the functionalized oligonucleotide.

[0019] Thus, in some embodiments, the nucleic acid sequence of the oligonucleotide sequence of the circular DNA molecule has less than 80% sequence identity with the nucleic acid sequence of the cleavage domain and / or other oligonucleotide sequences in the circular DNA molecule. Preferably, the oligonucleotide sequence of the circular DNA molecule has less than 70%, 60%, 50% or less than 40% sequence identity with the nucleic acid sequence of the cleavage domain and / or other oligonucleotide sequences in the circular DNA molecule. Sequence identity can be determined by any suitable method known to those skilled in the art, for example, by using the BLAST alignment algorithm.

[0020] Thus, the term "oligonucleotide sequence" refers to, without being particularly limiting, the template sequence used to generate functionalized single-stranded oligonucleotide or oligonucleotide of the present invention or its complement. In this regard, when the circular DNA molecule is single-stranded, the sequence of the circular DNA molecule is the reverse complement of the repeated (tandem) sequence in the RCA product obtained in step (b). When the circular DNA molecule is double-stranded, it contains both the repeated sequence and its reverse complement in the RCA product obtained in step (b). Thus, the process of processing the circular DNA to obtain the RCA template may include a step of cleaving the strand of the circular DNA molecule that contains the sequence that will be repeated in the RCA product.

[0021] The present invention can be used to create functionalized single-stranded oligonucleotides of any desired length. As shown in the examples, the method can be used to create functionalized single-stranded oligonucleotides of lengths far beyond those that can be accurately created using solid-phase synthesis methods. Thus, the oligonucleotide sequence (and thus the functionalized oligonucleotide created by the method) can be from about 6 to about 10,000 nucleotides in length. Thus, in some embodiments, the method can be viewed as creating a functionalized polynucleotide. In this regard, the size boundary between "oligonucleotide" and "polynucleotide" is not clear to those skilled in the art. For example, a sequence of more than 400 nucleotides may be referred to as a polynucleotide. Thus, the terms oligonucleotide and polynucleotide are used interchangeably herein to refer to a nucleotide sequence within the size range specified above. However, when the term "polynucleotide" is used, it will generally refer to a sequence containing more than 400 nucleotides. Thus, in some embodiments, the method and use can be viewed as a process for creating a plurality of single-stranded functionalized DNA molecules.

[0022] In some embodiments, the oligonucleotide sequence may be about 6 to about 750 nucleotides in length, and may include about 6 to about 500 nucleotides in length, e.g., about 8 to about 400 nucleotides in length, about 8 to about 300 nucleotides in length, about 8 to about 250 nucleotides in length, about 10 to about 200 nucleotides in length, about 10 to about 150 nucleotides in length, about 12 to about 100 nucleotides in length, about 12 to about 75 nucleotides in length, about 14 to about 70 nucleotides in length, about 14 to about 60 nucleotides in length, etc. In some embodiments, the oligonucleotide sequence contains about 10 to 400, 11 to 390, 12 to 380, 13 to 370, 14 to 360, or 15 to 350 nucleotides in length.

[0023] As noted above, the present invention is particularly useful for producing longer oligonucleotides containing about 30 or more nucleotides, such as about 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides. For example, oligonucleotides produced by the present invention may contain about 30-1000, 40-900, 50-800, 60-700, 70-600, 80-500, 90-450, or 100-400 nucleotides. In some embodiments, oligonucleotides (e.g., polynucleotides) produced by the present invention contain about 400-10,000, 500-9,000, 600-8,000, 700-7,000, 800-6,000, 900-5,000, or 1,000-4,000 nucleotides, and may contain, for example, about 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500 or more nucleotides.

[0024] In some embodiments, the circular DNA molecule comprises multiple oligonucleotide sequences, each of which is bordered by a cleavage domain. The oligonucleotide sequences may be the same or different from each other, or a combination thereof. Thus, in some embodiments, the circular DNA molecule may comprise more than one copy of the same oligonucleotide sequence as defined below, for example, 2, 3, 4, 5 copies, etc. In some embodiments, the circular DNA molecule may comprise one copy of each of the multiple different oligonucleotide sequences, for example, 2, 3, 4, 5 copies, etc., of the different oligonucleotide sequences defined below. In some embodiments, the circular DNA molecule may comprise one or more copies of the multiple different oligonucleotide sequences. As further described below, the present invention may be used to generate multiple functionalized oligonucleotides with controlled stoichiometry based on the number of copies of the oligonucleotide sequence in the circular DNA molecule.

[0025] It will be apparent that multiple oligonucleotide sequences may be present in any order within the circular DNA molecule. For example, multiple copies of the same oligonucleotide sequence may be directly adjacent to each other within the circular DNA molecule (separated only by a cleavage domain that borders the oligonucleotide sequence). Alternatively, multiple copies of the same oligonucleotide sequence may be interleaved with different oligonucleotide sequences. The circular DNA molecule may be designed (e.g., the order of multiple oligonucleotide sequences) to avoid or minimize interactions between repeated sequences within the RCA product that may interfere with the creation or cleavage of the RCA product as defined above.

[0026] The term "plurality" as used herein means two or more depending on the context of the invention, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 or more, 50, 100, 150, 200, 250 or more, etc. For example, a circular DNA molecule may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 or more oligonucleotide sequences, such as 2-100, 3-90, 4-80, 5-70, 6-60, 7-50, 8-40, 9-30 or 10-20 oligonucleotide sequences. In some embodiments, the method of the present invention may generate at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 or more, 50, 100, 150, 200, 250 or more functionalized single-stranded oligonucleotides, i.e., oligonucleotides with different sequences and / or structures. In this regard, different functionalized oligonucleotides may be generated from the same template oligonucleotide sequence, since the functionalized nucleotides may be randomly incorporated into the RCA product, i.e., when the functionalized nucleotides are present in a relative amount less than 100% as described above. Furthermore, when the circular DNA molecule contains multiple different oligonucleotide sequences, each template will result in multiple different functionalized single-stranded oligonucleotides. Furthermore, since the RCA reaction may utilize multiple circular DNA molecules, the reaction may generate multiple functionalized single-stranded oligonucleotides, e.g., 10 3 , 104 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 This results in one or more copies of each functionalized single-stranded oligonucleotide.

[0027] The term "different" refers to oligonucleotide sequences or functionalized single-stranded oligonucleotides that contain one or more different nucleotides. Thus, different oligonucleotide sequences can differ by one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides, for example, 20, 30, 40, 50, 60, 70, 80, 90 or more nucleotides. The difference can be the length and / or sequence of the oligonucleotide sequence. In another view, different oligonucleotide sequences have less than 100% sequence identity with each other, such as less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50% with each other. Different functionalized single-stranded oligonucleotides can contain the same nucleotide sequence, but the position of functionalized nucleotides or the type of functionalized nucleotide at a particular position within the oligonucleotide can be different (for example, when a combination of functionalized nucleotides is used in RCA reaction). Alternatively or additionally, the different functionalized single stranded oligonucleotides may differ in terms of their oligonucleotide sequence as defined above.

[0028] Thus, several different single-stranded functionalized oligonucleotides may be simultaneously produced using the present invention.In particular, by modifying the sequence of circular DNA molecule, in particular by controlling the number of copies of each different oligonucleotide sequence present, it is possible to control the stoichiometry of the single-stranded functionalized oligonucleotides that are ultimately produced by this method.However, since the functionalized single-stranded oligonucleotides obtained from each oligonucleotide sequence may be different (due to the random incorporation of functionalized nucleotides in RCA products), the stoichiometry of functionalized single-stranded oligonucleotides can be controlled in terms of oligonucleotide number based on a specific oligonucleotide sequence.Therefore, a circular DNA molecule may contain multiple copies of multiple different oligonucleotide sequences.

[0029] The term "cleavage domain" as used herein generally refers to a domain in a circular DNA molecule that results in a domain in an RCA product that can be specifically cleaved to release functionalized single-stranded oligonucleotide.Thus, the cleavage domain in a circular DNA molecule may be capable of cleavage directly, or may simply code for a cleavage domain that is functional only in the RCA product or functional under certain conditions, such as when contacted with a cofactor.

[0030] "Cleavage" includes any means of breaking a covalent bond. Thus, in the context of the present invention, cleavage includes cleavage of a covalent bond within a nucleotide chain (i.e., strand scission or strand splitting), for example, by cleavage of a phosphodiester bond.

[0031] Thus, in some embodiments, a cleavage domain can include a sequence that is recognized by one or more enzymes that can cleave a nucleic acid molecule, i.e., break the phosphodiester bond between two or more nucleotides.

[0032] Thus, in some embodiments, the present invention provides a method of making a single stranded functionalized oligonucleotide, the method comprising: (a) providing a circular DNA molecule comprising an oligonucleotide sequence flanked by cleavage domains; (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule of (a) as a template and one or more functionalized nucleotides (dNTPs); (c) enzymatically cleaving the product of the RCA reaction at a cleavage domain to release a single-stranded functionalized oligonucleotide.

[0033] Thus, in some embodiments, step (c) comprises contacting the product of the RCA reaction with a cleavage enzyme.

[0034] For example, the cleavage domain may comprise a restriction endonuclease (restriction enzyme) recognition sequence. Restriction enzymes cut double-stranded or single-stranded DNA at specific recognition nucleotide sequences known as restriction sites, and suitable enzymes are well known to those skilled in the art. For example, it may be particularly advantageous to use rare-cutting restriction enzymes, i.e., enzymes with long recognition sites (at least 8 base pairs in length), to facilitate the design of oligonucleotide sequences within the circular DNA molecule, e.g., to avoid inclusion of cleavage recognition sites within the oligonucleotide sequence.

[0035] In some embodiments, the cleavage domain may comprise a sequence recognized by a type II restriction endonuclease, more preferably a type IIs restriction endonuclease. While any suitable cleavage domain and cleavage enzyme may be used in the present invention, in some embodiments, the cleavage enzyme that recognizes the cleavage domain adjacent to the oligonucleotide sequence may be BseGI, BtsCI or an isoschizomer thereof, such as BstF51 or Fokl. Other representative enzymes that may be used include BsrDI, BtsI, BtsIMutl, MlyI or an isoschizomer thereof.

[0036] Thus, in some embodiments, cleaving the product of the RCA reaction comprises contacting the RCA product with a cleavage enzyme under suitable conditions to selectively cleave the RCA product at the cleavage domain.

[0037] In some embodiments, the cleavage domain can be made functional (activated) in the RCA product by the addition of another component, i.e., the RCA product can be engineered to include a functional cleavage domain, such as a restriction endonuclease recognition sequence. For example, this can be achieved by hybridizing an oligonucleotide (referred to herein as a "restriction oligonucleotide") to the cleavage domain of the RCA product to form a duplex. At least a portion of the formed duplex will contain a restriction endonuclease recognition site, which can be cleaved to release the functionalized oligonucleotide. This can be particularly advantageous in embodiments where the functionalized nucleotide incorporated in the RCA product, particularly the cleavage domain, can interfere with (e.g., reduce efficiency of) the activity of the cleavage enzyme.

[0038] Thus, in some embodiments, cleaving the product of the RCA reaction may comprise contacting the RCA product with a restriction oligonucleotide and a cleavage enzyme. The restriction oligonucleotide and the cleavage enzyme may be contacted with the RCA product simultaneously or sequentially.

[0039] In some embodiments, the cleavage domain may be cleaved by means other than a cleavage enzyme, for example, the cleavage domain may comprise a self-cleaving oligonucleotide sequence, such as a DNAzyme nuclease.

[0040] Thus, in some embodiments, the present invention provides a method of making a single stranded functionalized oligonucleotide, the method comprising: (a) providing a circular DNA molecule comprising an oligonucleotide sequence flanked by a self-cleaving cleavage domain (e.g., a cleavage domain comprising a DNAzyme nuclease); (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule of (a) as a template and one or more functionalized dNTPs; (c) cleaving the product of the RCA reaction at the cleavage domain to release a single-stranded functionalized oligonucleotide; Step (c) involves activating a self-cleaving cleavage domain in the product of the RCA reaction.

[0041] Suitable self-cleaving sequences are known to those skilled in the art. In embodiments utilizing a self-cleaving sequence, the cleavage domain may only be functional (active) in the RCA product. Alternatively, the self-cleaving sequence may be functional under certain conditions, and thus the method may include subjecting the RCA product to conditions that facilitate cleavage of the RCA product at the cleavage domain, i.e., conditions that activate the self-cleaving sequence, e.g., conditions that contact the RCA product with a cofactor, such as a metal ion, that is required for the self-cleavage activity. In another aspect, cleaving the product of the RCA reaction may include subjecting the RCA product to conditions that facilitate cleavage of the RCA product at the cleavage domain, i.e., conditions that activate the self-cleaving sequence, e.g., conditions that contact the RCA product with a cofactor, such as a metal ion, that is required for the self-cleavage activity.

[0042] In some embodiments, the cleavage domain comprises or consists of a sequence capable of forming a hairpin structure. A hairpin structure may also be known as a hairpin loop or a stem loop, and these terms are used interchangeably herein. A hairpin is an intramolecular base-pairing pattern that can occur in a single-stranded DNA or RNA molecule. A hairpin occurs when two regions of the same strand that are normally complementary in nucleotide sequence when read in opposite directions base pair (hybridize) to form a double-stranded stem (duplex) and an unpaired or single-stranded loop. The resulting structure may be described as a lollipop shape.

[0043] Thus, in some embodiments where the cleavage domain comprises or consists of a sequence capable of forming a hairpin structure, the cleavage domain comprises a sequence that is self-complementary. As the RCA product is extended, hybridization of these self-complementary regions results in a hairpin structure, and the double-stranded portion of the hairpin structure contains a sequence that is recognized by the cleavage enzyme. Thus, cleavage of the double-stranded portion of the hairpin structure in the RCA product results in the release of a functionalized single-stranded oligonucleotide and a hairpin structure (i.e., an oligonucleotide that forms a hairpin structure).

[0044] As used herein, the term "hybridization" or "hybridizing" refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex by Watson-Crick base pairing. Two nucleotide sequences are "complementary" to one another if the molecules share base-pair-organized homology. "Complementary" nucleotide sequences will combine with specificity to form a stable duplex under appropriate hybridization conditions. For example, two sequences are complementary if a section of a first sequence can bind to a section of a second sequence in an antiparallel sense, with the 3'-end of each sequence binding to the 5'-end of the other, and each A, T(U), G, and C of one sequence then aligning with the T(U), A, C, and G of the other, respectively. RNA sequences may also contain complementary G=U or U=G base pairs. Thus, two sequences need not have perfect homology to be "complementary" under the present invention. Typically, two sequences are sufficiently complementary when at least about 90% (preferably at least about 95%) of the nucleotides share base-pair organization over the defined length of the molecule.

[0045] When the RCA product is cleaved, a functionalized oligonucleotide is released. The released functionalized oligonucleotide may consist of the oligonucleotide sequence alone (i.e., without any additional nucleotides) or may contain one or more additional nucleotides from the cleavage domain flanking the oligonucleotide sequence at one or both ends. Thus, in some embodiments, the functionalized oligonucleotide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides from the cleavage domain at one or both ends. Preferably, the sequence of the circular DNA molecule is designed such that the functionalized oligonucleotide released when the RCA product is cleaved does not contain any additional nucleotides from the cleavage domain. In another view, the cleavage domain flanking the oligonucleotide sequence may be located within the circular DNA molecule such that its cleavage in the RCA product results in the release of a functionalized oligonucleotide that does not contain any additional nucleotides from the cleavage domain.

[0046] Because the cleavage enzyme may cleave the nucleic acid molecule at a position outside the cleavage enzyme recognition sequence, one or more nucleotides may be present between the cleavage domain and the oligonucleotide sequence. Alternatively, the cleavage domain may contain nucleotide sequences in addition to the cleavage enzyme recognition sequence to ensure that cleavage results in the release of a complete functionalized single-stranded oligonucleotide, preferably without any additional nucleotides (e.g., nucleotides that form part of the cleavage domain).

[0047] Thus, the term "bordered" refers to a cleavage domain that is directly or indirectly adjacent to an oligonucleotide sequence. In another view, the cleavage domain is located at either end of the oligonucleotide sequence, i.e., the cleavage domain is upstream and downstream (5' and 3' ends) of the oligonucleotide sequence. In some embodiments, the cleavage site of the cleavage domain (e.g., the site where the cleavage enzyme cleaves the cleavage domain) is directly adjacent to the end of the oligonucleotide sequence to which it is bordered. In some embodiments, the oligonucleotide sequence and the cleavage domain sequence may overlap, i.e., the end of the oligonucleotide sequence may form part of the cleavage domain, e.g., when the cleavage site is an internal site within the cleavage domain, i.e., the cleavage domain may form an end or part of an end of the oligonucleotide sequence. Thus, in some embodiments, one or more nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, may be present between the cleavage domain and the oligonucleotide sequence (i.e., between the ends of the sequence). In some embodiments, the cleavage domain and the oligonucleotide sequence may overlap by one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, nucleotides.

[0048] The size of the cleavage domain in the circular DNA molecule is not particularly limited and will depend on the type of cleavage domain mentioned above. The cleavage domain may be designed or selected such that the length of the cleavage domain is different from the length of the oligonucleotide sequence, so that the functionalized single-stranded oligonucleotide sequence can be easily purified once the RCA product is cleaved at the cleavage domain. Thus, in some embodiments, the cleavage domain may be selected to be shorter than the oligonucleotide sequence in the circular DNA molecule. When the circular DNA molecule contains multiple oligonucleotide sequences of different lengths, the cleavage domain may be selected to be shorter than the shortest oligonucleotide sequence in the circular DNA molecule. In other embodiments, the cleavage domain may be selected to be longer than the oligonucleotide sequence in the circular DNA molecule. When the circular DNA molecule contains multiple oligonucleotide sequences of different lengths, the cleavage domain may be selected to be longer than the longest oligonucleotide sequence in the circular DNA molecule. In some embodiments, the length of the cleavage domain and the oligonucleotide sequence differ by at least two nucleotides, such as at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0049] If the desired oligonucleotide sequence and the cleavage domain are approximately the same length, the cleavage domain may be extended so that it can be separated from the final single-stranded functionalized oligonucleotide by size. For example, an additional region of self-complementarity may be added to the end of the cleavage domain to extend the length of the stem region of the hairpin structure. Alternatively or additionally, nucleotides may be added to the loop region of the hairpin structure.

[0050] In some embodiments, the cleavage domain can be about 4-50 nucleotides in length, such as about 5-45, 6-45, 7-35, or 8-30 nucleotides in length. In some embodiments, the cleavage domain can range from about 10-25 nucleotides in length, including about 12-22 or about 14-20. However, it will be apparent that a cleavage domain of any suitable length may be used in the present invention so long as it meets the above functional requirements.

[0051] The cleavage domains flanking the oligonucleotide sequence may be the same or different.Advantageously, the cleavage domains flanking the oligonucleotide sequence are identical, so that a single cleavage step is sufficient to release all of the functionalized single-stranded oligonucleotides.For example, in some embodiments, cleaving the RCA product comprises contacting the RCA product with a single cleavage enzyme under conditions suitable for cleaving the cleavage domain in the RCA product.

[0052] The appropriate conditions for cleaving the cleavage domain in the RCA product will depend on the means used to achieve cleavage.For example, when cleavage is achieved using a cleavage enzyme such as a restriction endonuclease, the conditions will vary depending on the enzyme selected, and suitable conditions are well known to those skilled in the art, for example, the cleavage step can follow the manufacturer's instructions.Similarly, when cleavage is achieved using a self-cleaving sequence such as a DNAzyme, conditions suitable for a particular sequence can be used.Examples of suitable conditions that can be used for the cleavage step are described below.

[0053] For example, a cleavage enzyme, such as a restriction endonuclease, can specifically bind to a cleavage recognition site and selectively (e.g., specifically) cleave a nucleic acid in a variety of buffers, such as phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), HEPES-buffered saline (HBS), and Tris-buffered saline (TBS) both with and without EDTA. Cleavage can occur over a wide range of temperatures, e.g., 0-70° C., and at a pH range of about 3.0-10.0, e.g., 4.0-9.0, 5.0-8.0. Other suitable conditions can be readily determined by one of skill in the art.

[0054] The method of the present invention involves performing rolling circle amplification using a circular DNA molecule as a template. Rolling circle amplification (RCA) is well known to those skilled in the art and is described in Dean et al., 2001 (Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification, Genome Research, 11, pp. 1095-1099), the disclosure of which is incorporated herein by reference. In brief, RCA involves the synthesis of a nucleic acid molecule using a circular single-stranded nucleic acid molecule, such as a circular or circular oligonucleotide, as a rolling circle template (RCA template) and a strand-displacing polymerase to extend a primer hybridized to the template. The addition of polymerase and nucleotides initiates the synthesis reaction, i.e., the polymerase reaction. Since the rolling circle template is infinite, the resulting product is a long single-stranded nucleic acid molecule composed of tandem repeats complementary to the rolling circle template.

[0055] A typical RCA reaction mixture includes a circular DNA molecule and one or more primers that are used in a primer extension reaction; for example, RCA may be templated by a single primer to generate a single concatemeric product or multiple primers that each anneal to different regions of the circular oligonucleotide to generate multiple concatemeric products per round. The oligonucleotide primer that the circular nucleic acid can be contacted with will be of sufficient length to obtain hybridization to the circular DNA molecule under annealing conditions. In some embodiments, the primer may be provided by cutting (e.g., nicking) one strand of the double-stranded circular DNA molecule obtained in step (a).

[0056] In addition to the components described above, the reaction mixtures used in the present invention will generally include a polymerase (defined further below, e.g., phi29 DNA polymerase), one or more functionalized nucleotides, one or more conventional nucleotides, and other components required for a DNA polymerase reaction as described below. The desired polymerase activity may be provided by one or more intrinsic polymerase enzymes.

[0057] The RCA reaction mixture may further comprise an aqueous buffer medium comprising a source of monovalent ions, a source of divalent cations, and a buffering agent. 4 Any convenient source of monovalent ions may be utilized, such as Cl, ammonium sulfate, etc. The divalent cation may be magnesium, manganese, zinc, etc., and the cation will generally be magnesium. MgCl 2 Any convenient source of magnesium cations may be utilized, including magnesium acetate, magnesium phosphate, etc. 2+ The amount of may range from 0.5 to 10 mM, but will preferably be in the range of about 3 to 6 mM, and ideally about 5 mM. Representative buffering agents or salts that may be present in the buffer include Tris, Tricine, HEPES, MOPS, etc., and the amount of buffering agent will generally range from about 5 to 150 mM, usually about 10 to 100 mM, more usually about 20 to 50 mM, and in certain preferred embodiments, the buffering agent will be present in an amount sufficient to obtain a pH range of about 6.0 to 9.5. Other agents that may be present in the buffer medium include chelating agents such as EDTA, EGTA, etc.

[0058] In embodiments in which a single strand of a double-stranded circular DNA molecule is cleaved (e.g., nicked) to obtain an RCA template (and a primer for the RCA reaction), it may be useful to include a single-stranded binding protein in the RCA reaction mixture. For example, E. coli single-stranded DNA binding protein is used to increase the yield and specificity of primer extension reactions and PCR reactions (U.S. Patent Nos. 5,449,603 and 5,534,407). The gene 32 protein of T4 phage (a single-stranded DNA binding protein) apparently improves the ability to amplify larger DNA fragments (Schwartz et al., Nucl. Acids Res. 18:1079 (1990)), enhances DNA polymerase fidelity (Huang, DNA Cell. Biol. 15: 589-594 (1996)), and most importantly, prevents DNA polymerase from switching templates to, for example, already made single-stranded DNA and synthesizing double-stranded DNA (Ducani et al., Nucl. Acids Res. 42: 10596 (2014)). When utilized, such proteins would be used to achieve concentrations in the reaction mixture that range from about 0.01 ng / μL to about 1 μg / μL; from about 0.1 ng / μL to about 100 ng / μL, etc.; including from about 1 ng / μL to about 10 ng / μL.

[0059] The RCA reaction ultimately produces a polynucleotide product that contains adjacent (tandem) repeats of the complementary sequence of the circular DNA molecule. This product may be known as a concatemer, RCA product, or "RCP." Thus, the RCA product contains a linear sequence of an oligonucleotide sequence (or specifically the reverse complement of the oligonucleotide sequence of the circular DNA molecule template) that contains functionalized nucleotides, flanked by cleavage domains.

[0060] As described above, when the circular DNA molecule is single stranded, the RCA reaction mixture may include one or more oligonucleotide primers to initiate the RCA polymerization reaction. The primers will be of sufficient length to obtain hybridization to the circular DNA molecule under annealing conditions. The primers will generally be at least 10 nucleotides in length, usually at least 15 nucleotides in length, more usually at least 16 nucleotides in length, and may be as long as 30 nucleotides in length or longer, with the length of the primers generally ranging from 18-50 nucleotides in length, usually about 20-35 nucleotides in length.

[0061] The primer may anneal to any region within the circular DNA molecule. In some embodiments, the circular DNA molecule may contain a specific domain (RCA primer binding site) to which the primer can hybridize. In a representative embodiment, the circular DNA molecule may contain a sequence that can function as an RCA primer binding site, rather than an oligonucleotide sequence, between the cleavage domains that are flanked by the oligonucleotide sequence. Similar to the cleavage domains described above, the RCA primer binding site may be designed to be of a different length than the oligonucleotide sequence to ensure that it can be readily separated from the functionalized single-stranded oligonucleotide upon cleavage of the RCA product. It will be apparent that in a circular DNA molecule that contains multiple oligonucleotide sequences flanked by cleavage domains, the RCA primer binding site may be between any two cleavage domains.

[0062] The term "annealing conditions" refers to the conditions under which two nucleic acid molecules that contain complementary nucleotide sequences will specifically hybridize to each other.Various parameters affect hybridization, including temperature, salt concentration, nucleic acid concentration, composition and length, and buffer composition.Those skilled in the art can routinely determine the appropriate annealing conditions for a particular primer / template combination for RCA reaction.

[0063] As mentioned above, in some embodiments, the circular DNA molecule is double-stranded, and the method includes an additional step of cleaving one strand of the circular DNA molecule to prepare an RCA template before the RCA reaction is performed. It will be clear that the step of cleaving one strand of the circular DNA molecule can replace the step of providing a primer to initiate the RCA reaction, i.e., the cleaved strand serves as an RCA primer. Therefore, in another view, the method includes an additional step of cleaving one strand of the circular DNA molecule to prepare an RCA template and a primer before the RCA reaction is performed, i.e., the introduction of a single-strand break in the circular DNA molecule creates a 3' end that can act as a primer for the RCA reaction. However, in some embodiments, it may be advantageous to provide one or more RCA primers in the reaction mixture in addition to cleaving one strand of the double-stranded circular DNA molecule, for example to increase the number of RCA products obtained per turn.

[0064] In some embodiments, the step of cleaving a single strand of the circular DNA molecule to prepare an RCA template comprises cleaving the single strand of the circular DNA molecule with a cleavage enzyme, which results in a single-stranded break (nick).

[0065] In some embodiments, it may be advantageous to cut a single strand of a circular DNA molecule multiple times in close proximity to facilitate the binding of DNA polymerase to the 3' end generated by the cut. Thus, a single strand of a circular DNA molecule may be cut more than once, i.e., two or more nicks may be generated close to each other. Preferably, the nicks are generated within 20 nucleotides of each other, more preferably within 10 nucleotides, more preferably within 5 nucleotides, for example, within 1, 2, 3, 4 or 5 nucleotides of each other.

[0066] In some embodiments, the cleavage enzyme used to cleave one strand of the double-stranded circular DNA molecule is a nickase. Nickases are endonucleases that cleave only one strand of a DNA duplex. Some nickases introduce single-stranded nicks only at specific sites on the DNA molecule by binding to and recognizing a specific nucleotide recognition sequence. Several naturally occurring nickases have been discovered, and the sequence recognition properties of at least four have now been determined. Nickases are described in U.S. Patent No. 6,867,028, which is incorporated herein by reference in its entirety, and any suitable nickase may be used in the methods of the present invention. In some embodiments, the cleavage enzyme (nickase) may be Nb.BsrDI, Nt.BspQI, or a combination thereof.

[0067] In some embodiments utilizing a nickase enzyme, the nickase enzyme may be removed from the assay or inactivated after cleavage of the circular DNA molecule to prevent unwanted cleavage of the RCA product.

[0068] A cleavage enzyme (e.g., a nickase) that cleaves a single strand of a circular DNA molecule may cleave at any site within the circular DNA molecule. In some embodiments, the circular DNA molecule may contain a specific domain (single-stranded cleavage site or domain, e.g., a nickase site) where the cleavage enzyme can act. In a representative embodiment, the circular DNA molecule may contain a sequence between the cleavage domains that can function as a single-stranded cleavage site or domain, rather than an oligonucleotide sequence. Due to the fact that there is no sequence (single-stranded cleavage site or domain) recognized by the cleavage enzyme within the oligonucleotide sequence, it is ensured that the oligonucleotide generated from the 5' end of the RCA product is not truncated. Similar to the cleavage domain and RCA primer binding site described above, the single-stranded cleavage site or domain may be designed to be of a different length than the oligonucleotide sequence to ensure that it can be readily separated from the functionalized single-stranded oligonucleotide upon cleavage of the RCA product. Thus, in some embodiments, the RCA primer binding site also functions as a single-stranded cleavage site or domain, or vice versa.

[0069] Any DNA polymerase with at least some strand displacement activity can be used in the RCA reaction of the present invention. The strand displacement activity ensures that as the polymerase extends around the circular DNA molecule, it can displace the primer sequence and the extending product and continue to "spin" around the template. In embodiments where the nicked strand of the circular DNA molecule forms the primer for the RCA extension, the strand displacement activity ensures that the polymerase can displace the nicked strand. Suitable DNA polymerase enzymes with at least some strand displacement activity include phi29 DNA polymerase, E. coli DNA polymerase I, Bsu DNA polymerase (large fragment), Bst DNA polymerase (large fragment) and Klenow fragment. As used herein, the term "DNA polymerase" includes not only naturally occurring enzymes, but also all such modified derivatives, including derivatives of naturally occurring DNA polymerase enzymes. For example, in some embodiments, the DNA polymerase can be modified to remove 5'-3' exonuclease activity.

[0070] In particular, preferred DNA polymerase enzymes for use in the present invention include phi29 DNA polymerase, Bst DNA polymerase and derivatives, such as sequence modified derivatives or mutants thereof.

[0071] Sequence modified derivatives or variants of DNA polymerase enzymes include variants that retain at least some of the functional activities of the wild-type sequence, such as DNA polymerase activity and strand displacement activity. Mutations can affect the activity profile of the enzyme under different reaction conditions, such as temperature, template concentration, primer concentration, etc., e.g., enhance or decrease the polymerization rate. Mutations or sequence modifications may also affect the exonuclease activity and / or thermostability of the enzyme.

[0072] As noted above, the RCA reaction of the present method is generally carried out using a mixture of functionalized and conventional nucleotides.

[0073] As used herein, the term "conventional nucleotide" refers to a deoxynucleotide that contains one of the four bases found in DNA; adenine, guanine, cytosine, and thymine. The term "conventional nucleotide" thus encompasses, for example, dATP, dGTP, dCTP, and dTTP. Although uracil is not commonly found in DNA in nature, dUTP can easily be used in place of or in addition to dTTP. Thus, in the context of the present invention, dUTP may be considered a "conventional" nucleotide. Typically, the reaction mixture will contain at least three of the four different types of dNTPs that correspond to the four naturally occurring bases present, namely dATP, dTTP, dCTP, and dGTP. However, as noted above, dUTP may be used in place of or in addition to dTTP. Additionally, in some embodiments, the reaction mixture may contain four or five dNTPs, namely dATP, dCTP, dGTP, dTTP, and / or dUTP. In the subject methods, each dNTP will generally be present in an amount ranging from about 10 to 5000 μM, usually about 20 to 1000 μM. Each dNTP may be present in different amounts, or equal amounts of each dNTP may be used.

[0074] The term "functionalized nucleotide" or "functionalized dNTP" refers to a nucleotide that includes a modification compared to an unmodified conventional nucleotide, which modification provides the functionalized nucleotide and / or an oligonucleotide that includes at least one functionalized nucleotide with additional or selective properties or characteristics, i.e., compared to the corresponding conventional nucleotide. For example, the modification can render the nucleotide detectable, e.g., by incorporation of a label, or capable of interacting and / or reacting with another moiety, i.e., a moiety with which the corresponding conventional nucleotide does not interact or react. In some embodiments, the modification can render the oligonucleotide containing the nucleotide resistant to degradation, e.g., chemical and / or enzymatic degradation (e.g., nuclease degradation), or alter the metabolism of the nucleotide.

[0075] The terms "functionalized single-stranded oligonucleotide", "single-stranded functionalized oligonucleotide" and "functionalized oligonucleotide" are used interchangeably herein and refer to a single-stranded oligonucleotide that contains at least one functionalized nucleotide. Thus, a functionalized oligonucleotide has additional or alternative properties or characteristics compared to a corresponding oligonucleotide that contains only conventional nucleotides. For example, the incorporation of one or more functionalized nucleotides can make the oligonucleotide detectable, for example by incorporation of a label, or capable of interacting and / or reacting with another component, i.e., a component with which a corresponding oligonucleotide containing only conventional nucleotides does not interact or react. In some embodiments, the modification may render the oligonucleotide resistant to degradation, for example chemical and / or enzymatic degradation (e.g., nuclease degradation), or may alter the metabolism of the oligonucleotide. In some embodiments, the modification may improve the stability of the oligonucleotide, for example, improving the stability of the duplex formed by the oligonucleotide, such as the thermostability (e.g., melting temperature) of the duplex. In some embodiments, the incorporation of one or more functionalized nucleotides can enable the oligonucleotide to form secondary or tertiary structures that are not formed by a corresponding oligonucleotide containing only conventional nucleotides.

[0076] Thus, in the context of functionalized single-stranded oligonucleotides, the term "single-stranded" is understood to refer to an oligonucleotide that is single-stranded under denaturing conditions, i.e., after application of heat or a suitable chemical denaturant, has only one continuous backbone (one strand). As mentioned above, this does not prevent the functionalized single-stranded oligonucleotide from forming secondary or tertiary structures. For example, the functionalized single-stranded oligonucleotide may contain a self-complementary region and thus be capable of forming a hairpin or stem-loop structure mediated by a region of the functionalized single-stranded oligonucleotide that hybridizes to a complementary region in another part of the same oligonucleotide.

[0077] Conventional functionalization of nucleotides may be achieved by altering or modifying the structure of any part of the nucleotide. Thus, functionalized nucleotides may contain modifications, e.g., chemical modifications, on the nucleobase, sugar, or groups involved in the linkage between nucleotides. In some preferred embodiments, functionalized nucleotides contain modifications, e.g., chemical modifications, on the nucleobase. Modifications at various positions are described in more detail below, and it is believed that any of the specific positions described below can be modified with any of the functional groups described below.

[0078] For example, substitution of the C5 position in pyrimidines (i.e., cytosine, thymine, and uracil) with a small, rigid, hydrophobic group may improve the base stacking interactions of oligonucleotides that include functionalized nucleotides containing the substituted pyrimidines and / or stabilize duplexes formed thereby. The small, rigid, hydrophobic group may be an alkynyl group, such as methyl, ethynyl, propynyl, or a halogen group, such as fluoro, chloro, or bromo. Thus, in some embodiments, the functionalized nucleotide includes a pyrimidine with a substitution at the C5 position, such as an alkynyl group or a halogen as defined above.

[0079] In some embodiments, the modification that can make a nucleotide detectable can involve the incorporation of a label into the nucleotide. Any label can find utility in the present invention and can be a molecule that directly or indirectly gives a signal. For example, a label that gives a direct signal can be a fluorescent molecule, i.e., the functionalized nucleotide can be a fluorescently labeled nucleotide. A label that gives an indirect signal can be, for example, a biotin molecule, i.e., the labeled nucleotide can be a biotin-labeled nucleotide, which requires an additional step to obtain a signal, for example, adding streptavidin conjugated to an enzyme that can act on a chemical substrate to obtain a detectable signal, for example, a visually detectable color change. In some embodiments, the label is incorporated (conjugated) into the nucleic acid base.

[0080] Thus, in some embodiments, the functionalized nucleotide comprises a biotin group conjugated to the nucleobase. In some embodiments, the biotin group may be indirectly conjugated to the nucleobase, for example, via a linker or linking domain, and suitable linkers can be easily selected from those well known to those skilled in the art. For example, the linker can be selected to promote the interaction between biotin and streptavidin, i.e., to minimize or prevent steric hindrance. In some embodiments, the biotin group is conjugated to the pyrimidine at the C5 position. In a representative embodiment, the biotin containing functionalized nucleotide may be biotin-16-aminoallyl-2'-dUTP, biotin-16-aminoallyl-2'-dTTP, or biotin-16-aminoallyl-2'-dCTP.

[0081] In some embodiments, the nucleotide may be labeled with a sterol group, i.e., the nucleotide may include a sterol group, hi some embodiments, the nucleotide may be labeled with or include a cholesterol group.

[0082] A directly detectable label is one that can be directly detected without the use of additional reagents, and an indirectly detectable label is one that can be detected by utilizing one or more additional reagents, for example, when the label is a member of a signal generating system that is composed of two or more components. In many embodiments, the label is a directly detectable label, and directly detectable labels of interest include, but are not limited to, fluorescent labels, colored labels, radioisotope labels, chemiluminescent labels, and the like. Any spectrophotometric or optically detectable label may be used. In other embodiments, the label may indirectly produce a signal, i.e., require the addition of an additional component to generate a signal. For example, the label may be capable of binding a molecule that is conjugated to a molecule that provides a signal.

[0083] In some embodiments, the functionalized nucleotide is a fluorescently labeled nucleotide. Fluorescent labels require excitation to obtain a detectable signal, but the source of excitation is derived from the instrument / device used to detect the signal, so fluorescent labels may be viewed as directly signaling labels.

[0084] Fluorescent molecules that can be used to label nucleotides are well known to those skilled in the art. Fluorophores have been identified with excitation and emission spectra ranging from UV to near infrared wavelengths. Thus, fluorophores can have excitation and / or emission wavelengths in the UV, visible or infrared spectral range.

[0085] The fluorophore may be a protein, a peptide, a small organic compound, a synthetic oligomer or a synthetic polymer. In some embodiments, the fluorophore is a small organic compound, for example an organic compound with a molecular weight of 5000 Da or less. Thus, in some embodiments, the fluorophore has a molecular weight of 4000 Da or less, such as 3500 Da, 3000 Da, 2500 Da, 2250 Da, 2000 Da, 1900 Da, 1800 Da, 1700 Da, 1600 Da, 1500 Da or less.

[0086] Thus, fluorophores include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine), squaraine derivatives (e.g., ring-substituted squaraines, Seta, SeTau), naphthalene derivatives (e.g., dansyl or prodan derivatives), coumarin derivatives, oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole and benzoxadiazo). The dye may be an anthracene derivative (such as anthraquinones, including DRAQ5, DRAQ7, and CyTRAK Orange), a pyrene derivative (such as Cascade Blue), an oxazine derivative (such as Nile Red, Nile Blue, Cresyl Violet, Oxazine 170), an acridine derivative (such as Proflavine, Acridine Orange, Acridine Yellow), an arylmethine derivative (such as Auramine, Crystal Violet, Malachite Green), or a tetrapyrrole derivative (such as Porphyrin, Phthalocyanine, Bilirubin, etc.).

[0087] Specific examples of fluorophores or fluorophore series that may find utility in the present invention include Alexa Fluor (Alexa Fluor 488, Alexa Fluor 647, etc.), Atto, cyanine (Cy), indocyanine, sulfocyanine, DyLight, Abberior STAR, Chromeo, Oregon Green, fluorescein, Texas Red, rhodamine, silicon rhodamine (SiR), squaraine, FluoProbes, tetrapyrrole, Bodipy, HiLyte, Quasar, CAL fluor, coumarin, Seta, CF, Tracey, IRDye, CruzFluor, Tide Fluor, Oyster, iFluor, Chromis, and Brilliant Violet, as well as fluorescent derivatives or analogues thereof.

[0088] In some embodiments, the functionalized nucleotide contains a cyanine fluorescent label, such as Cy3. In some particular embodiments, the functionalized nucleotide is a Cy3-labeled dATP, such as 7-propargylamino-7-deaza-ATP-Cy3.

[0089] In some embodiments, the functionalized nucleotide contains an atto fluorescent label, such as atto-488. In some particular embodiments, the functionalized nucleotide is dATP labeled with atto-488, such as 7-propargylamino-7-deaza-ATP-Atto-488.

[0090] In some embodiments, modifications that can render a nucleotide reactive include the incorporation of a reactive group, e.g., a group that can form a covalent bond with another chemical group, e.g., a chemical group on a molecule or moiety that is to be conjugated to the functionalized oligonucleotide, such as a label as defined herein. Potential reactive groups include nucleophilic functional groups (alkyne, alkenyl, amine, alcohol, thiol, hydrazide, azide), electrophilic functional groups (aldehyde, ester, vinyl ketone, epoxide, isocyanate, maleimide), functional groups capable of cycloaddition reactions, disulfide bond formation, or binding to metals. Specific examples include ethyne (acetylene), propyne, 1-butyne, 2-butyne azide, vinyl (ethenyl), propenyl, 1-butenyl, primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonate, oxycarbonylimidazole, nitrophenyl esters, trifluoroethyl esters, glycidyl ethers, vinyl sulfones, azides, and maleimides.

[0091] In some embodiments, modifications that can render a nucleotide reactive include the incorporation of a reactive group that can react with another chemical group, for example, a chemical group on a molecule or component that is to be conjugated to the oligonucleotide functionalized by click chemistry. As used herein, the term "click chemistry" generally refers to reactions that are modular, comprehensive, produce high yields, can be removed by non-chromatographic methods, are stereospecific (but not necessarily enantioselective), and produce only harmless by-products. See, for example, Angew. Chem. Int. Ed., 2001, 40(11):2004-2021, incorporated herein by reference in its entirety. In some cases, click chemistry can describe pairs of functional groups that can selectively react with each other under mild aqueous conditions. Thus, click chemistry groups are suitable for conjugating additional functional groups to the oligonucleotides of the present method. Common click chemistry reactions include azide-alkyne cycloaddition, alkyne-nitrone cycloaddition, alkene-tetrazine reaction, and alkene-tetrazole reaction. Thus, a functionalized nucleotide may contain an azide group, an alkyne group, an alkene group, a nitrone group, a tetrazine group or a tetrazole group.

[0092] A specific example of a click chemistry reaction can be the Huisgen 1,3 dipolar cycloaddition of azides and alkynes, i.e., the copper-catalyzed reaction of alkynes with azides to form five-membered heteroatomic rings called 1,2,3-triazoles. The reaction can also be known as Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), Cu(I) click chemistry or Cu+ click chemistry. The catalyst for click chemistry can be a Cu(I) salt or a Cu(I) salt made in situ by reducing a Cu(II) reagent to a Cu(I) reagent with a reducing agent (Pharm Res. 2008, 25(10): pp. 2216-2230). Known Cu(II) reagents for click chemistry can include, but are not limited to, Cu(II)(TBTA) complex and Cu(II)(THPTA) complex. TBTA, tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known as tris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I) salts. THPTA, tris-(hydroxypropyltriazolylmethyl)amine, can be another example of a stabilizer for Cu(I). Other conditions using copper-free click chemistry can also be accomplished to construct 1,2,3-triazole rings (e.g., cycloalkynes such as cyclooctyne or cyclononyne) from azides and alkynes, such as by strain-promoted azide-alkyne click chemistry (SPAAC, see, e.g., Chem. Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90), each of which is incorporated herein by reference in its entirety.

[0093] In some embodiments, the functionalized nucleotide contains a reactive group within the sugar group, e.g., a modification at the 2-position of the deoxyribose sugar, such as replacing a hydrogen with a fluoro, chloro, bromo, or azido group, hi some embodiments, the functionalized nucleotide is a 2'-azido dNTP, e.g., a 2'-azido dATP.

[0094] In some embodiments, the functionalized nucleotide contains a reactive group at the nucleobase, in particular selected from an alkyne, alkenyl, thio or halogen group. As mentioned above, the functionalized nucleotide may contain a pyrimidine containing a substitution at the C5 position. In some embodiments, the alkyne group is an ethyne, for example, the functionalized nucleotide is an ethynyl-dNTP, such as 5'-ethynyl-dUTP. Alternatively, the alkyne group may be a propynyl group, for example, the functionalized nucleotide is a propynyl-dNTP, such as 5'-propynyl-dUTP. In some embodiments, the alkenyl group is vinyl (ethenyl), for example, the functionalized nucleotide is a vinyl-dNTP, such as 5'-vinyl-dUTP. In some embodiments, the functionalized nucleotide is a thio-dNTP, such as 4'-thio-dTTP. In some embodiments, the halogen group is a bromine, for example, the functionalized nucleotide is a bromo-dNTP, such as 5'-bromo-dUTP. Incorporation of halogen groups into functionalized oligonucleotides can be used to facilitate nucleophilic aromatic displacement or UV-mediated cross-linking, for example with proteins. Thus, in some embodiments, functionalized oligonucleotides produced by the present method may be further modified to include aromatic groups or conjugated to other molecules, for example proteins or peptides, by UV-mediated cross-linking.

[0095] In some embodiments, functionalized nucleotides may contain groups capable of interacting with another component, such as groups that interact through non-covalent bonds.For example, nucleotides may be modified to incorporate a portion or moiety of a cognate binding pair, such as an affinity binding partner, such as biotin or a hapten, capable of binding to a binding partner, i.e., a cognate binding partner, such as streptavidin or an antibody.Such functionalized nucleotides may find utility, for example, in the creation of functionalized oligonucleotides that can be immobilized on solid supports.

[0096] In some embodiments, the functionalized nucleotide contains a modification that renders the oligonucleotide containing the nucleotide resistant to degradation, e.g., chemical and / or enzymatic degradation (e.g., nuclease degradation). In some embodiments, the nucleotide contains a modification of the sugar group, e.g., a modification at the 2-position of the deoxyribose sugar, such as replacing a hydrogen with a fluoro, chloro, O-methyl, or O-ethyl group. Thus, in some embodiments, the functionalized nucleotide is a 2'-fluoro-dNTP, e.g., 2-fluoro-UTP. In some embodiments, the functionalized nucleotide contains an O-Me group, e.g., 2'-O-methyl-ATP. In some embodiments, the nucleotide contains a modification in the phosphate group that forms the internucleotide linkage, such as replacing an oxygen with a sulfur, e.g., the nucleotide contains a phosphorothioate group. Thus, in some embodiments, the functionalized nucleotide is a nucleotide thiotriphosphate, such as 2-deoxythymidine-5'-O-(1-thiotriphosphate), 2-deoxycytidine-5'-O-(1-thiotriphosphate), 2-deoxyuridine-5'-O-(1-thiotriphosphate), 2-deoxyadenosine-5'-O-(1-thiotriphosphate), or 2-deoxyguanosine-5'-O-(1-thiotriphosphate). In some embodiments, the functionalized nucleotide contains a modification in the nucleobase, such as an amino, methyl, ethyl, or propynyl modification, such as 2-amino-dATP, 5-methyl-dCTP, C-5 propynyl-dCTP, or C-5 propynyl-dUTP.

[0097] In some embodiments, the functionalized nucleotide contains a modification that affects the thermostability of the oligonucleotide. In some embodiments, the nucleotide contains a locked ribose sugar, i.e., contains an additional covalent bond between the 2' oxygen and the 4' carbon of the pentose ring. In some embodiments, the nucleotide is an LNA (locked nucleic acid) nucleotide, i.e., an LNA-NTP, such as LNA-ATP. The locked ribose conformation enhances base stacking, thus increasing the melting temperature of the oligonucleotide containing the LNA nucleotide.

[0098] Thus, in some embodiments, functionalized nucleotides that can be used in the present invention include: nucleotides containing (internalized) alkyne or azide groups, fluorescently labeled nucleotides, nucleotides containing sterol groups, nucleotides containing polyether groups, nucleotides containing metal complexes, nucleotides containing vinyl groups, nucleotides containing thiol groups, thionated nucleotides, nucleotides modified to increase nuclease resistance, nucleotides containing chemical groups capable of participating in click chemistry, nucleotides that affect (e.g., increase) the thermostability of oligonucleotides (e.g., LNA nucleotides), or combinations thereof. The incorporation of these functionalized nucleotides into single-stranded oligonucleotides of the present invention can provide a variety of useful functions. For example, single-stranded oligonucleotides containing fluorophores can be used as sequence-specific fluorescent probes. The inclusion of thiolated nucleotides in single-stranded oligonucleotides allows the oligonucleotides to be labeled with thiol-reactive molecules and used as probes for molecular detection of such thiol-reactive molecules.

[0099] In some embodiments, the functionalized nucleotide that can be used in the present invention does not include the nucleotide that contains digoxigenin group.Alternatively, in addition to some embodiments, the functionalized nucleotide is not a digoxigenin-labeled nucleotide.In particular, in some embodiments, the functionalized nucleotide is not digoxigenin-11-dUTP.

[0100] In a preferred embodiment, the functionalized dNTP is a nucleotide that includes an alkyne or vinyl group, e.g., a modified nucleobase that contains an alkyne (e.g., ethynyl) or vinyl group, e.g., a nucleotide that includes a pyrimidine with an alkyne or vinyl group at the C5 position. In another preferred embodiment, the functionalized dNTP is a nucleotide that includes an azide group, e.g., a nucleotide that includes a modified sugar that contains an azide group, e.g., a nucleotide that includes an azide group at the 2-position of the deoxyribose sugar.

[0101] The reaction mixture of the RCA reaction must contain a combination of components capable of generating RCA products from a template circular DNA molecule. For example, the nucleotides present in the reaction mixture (e.g., a mixture of conventional and functionalized nucleotides) must be capable of hybridizing to the respective nucleotides in the circular DNA template to enable rolling circle amplification. The relative amounts of functionalized and conventional nucleotides present in the reaction mixture may vary depending on the identity of the functionalized nucleotide and the polymerase. Furthermore, the relative amounts of each nucleotide present in the reaction mixture may be used to control the incorporation of functionalized nucleotides into the RCA product. For example, by increasing the concentration of functionalized nucleotides (or decreasing the proportion of conventional nucleotides), the proportion of functionalized nucleotides in the RCA product may be greater (e.g., when functionalized nucleotides are used in combination with equivalent conventional nucleotides). Conversely, by decreasing the concentration of functionalized nucleotides (or increasing the proportion of conventional nucleotides), the proportion of functionalized nucleotides in the RCA product may be lower.

[0102] Thus, functionalized nucleotides may be used in addition to or instead of conventional nucleotides that hybridize to the same DNA base (nucleotide) in the template DNA. In some embodiments, the reaction mixture may contain only one type of functionalized nucleotide. In some embodiments, a combination of different functionalized nucleotides may be used in the same reaction. In some embodiments, all of the functionalized nucleotides in the reaction mixture contain the same type of functional group, such as an alkyne group. In some embodiments, the functionalized nucleotides in the reaction mixture contain different types of functional groups. As an example, different types of functionalized nucleotides may be capable of hybridizing to the same DNA base (nucleotide), such as a dATP nucleotide functionalized with a fluorophore and a second dATP nucleotide functionalized with a sterol group. In another representative example, different types of functionalized nucleotides may be capable of hybridizing to different DNA bases (nucleotides), such as a dATP nucleotide functionalized with a fluorophore and a dTTP nucleotide functionalized with a sterol group (or a fluorophore different from the fluorophore on the dATP nucleotide). Thus, any combination of functionalized and conventional nucleotides may be used in the present invention. In a preferred embodiment, the RCA reaction mixture contains only one type of functionalized nucleotide.

[0103] The amount of functionalized nucleotides present in a reaction mixture can be measured as a relative percentage of the total nucleotides capable of hybridizing to a particular DNA base (nucleotide). Alternatively, this value can be considered as the percentage that the functionalized nucleotide replaces the corresponding conventional nucleotide. For example, the use of equal amounts of conventional dATP and fluorophore-modified dATP could be expressed as 50% of the total dATP nucleotides being modified (functionalized), or 50% replacement of conventional dATP nucleotides by functionalized dATP nucleotides.

[0104] The relative amount of functionalized nucleotide in RCA reaction mixture can be varied to control the frequency of functionalized nucleotide in final single-stranded oligonucleotide.In some embodiments, functionalized nucleotide can represent up to about 5% of the total nucleotides that can hybridize to specific DNA base (nucleotide), for example, about 1%, 2%, 3%, 4% or 5%.Alternatively, in some embodiments, functionalized nucleotide can represent a higher percentage, such as 25%, 50%, 75% or 100% of the total nucleotides that can bind to specific DNA base (nucleotide).

[0105] The relative amount of functionalized nucleotide present can vary for many reasons. For example, some functionalized nucleotides, such as Cy3-modified dATP, are not commercially available in high concentrations. Furthermore, as shown in the examples, the inventors have determined that the inclusion of functionalized nucleotides can affect the yield of functionalized single-stranded oligonucleotides produced by the present invention, for example, the use of high relative amounts of functionalized nucleotides can inhibit the activity of the DNA polymerase involved in the RCA reaction (e.g., reducing the efficiency with which the RCA product is synthesized), or the cleavage enzyme involved in the cleavage of the RCA product and the release of single-stranded functionalized oligonucleotide (e.g., reducing the efficiency with which the RCA product is cleaved).

[0106] In particular, however, the inventors have surprisingly found that high yields of functionalized single-stranded oligonucleotides can be achieved even when using high relative amounts, for example, up to about 75%, such as up to about 70%, 65%, 60%, 55% or 50%, of functionalized oligonucleotides. In some embodiments, it may be possible to use more than 75%, such as about 80%, 85%, 90%, 95% or 100%, of functionalized oligonucleotides. In particular, the inventors have unexpectedly found that functionalized nucleotides containing alkyne or vinyl groups in the nucleobase are particularly useful in the present invention, since they can be efficiently incorporated into the RCA product. Furthermore, although the RCA product could be easily cleaved to produce functionalized single-stranded oligonucleotides, as described below, in some embodiments, a larger amount of cleavage enzyme may be required compared to the amount required to cleave the equivalent RCA product containing only conventional nucleotides.

[0107] Similarly, the inventors have discovered that functionalized nucleotides containing an O-methyl group on the deoxyribose sugar can fully substitute for conventional nucleotides in RCA reactions and still produce RCA products. Moreover, the inventors have surprisingly found that incorporation of these nucleotides into the RCA product does not affect the formation of a cleavage domain (e.g., a hairpin cleavage domain) or its enzymatic cleavage (e.g., by a restriction endonuclease).

[0108] Thus, the relative amount of functionalized nucleotides present in the reaction mixture may be adjusted to optimize the yield of the desired functionalized single-stranded oligonucleotide or optimize the production of the RCA product. Such modifications are within the purview of one of skill in the art based on the methods described in the Examples below. Thus, in some embodiments, the relative amount of functionalized nucleotides in the reaction mixture may be about 1-5%, 1-10%, 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 25-100%, 50-75%, 50-100%, or 75-100%.

[0109] In addition to the relative amount of functionalized nucleotides in the reaction mixture, and the absolute amount of nucleotides (both conventional and functionalized) after generation of the RCA product in step (b), the method includes a step of cleaving the RCA product at the cleavage domain to release a single-stranded functionalized oligonucleotide. As mentioned above, the step of cleaving the RCA product can be achieved by contacting the RCA product with a cleavage enzyme under suitable conditions to selectively cleave the RCA product at the cleavage domain.

[0110] The term "release" as used in this context refers to cleaving the RCA product at the cleavage domains flanking the oligonucleotide sequence to release or separate the functionalized oligonucleotide from the cleavage domains. It is desirable that the release of a given functionalized oligonucleotide will include cleavage at both of the cleavage domains flanking the oligonucleotide sequence.

[0111] It is not necessary that cleavage occurs at all of the cleavage domains in the RCA product to generate functionalized single-stranded oligonucleotides. As described above, the incorporation of functionalized nucleotides in the RCA product, particularly in the cleavage domains, may reduce the efficiency of the cleavage process. Nevertheless, cleavage of a portion of the cleavage domains will result in the release of a portion of the functionalized single-stranded oligonucleotides. Thus, in some embodiments, cleaving the RCA product results in cleavage of at least about 30%, such as at least about 35%, 40%, 45%, 50%, 60%, 70% or 80% of the cleavage domains in the RCA product. In some embodiments, cleaving the RCA product results in cleavage of at least about 90%, such as 95% or more of the cleavage domains in the RCA product.

[0112] In another aspect, in some embodiments, cleaving the RCA product results in the release of at least about 30%, such as at least about 35%, 40%, 45%, 50%, 60%, 70% or 80% of the functionalized single-stranded oligonucleotides contained in the RCA product. In some embodiments, cleaving the RCA product results in the release of at least about 90%, such as 95% or more of the functionalized single-stranded oligonucleotides contained in the RCA product.

[0113] Once the single-stranded functionalized oligonucleotide has been released, it may be desirable to isolate, separate or purify it from the cleavage reaction mixture (e.g., reaction components and / or degradation products, such as the cleavage domain, uncleaved RCA products, etc.) for use in other applications.

[0114] Thus, in some embodiments, the methods of the invention further comprise the step of isolating, separating or purifying the functionalized single-stranded oligonucleotide, which may be performed by any suitable method known to those of skill in the art.

[0115] In some embodiments, after the isolation, separation or purification step, the functionalized single-stranded oligonucleotide is preferably substantially free of any contaminating components (e.g., reaction components and / or degradation products, such as cleavage domains, uncleaved RCA products, etc.) that may result from the isolation procedure or materials or components used in its preparation. In some embodiments, the functionalized single-stranded oligonucleotide is purified to a purity of greater than about 50 or 60%, such as greater than about 70, 80 or 90%, greater than about 95 or 99%, etc., as assessed mass / mass (dry mass). Such purity levels may include degradation products of the functionalized single-stranded oligonucleotide.

[0116] In some embodiments, it may be useful to prepare an enriched preparation of functionalized single-stranded oligonucleotides having a lower purity, e.g., containing less than about 50%, e.g., less than about 40 or 30%, of the functionalized single-stranded oligonucleotide of interest.

[0117] As mentioned above, the present invention can result in a mixture or a plurality (e.g., a library) of functionalized single-stranded oligonucleotides. Thus, in some embodiments, it may be desirable to further separate the functionalized single-stranded oligonucleotides, for example, by size, to obtain a specific functionalized single-stranded oligonucleotide (i.e., to isolate a specific functionalized single-stranded oligonucleotide) or to generate a subgroup or sublibrary of functionalized single-stranded oligonucleotides. Any suitable means may be utilized to separate the mixture of functionalized single-stranded oligonucleotides to isolate a specific functionalized single-stranded oligonucleotide or a subgroup or sublibrary of functionalized single-stranded oligonucleotides.

[0118] Thus, in some embodiments, the method includes the further step of separating functionalized single-stranded oligonucleotides from the mixture (e.g., library) of functionalized single-stranded oligonucleotides obtained by the above method to isolate a particular functionalized single-stranded oligonucleotide or a subgroup of functionalized single-stranded oligonucleotides.

[0119] For example, the products of the cleavage reaction may be separated by size using gel electrophoresis using agarose or polyacrylamide gels. The desired functionalized oligonucleotides may then be isolated from the gel and, if necessary, further purified according to methods known to those skilled in the art. Other methods for purifying, isolating or separating the functionalized oligonucleotides of the present invention utilize chromatography (e.g., HPLC, size exclusion, ion exchange, affinity, hydrophobic interaction, reverse phase) or capillary electrophoresis.

[0120] As mentioned above, the functionalized oligonucleotides produced by the above method may contain a reactive group capable of reacting with another chemical group, for example, a chemical group on the molecule or component to be conjugated to the functionalized oligonucleotide, for example, by click chemistry. For example, the step of conjugating additional molecules or components (which themselves contain or can be seen as functional groups) to the single-stranded oligonucleotide may be particularly useful for incorporating large or bulky groups, such as groups that may inhibit or reduce the efficiency of the method if present in the functionalized nucleotide used in the RCA reaction. Thus, for example, functionalized oligonucleotides may be produced that contain molecules or components that cannot be directly incorporated by polymerase during the RCA reaction or that would be incorporated only with low efficiency or yield. Additionally or alternatively, subjecting the functionalized oligonucleotides obtained by the method to further conjugation steps may increase the structural diversity present in the functionalized oligonucleotide library.

[0121] Thus, in some embodiments, the method further comprises conjugating a molecule or moiety to the functionalized oligonucleotide via a functional (e.g. reactive) group in the oligonucleotide, such as by click chemistry. In some preferred embodiments, the molecule or moiety is conjugated to the functionalized oligonucleotide via an alkyne, vinyl or azide group (i.e., an alkyne, vinyl or azide group in the functionalized nucleotide that is incorporated into the RCA product in the methods defined herein).

[0122] In the context of the present invention, the term "conjugation" with respect to linking or connecting a molecule or moiety to a functionalized oligonucleotide (e.g. an alkyne, vinyl or azide group in said functionalized oligonucleotide) refers to connecting said molecule or moiety to said oligonucleotide by a covalent bond. In particular, this conjugation may occur by a click chemistry reaction.

[0123] An example of a specific click chemistry reaction that can be used to conjugate additional molecules or components to the single-stranded functionalized oligonucleotide of the present invention that contains one or more alkyne groups is azide-alkyne cycloaddition. To achieve the desired conjugation, the oligonucleotide that contains an alkyne group can be incubated with a molecule or component that contains an azide group (e.g., a label such as a fluorophore) for a suitable time. The azide-alkyne cycloaddition reaction generally uses a copper catalyst, particularly a copper(I) catalyst. In some embodiments, the oligonucleotide and the azide-containing molecule or component can be incubated in the presence of copper sulfate. A reducing agent can also be used to generate an active copper(I) catalyst. The reducing agent can be, for example, sodium ascorbate.

[0124] A further representative example of conjugating additional molecules or components to the single-stranded functionalized oligonucleotide of the present invention that contains one or more vinyl groups may utilize the alkene-tetrazine reaction. This reaction has the advantage of being copper-free. Moreover, this reaction is completely orthogonal to the above-mentioned alkyne-azide click chemistry reaction. Thus, a single-stranded functionalized oligonucleotide that contains both an alkyne group and a vinyl group may be subjected to two click chemistry reactions independently to conjugate two different additional molecules or components to the same oligonucleotide.

[0125] Thus, in some embodiments, the present invention can be viewed as providing a two-step method for making functionalized single-stranded oligonucleotides, comprising a first step of incorporating a functionalized nucleotide (e.g., containing a group capable of participating in a click chemistry reaction, e.g., a reactive group such as an alkyne, vinyl, or azide group) into the oligonucleotide using the methods described herein, and a second step of conjugating an additional molecule or moiety to the single-stranded oligonucleotide via the functional group in the functionalized nucleotide.

[0126] It will be apparent that any desired molecule or moiety (i.e., entity) can be conjugated to the functional group in the single-stranded oligonucleotide produced by the present method. Such a molecule or moiety simply requires the presence of a group (e.g., a reactive group) capable of reacting with the functional group in the oligonucleotide to form a covalent bond. In some embodiments, the molecule or moiety may be a nucleic acid molecule, a protein, a peptide, a small molecule organic compound (e.g., a sterol such as cholesterol), a fluorophore, a metal-ligand complex, a polysaccharide, a nanoparticle, a nanotube, a polymer, a cell, an organelle, a vesicle, a virus, a virus-like particle, or any combination thereof.

[0127] The cell may be a prokaryotic or eukaryotic cell, hi some embodiments, the cell is a prokaryotic cell, such as a bacterial cell.

[0128] In some embodiments, the functionalized oligonucleotides may be conjugated or fused to compounds or molecules having a therapeutic or prophylactic effect, such as antibiotics, antivirals, vaccines, antitumor drugs such as radioactive compounds or isotopes, cytokines, toxins, oligonucleotides, nucleic acids encoding genes or nucleic acid vaccines.

[0129] In some embodiments, functionalized oligonucleotides (e.g., aptamers) may be conjugated or fused to labels, such as radioactive labels, fluorescent labels, luminescent labels, chromophoric labels, and enzymes that generate substances and detectable substrates, such as horseradish peroxidase, luciferase, or alkaline phosphatase. This detection may be applied to numerous assays where antibodies are traditionally used, including Western blotting / immunoblotting, histochemistry, enzyme-linked immunosorbent assay (ELISA) or flow cytometry (FACS) formats. Labels for magnetic resonance imaging, positron emission tomography probes, and boron-10 for neutron capture therapy may be conjugated to the functionalized oligonucleotides described herein.

[0130] In some embodiments, the molecule or moiety may be selected from the group consisting of: a fluorophore, a sterol (e.g., cholesterol), a polyether, a metal complex, a thiol-containing molecule, a molecule containing a group that confers increased nuclease resistance, and a molecule containing a group capable of participating in a click chemistry reaction.

[0131] It will be apparent that the molecule or component conjugated to the functionalized oligonucleotide may interact with other molecules, and such interactions may be covalent or non-covalent interactions. For example, a peptide conjugated to an oligonucleotide may non-covalently interact with its cognate binding partner, such as an antibody. In a further example, a molecule containing a group capable of participating in a click chemistry reaction may be conjugated to another molecule or component as defined above by reaction with a reactive group of said molecule or component to form a covalent complex.

[0132] The circular DNA molecule obtained in step (a) of the method may be produced by any suitable means, a variety of means being well known to those skilled in the art. Thus, the method of the present invention may comprise an additional step prior to step (a) of providing a circular DNA molecule.

[0133] For example, a method for generating a circular DNA molecule may include designing in silico a sequence that includes one or more desired oligonucleotide sequences (referred to herein as "pseudogenes") flanked by cleavage domains. Thus, the method may utilize computer-implemented methods for designing pseudogenes, such as those described in the literature, e.g., Ducani et al., 2013, supra.

[0134] As used herein, the term "pseudogene" refers to a nucleotide sequence that comprises one or more desired oligonucleotide sequences flanked by cleavage domains. Alternatively, this sequence may be referred to herein as a "nucleic acid construct."

[0135] The in silico designed pseudogene sequence can be generated (e.g., synthesized) using commercially available gene synthesis methods or any other suitable means known to those of skill in the art, such as assembly PCR. Thus, the method may include a step of generating a pseudogene.

[0136] The pseudogene may be generated as a single-stranded or double-stranded molecule. The pseudogene may be circularized using any suitable means known to those skilled in the art to obtain the circular DNA molecule of step (a). For example, the double-stranded molecule may be ligated using a ligase enzyme as described below. In this regard, it will be understood that at least one of the 5' ends of the pseudogene is phosphorylated to allow ligation to occur. In embodiments where the step of generating the pseudogene results in a DNA molecule with a non-phosphorylated 5' end, the method may include the further step of phosphorylating the 5' end of the pseudogene using a kinase enzyme such as, for example, T4 polynucleotide kinase.

[0137] Similarly, if the pseudogene is obtained as a single-stranded molecule, it may be circularized using a suitable ligase enzyme capable of catalyzing intramolecular ligation of single-stranded oligonucleotide molecules, such as CircLigase. The method may also include the further step of phosphorylating the 5' end of the pseudogene to facilitate the ligation reaction, for example using a kinase enzyme, such as T4 polynucleotide kinase.

[0138] In some embodiments, it may be advantageous to insert the pseudogene into a plasmid that can be replicated in bacteria such as E. coli. Suitable plasmid sequences are well known to those skilled in the art. This allows the sequence of the pseudogene to be confirmed (e.g., by sequencing) and any errors in the sequence to be corrected, for example, by repeated sequencing and mutagenesis using any suitable method.

[0139] The insertion of a pseudogene into a plasmid also facilitates the generation of a significant number of copies of a circular DNA molecule. For example, a single bacterial colony containing a plasmid containing a sequence-verified pseudogene may be grown to amplify the plasmid, which may then be purified from the bacteria using any suitable means known to those skilled in the art.

[0140] The pseudogene sequence is then excised from the plasmid using, for example, the restriction enzymes described above. The excised linear pseudogene sequence can be purified using PAGE and gel extraction or other suitable methods. The purified linear pseudogene sequence can then be recircularized using a suitable ligase enzyme, such as T4 ligase, to form the circular DNA molecule obtained in step (a) of the method.

[0141] In addition to amplification, the method of transfecting bacteria with the pseudogene containing plasmid also allows for the creation of bacterial glycerol stocks: Bacteria containing the desired pseudogene plasmid can be prepared in glycerol, frozen, and stably stored for long periods of time.

[0142] Thus, in some embodiments, step (a) of the method comprises: (i) cloning a linear DNA molecule comprising an oligonucleotide sequence flanked by cleavage domains into a DNA plasmid; (ii) amplifying the plasmid; (iii) excising a portion of the plasmid containing the DNA molecule that includes the oligonucleotide sequence flanked by the cleavage domain; (iv) circularizing the portion of the plasmid obtained in step (iii).

[0143] In some embodiments, step (ii) comprises transfecting the DNA plasmid into a bacterium and propagating the bacterium.

[0144] In some embodiments, the linear DNA molecule comprising the oligonucleotide sequence flanked by cleavage domains further comprises a 5' terminal region and a 3' terminal region, each of which comprises a cleavage domain, and step (iii) comprises cleaving the cleavage domain within the terminal regions with a cleavage enzyme.

[0145] Any suitable cleavage enzyme as defined herein may be used to excise the pseudogene from the plasmid, in some embodiments the cleavage enzyme is BsmBI or Bsal or an isoschizomer thereof.

[0146] In some specific embodiments of the invention, the method uses phi29 DNA polymerase or a derivative thereof and a functionalized nucleotide comprising a modified nucleobase. In some embodiments, the functionalized nucleotide comprises a reactive group in the nucleobase, such as a reactive group capable of participating in a click chemistry reaction. In some embodiments, the reactive group in the nucleobase is an alkyne or vinyl group as defined above. In some embodiments, the relative amount of functionalized nucleotide in the RCA mixture is about 25-100%, such as 25-75%, 50-100%, or 75-100%, or any value within these ranges. In some embodiments, the cleavage domain is capable of forming a hairpin structure in the RCA product as defined above.

[0147] In a further aspect, the present invention provides a kit, in particular a kit for use in preparing functionalized single stranded oligonucleotides, said kit comprising: (i) a circular DNA molecule comprising an oligonucleotide sequence flanked by a cleavage domain; and optionally (ii) one or more cleavage enzymes that cleave the cleavage domain of (i); and / or (iii) contains functionalized dNTPs.

[0148] In some embodiments, the present invention provides a method for the preparation of a method for treating atopic dermatitis comprising: (i) a circular DNA molecule comprising an oligonucleotide sequence flanked by a cleavage domain; the cleavage domain comprises or consists of a sequence capable of forming a hairpin structure, the double-stranded portion of the hairpin structure comprising a sequence recognized by a cleavage enzyme; and (ii) a functionalized dNTP (e.g., as defined herein); and optionally (iii) one or more cleavage enzymes that cleave the cleavage domain of (i);

[0023] In accordance with the present invention, there is provided a kit for use in the methods described herein comprising:

[0149] In some embodiments, the kit may include a DNA polymerase enzyme capable of performing rolling circle amplification, i.e., that contains at least some strand displacement activity as defined herein. For example, the kit may include phi29 polymerase or a derivative thereof, or Bst DNA polymerase or a derivative thereof.

[0150] In some embodiments, the kit may include one or more nickases. For example, the kit may include Nb.BsrDI, Nt.BspQI, or a combination thereof.

[0151] In some embodiments, the kit may include one or more single-stranded binding proteins as defined herein. For example, the kit may include E. coli single-stranded DNA binding protein, gene 32 protein of T4 phage, or a combination thereof.

[0152] In some embodiments, the kit may include one or more molecules or components that are conjugated to the functionalized single-stranded oligonucleotides defined above.

[0153] The circular DNA molecule, the cleavage enzyme and the functionalized dNTPs of the kit are as described above.

[0154] In a further aspect, the present invention provides a single-stranded functionalized oligonucleotide obtainable by the methods described herein.

[0155] In some embodiments, the single-stranded functionalized oligonucleotide contains at least 20 nucleotides. For example, the single-stranded functionalized oligonucleotide may contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides. In some preferred embodiments, the single-stranded functionalized oligonucleotide contains at least 50 nucleotides, such as at least about 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides. In some embodiments, the single-stranded functionalized oligonucleotide contains about 50-1000, 55-900, 60-800, 65-700, 70-600, 80-500, 90-450 or 100-400 nucleotides. In some embodiments, the single stranded functionalized oligonucleotide contains about 400-10000, 500-9000, 600-8000, 700-7000, 800-6000, 900-5000, or 1000-4000 nucleotides, such as about 500, 1000, 1500, 2000, 2500, 3000, 3500 or more nucleotides.

[0156] In some embodiments, at least 5% of the nucleotide residues of the single-stranded functionalized oligonucleotide are functionalized nucleotides, i.e., nucleotides containing a functional group as defined herein. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the nucleotide residues of the single-stranded functionalized oligonucleotide are functionalized nucleotides. Thus, in some embodiments, about 5-100% of the nucleotide residues of the single-stranded functionalized oligonucleotide are functionalized nucleotides, for example, about 10-95%, 15-90%, 20-85%, 25-80%, 30-75%, 35-70%, 40-65% or 45-55% of the nucleotide residues of the single-stranded functionalized oligonucleotide are functionalized nucleotides.

[0157] In some embodiments, the single-stranded functionalized oligonucleotide contains at least one internal functionalized nucleotide, i.e., a functionalized nucleotide that is not at the 5' or 3' end of the oligonucleotide. In some embodiments, the single-stranded functionalized oligonucleotide contains 2, 3, 4, 5, 6, 7, 8, 9, 10 or more internal functionalized nucleotides, such as 15, 20, 25, 30, 35, 40, 45, 50 or more internal functionalized nucleotides.

[0158] A single-stranded functionalized oligonucleotide may contain any one or more of the functionalized nucleotides defined herein. In some embodiments, a chain-functionalized oligonucleotide comprises a functionalized nucleotide containing a functional group selected from an alkyne group, an alkene group (e.g., a vinyl group), an azide group, a halogen group, an O-methyl group, a locked ribose sugar, or a combination thereof.

[0159] Thus, in one embodiment, the present invention provides a single-stranded functionalized oligonucleotide obtained by the method described herein, (i) the oligonucleotide contains at least 50 nucleotides; (ii) at least 5% of the nucleotide residues contain a functional group selected from an alkyne group, an alkene group (e.g., vinyl), an azide group, a halogen group, an O-methyl group, a locked ribose sugar, or a combination thereof, and preferred positions of the functional groups within the functionalized nucleotide residues are as defined above.

[0160] In yet a further aspect, the present invention provides a library comprising a plurality of single-stranded functionalized oligonucleotides obtained by the method described herein. The library of a plurality of single-stranded functionalized oligonucleotides may comprise one or more single-stranded functionalized oligonucleotides as defined above.

[0161] In a still further aspect, the present invention provides a method as defined herein for producing a pool of single-stranded functionalized oligonucleotides for use in single molecule fluorescence in situ hybridization (smFISH), wherein the functionalized nucleotides are fluorescently labeled nucleotides, and each single-stranded functionalized oligonucleotide in the pool contains about 15-30, preferably about 20-25 nucleotides. In a preferred embodiment, the functionalized nucleotides are labeled with the same fluorescent molecule.

[0162] As an alternative to the use of directly fluorescently labeled functionalized nucleotides, the method may include the use of nucleotides that contain a chemical group capable of participating in click chemistry, to which a fluorescent label can then be conjugated. Suitable fluorescent labels are well known to those skilled in the art and are defined above.

[0163] Thus, in a still further aspect, the present invention provides a method as defined herein for producing a pool of single-stranded functionalized oligonucleotides for use in single molecule fluorescence in situ hybridization (smFISH), wherein the functionalized nucleotides are nucleotides comprising a chemical group capable of participating in click chemistry, said method further comprising the step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry, each single-stranded functionalized oligonucleotide in the pool containing about 15-30, preferably about 20-25 nucleotides. The nucleotides comprising a chemical group capable of participating in click chemistry are as defined above and are nucleotides comprising an azide group, an alkyne group, an alkene group, a nitrone group, a tetrazine group or a tetrazole group or combinations thereof.

[0164] In the smFISH technique, single-stranded functionalized oligonucleotides act as hybridization probes to identify the presence and location of nucleic acid molecules containing specific target sequences to be detected. Thus, once a pool of single-stranded functionalized oligonucleotides for use in single molecule fluorescence in situ hybridization (smFISH) has been generated by the methods disclosed herein, the functionalized oligonucleotides can be hybridized to nucleic acid molecules containing the target sequences to be detected in the smFISH method.

[0165] When the single-stranded functionalized oligonucleotide comprises a functionalized nucleotide that includes a chemical group capable of participating in click chemistry, the step of conjugating a fluorescent label to the functionalized nucleotide by click chemistry may be performed before or after the functionalized oligonucleotide is hybridized to a nucleic acid molecule that includes a target sequence.

[0166] For example, functionalized oligonucleotides comprising functionalized nucleotides comprising chemical groups capable of participating in click chemistry may be subjected to a step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry, and after the fluorescent label is conjugated, may then be hybridized to a nucleic acid molecule comprising a target sequence. Alternatively, functionalized oligonucleotides comprising functionalized nucleotides comprising chemical groups capable of participating in click chemistry may first be hybridized to a nucleic acid molecule comprising a target sequence, and after hybridization to the target sequence, may then be subjected to a step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry.

[0167] Thus, in a still further aspect, the present invention provides a method as defined herein which is a method of making a pool of single-stranded functionalized oligonucleotides for use in single molecule fluorescence in situ hybridization (smFISH), wherein the functionalized nucleotides are nucleotides comprising a chemical group capable of participating in click chemistry, the method further comprising the step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry, wherein each single-stranded functionalized oligonucleotide in the pool contains about 15-30, preferably about 20-25 nucleotides, and wherein the step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry is performed before the functionalized oligonucleotides are hybridized to a nucleic acid molecule comprising a target sequence.

[0168] Similarly, the present invention also provides a method as defined herein of making a pool of single-stranded functionalized oligonucleotides for use in single molecule fluorescence in situ hybridization (smFISH), wherein the functionalized nucleotides are nucleotides that comprise a chemical group capable of participating in click chemistry, the method further comprising the step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry, wherein each single-stranded functionalized oligonucleotide in the pool contains about 15-30, preferably about 20-25 nucleotides, and the step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry is performed after the functionalized oligonucleotides have been hybridized to a nucleic acid molecule comprising a target sequence.

[0169] A "pool" of single-stranded functionalized oligonucleotides refers to multiple oligonucleotides with different sequences, i.e., sequences that hybridize to different (e.g., non-overlapping) sequences in a target (molecule to be detected). In some embodiments, the pool contains at least about 20 oligonucleotides, e.g., about 22, 25, 27, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 oligonucleotides. For example, in some embodiments, the pool contains about 30-96 oligonucleotides, e.g., about 36, 48, 60, 72, 84, or 96 oligonucleotides.

[0170] Viewed another way, in some embodiments, the pseudogene contains at least about 20 oligonucleotide sequences flanked by cleavage domains, e.g., about 22, 25, 27, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 oligonucleotide sequences flanked by cleavage domains. For example, in some embodiments, the pseudogene contains about 30-96 oligonucleotide sequences flanked by cleavage domains, e.g., about 36, 48, 60, 72, 84, or 96 oligonucleotide sequences flanked by cleavage domains.

[0171] The invention will now be described in more detail in the following non-limiting examples with reference to the following figures: [Brief description of the drawings]

[0172] [Figure 1] Photographs of agarose gels visualized using UV light after ethidium bromide staining (top) and fluorescent imaging using wavelengths corresponding to the emission wavelengths of the fluorophores (bottom). The agarose gels show functionalized single-stranded oligonucleotide products of the invention (containing 378 nucleotides) with the fluorophores ATTO-488 (A) or Cy3 (B). [Diagram 2]1 shows a negative of a photograph of an agarose gel visualized using UV light after ethidium bromide staining, showing functionalized single-stranded oligonucleotide products of the invention (containing 420 nucleotides) with various relative amounts of 5-EdUTP / dTTP nucleotides, i.e., 25%, 50%, 75%, and 100%, and 5-ethynyl-dUTP (5-EdUTP) generated using phi29 DNA polymerase (A) or Bst DNA polymerase (B). [Diagram 3] Photograph of an agarose gel visualized using UV light after ethidium bromide staining (left) and fluorescent imaging (right) using a wavelength corresponding to the emission wavelength of the Cy3 fluorophore. The agarose gel shows single-stranded oligonucleotide products of the invention (containing 420 nucleotides) containing 5-ethnyl-dUTP (5-EdUTP) or conventional dTTP that were subsequently reacted with a Cy3 fluorophore-azide molecule (N3-Cy3). Shaded boxes indicate the presence of the indicated species. [Figure 4] FIG. 1 shows a negative of a photograph of an agarose gel visualized using UV light after ethidium bromide staining. The agarose gel shows functionalized single-stranded oligonucleotide products of the invention (containing 420 nucleotides) containing: (A) 2'-fluoro-2'-deoxyuridine 5'-triphosphate (2'F-dUTP); (B) 2'-deoxythymidine-5'-O-(1-thiotriphosphate) (αthiol-dTTP); and (C) 2-dNTP alpha S nucleotides (alpha S-dATP, alpha S-dTTP, alpha S-dCTP, alpha S-dTTP and alpha S-dNTP mixture) made using various relative amounts of functionalized nucleotides. The right panel in A and B shows lanes corresponding to 100% functionalized nucleotides overexposed to show bands corresponding to RCA products. The right panel in C shows lanes corresponding to alpha S-dNTP mixture overexposed to show bands corresponding to RCA products. [Diagram 5]1 shows the negative of a photo of an agarose gel visualized using UV light after ethidium bromide staining. The agarose gel shows the oligonucleotides produced by the present invention subjected to various concentrations of DNase I, (A) shows the reaction product of the oligonucleotide containing only conventional nucleotides (natural ODN); (B) shows the reaction product of the oligonucleotide containing 2'-fluoro-2'-deoxyuridine-5'-triphosphate functionalized nucleotide (2'F-dUTP); and (C) shows the reaction product of the oligonucleotide containing 2'-deoxythymidine-5'-O-(1-thiotriphosphate) (S-ODN). [Figure 6] 1 shows a negative of a photograph of a denaturing PAGE gel visualized using UV light after SybrGold staining, showing functionalized single-stranded oligonucleotide products of the invention containing 5-vinyl-2'-deoxyuridine-5'-triphosphate (5-vinyl-dUTP) made using various relative amounts of 5-vinyl-dUTP nucleotides, i.e., 25%, 50%, 75% and 100%. [Figure 7] 1 shows a negative of a photograph of a denaturing PAGE gel visualized using UV light after SybrGold staining, which shows functionalized single-stranded oligonucleotide products of the invention containing 4-thiothymidine-5'-triphosphate (4-thio-dTTP) made using various relative amounts of 5-vinyl-dUTP nucleotides, i.e., 25%, 50%, 75% and 100%. [Figure 8] FIG. 1 shows an annotated version of the pseudogene sequence used to generate oligonucleotides having sequences corresponding to SEQ ID NOs: 1-13. Sequences recognized by the cleavage and nicking enzymes, the hairpin sequence and the final oligonucleotide sequence are identified. [Figure 9]2'-azido-dATP structure (A) and negative photographs of denaturing PAGE gels (B and C) visualized using UV light after SybrGold staining. The PAGE gels show functionalized single-stranded oligonucleotide products of the invention containing various relative amounts of 2'-azido-dATP nucleotides, i.e., 25%, 50%, 75% and 100%, and 2'-azido-dATP generated using phi29 DNA polymerase (B) or Bst DNA polymerase (C). [Figure 10] FIG. 1 shows the structure of biotin-16-aminoallyl-2′-dUTP (A) and a negative of a photograph of a denaturing PAGE gel visualized using UV light after SybrGold staining (B). The PAGE gel shows various relative amounts of biotin-16-AA-dUTP nucleotides, i.e., 25%, 50%, 75%, and 100%, as well as functionalized single-stranded oligonucleotide products of the invention containing biotin-16-aminoallyl-2′-dUTP generated using phi29 DNA polymerase. [Figure 11] Structures of 5'-bromo-2'-deoxyuridine-5'-triphosphate nucleotide (A) and 5'-propynyl-2'-deoxycytidine-5'-triphosphate nucleotide (B); negatives of photographs of denaturing PAGE gels (C, D, E and F) visualized using UV light after SybrGold staining. The PAGE gels show functionalized single-stranded oligonucleotide products of the invention containing various relative amounts of each functionalized nucleotide, i.e., 25%, 50%, 75% and 100%, and 5'-Br-dUTP (C and E) or 5'-propynyl-dCTP (D and F) generated using phi29 DNA polymerase or Bst DNA polymerase. [Figure 12]2 shows the structure of 2'-O-methyladenosine-5'-triphosphate nucleotide (A) and a negative of a photograph of a denaturing PAGE gel (B) visualized using UV light after SybrGold staining. The PAGE gel shows various relative amounts of 2'-OMe-ATP nucleotides, i.e., 25%, 50%, 75% and 100%, as well as functionalized single-stranded oligonucleotide products of the invention containing 2'-OMe-ATP generated using phi29 DNA polymerase. [Figure 13] Structure of LNA-adenosine-5'-triphosphate nucleotides (A) and negatives of photographs of denaturing PAGE gels (B and C) visualized using UV light after SybrGold staining. The PAGE gels show functionalized single-stranded oligonucleotide products of the invention containing various relative amounts of LNA-ATP nucleotides, i.e. 25%, 50%, 75% and 100%, and LNA-ATP generated using phi29 DNA polymerase (B) or Bst DNA polymerase (C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0173] Example 1 Enzymatic generation of single-stranded oligonucleotides containing fluorescent nucleotides. A single-stranded fluorescent oligonucleotide 378 nucleotides in length (SEQ ID NO:1) was enzymatically generated using phi29 DNA polymerase by incorporation of two different functionalized dATP nucleobases, one containing the fluorophore Cy3 (7-propargylamino-7-deaza-ATP-Cy3) and one containing the fluorophore ATTO-488 (7-propargylamino-7-deaza-ATP-ATTO-488).

[0174] A double-stranded circular DNA template containing SEQ ID NO:1 and a hairpin cleavage domain was prepared as described in Ducani et al., 2013, Nature Methods, pp. 647-652. The template (1 ng / μL) was nicked with Nb.BsrDI and Nt.BspQI (0.25 U / μL) and rolling circle amplification reactions (template DNA 0.1-0.25 ng / μL, phi29 DNA polymerase 0.25 U / μL, T4 gene32 0.1 μg) were performed several times with different ratios of natural dATP to functionalized dATP in each reaction (i.e., different relative amounts of functionalized dATP, 2%, 3% or 5%). The resulting RCA products were diluted 5-fold in deionized water and 1× digestion buffer (50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, 100 μg / mL BSA, pH 7.9 at 25° C.) and then digested with BtsCI restriction enzyme (0.5 U / μL) overnight at 50° C., and the digestion products were run on an agarose gel. Imaging was performed using emission wavelengths corresponding to the two fluorophores and by UV visualization after ethidium bromide staining.

[0175] The resulting images are shown in Figure 1A and Figure 1B for ATTO-488 and Cy3, respectively. It can be seen that by increasing the percentage of dATP-ATTO-488 nucleotides, oligonucleotides with higher fluorescence were obtained. However, when 5% of the dATP nucleotides were dATP-ATTO-488, the total amount of RCA product was reduced by approximately 60%.

[0176] Surprisingly, and in contrast to the use of dATP-ATTO-488, the percentage of dATP-Cy3 nucleotides present did not appear to affect the efficiency of phi29 DNA polymerase; single-stranded DNA products were visible at 5% dATP-Cy3 in the RCA mixture (Figure 1B). Furthermore, incorporation of modified nucleotides did not prevent or reduce the efficiency of the BtsCI restriction enzyme and, consequently, the release of the engineered hairpin containing the cleavage domain.

[0177] SEQ ID NO:1: [ka]

[0178] Example 2 Enzymatic generation of single-stranded oligonucleotides containing internalized alkyne groups, i.e., nucleotides with alkyne groups in the nucleobase A single-stranded oligonucleotide 420 nucleotides in length (SEQ ID NO:2) containing nucleotides bearing alkyne groups was enzymatically generated using phi29 DNA polymerase.

[0179] A double-stranded circular DNA template containing SEQ ID NO:2 and a hairpin cleavage domain was prepared as described in Ducani et al., 2013, Nature Methods, pp. 647-652. Nicking template RCA reactions were performed using conditions described in Example 1, but with increasing relative amounts of 5'-ethynyl-dUTP (5'-EdUTP), i.e., replacing dTTP with 5'-EdUTP such that the relative amount of 5'-EdUTP was 0% (control reaction), 25%, 50%, 75% or 100%. After amplification, the RCA products were digested with BtsCI restriction enzyme and loaded onto an agarose gel as described in Example 2.

[0180] The results in Figure 2A surprisingly show that even when 100% of the dTTP nucleotides were replaced with alkyne-functionalized dUTP, the RCA yield only dropped by 15-20%. Thus, Figure 2A also demonstrates that the RCA product was successfully and efficiently cleaved by BtsCI. The incorporation of the alkyne-functionalized dUTP nucleotide was confirmed by the fact that a lower mobility of the functionalized oligonucleotide was observed.

[0181] Additional experiments were performed substituting Bst DNA polymerase for phi29 DNA polymerase. Gel electrophoresis showed no change in amplification yields up to 50% of functionalized dUTP. The final product migrated compared to that of 25% of modified dUTP, confirming the incorporation of a modified nucleotide with a higher molecular weight than the corresponding natural nucleotide (dTTP) (Figure 2B).

[0182] SEQ ID NO:2: [ka]

[0183] Example 3 Click chemistry reaction for conjugating azide-fluorophores to single-stranded nucleotides containing internalized alkyne groups The successful incorporation of functionalized 5-ethynyl dUTP nucleotides into the oligonucleotides prepared in Example 2 was further demonstrated by performing a click chemistry reaction.

[0184] Functionalized oligonucleotides from reactions with 75% alkyne-functionalized dUTP nucleotides were incubated with Cy3-azide (50 μM). The click chemistry solution also contained copper sulfate (50 μM), sodium ascorbate (50 mM), and THPTA (250 μM) as catalysts. As a negative control, oligonucleotides made from the same template but using conventional dNTPs were also incubated with Cy3-azide. In addition, functionalized oligonucleotides containing internalized alkyne groups were also incubated in the absence of Cy3-azide. Reaction mixtures from the three reactions were run on an agarose gel and imaged (Figure 3). Fluorescent single-stranded oligonucleotides of the expected length were only observed for reactions containing functionalized oligonucleotides and fluorophore-azide. In addition, no visible DNA degradation due to the presence of copper sulfate was observed.

[0185] Example 4 Enzymatic generation of single-stranded oligonucleotides containing endonuclease-resistant nucleotides. Due to their ability to confer nuclease stability, both 2'-fluoro-2'-deoxyuridine-5'-triphosphate (2'F-dUTP) or 2'-deoxythymidine-5'-O-(1-thiotriphosphate) (phosphorothioate dTTP) have been used previously to modify DNA and RNA oligonucleotides for biomedical and therapeutic applications. These modified nucleotides were incorporated into single-stranded DNA oligonucleotides by RCA using the experimental scheme described above. Conventional dTTP nucleotides were replaced with increasing percentages of functionalized nucleotides from 0 to 100%. The tandem repeat RCA products were then digested by BtsCI restriction enzyme into separate 420 base single-stranded functionalized oligonucleotides (SEQ ID NO:2).

[0186] In experiments performed with 2′F-dUTP-functionalized nucleotides, comparable RCA yields were visible from 0 up to 75% functionalized nucleotides, with a sharp drop in yield observed at 100% functionalized nucleotides ( Figure 4 A).

[0187] In the phosphorothioate dTTP experiments, the RCA yield gradually decreased with increasing amounts of functionalized nucleotide, decreasing by approximately 65% ​​at 75% of functionalized nucleotides compared to the yield with conventional nucleotides alone (Figure 4B).

[0188] However, overexposure of the agarose gel showed that even with 100% of the nucleobases functionalized, functionalized single-stranded oligonucleotides were produced, see Figures 4A and 4B, right panels.

[0189] Other phosphorothioate dNTPs (denoted alphaS dNTPs) were tested in additional experiments, either sequentially or in combination (Figure 4C). Even in the final case, where 75% of all conventional nucleotides were replaced with the corresponding alphaS functionalized nucleotides, RCA products were synthesized and enzymatically cleaved to yield oligonucleotides visible on agarose gels.

[0190] The endonuclease resistance of functionalized oligonucleotides compared to control oligonucleotides made with conventional nucleotides alone was investigated. A 420 nt long control oligonucleotide made with conventional nucleotides alone, a 2'-F-dUTP functionalized oligonucleotide, and a phosphorothioate dTTP functionalized oligonucleotide (both made with 75% relative amount of functionalized nucleotides) were incubated with increasing concentrations of DNAse I (Figure 5A-C). The control oligonucleotide was completely digested with 18 mU / mL DNAse I, while both enzymatically made DNA oligonucleotides functionalized with 2'-F-dUTP and phosphorothioate dTTP were still visible on agarose gel after incubation with the same concentration of endonuclease.

[0191] Example 5 Enzymatic preparation of single-stranded oligonucleotides containing vinyl-containing nucleotides. Single-stranded DNA oligonucleotides (SEQ ID NO: 3-13) of 76-81 bases long functionalized with the thymidine analog 5-vinyl-2'-deoxyuridine-5'-triphosphate (5-vinyl-dUTP) were enzymatically generated by RCA reaction according to the experimental scheme described above. All of the oligonucleotides were encoded on a single pseudogene (SEQ ID NO: 16). The incorporation of such functionalized nucleotides into single-stranded oligonucleotides allows for the conjugation of tetrazine-like molecules to oligonucleotides using copper-free click chemistry. This alkene-tetrazine reaction may be fully orthogonal to the alkyne-azide click chemistry that has been previously performed.

[0192] Increasing the amount of functionalized nucleotide in the RCA reaction mixture compared to the conventional nucleotide dTTP resulted in successful incorporation of 5-vinyl-dUTP into the single-stranded RCA product and successful digestion of the hairpin structure. However, when the functionalized nucleotide completely replaced the conventional dTTP nucleotide, the activity levels of both phi29 DNA polymerase and the type II endonuclease used to cleave the RCA product were lower than in the absence of the functionalized nucleotide, and consequently, this resulted in a higher molecular weight band containing the undigested hairpin structure (Figure 6).

[0193] [Table 1]

[0194] Example 6 Enzymatic generation of single-stranded oligonucleotides containing thiolated nucleotides. Single-stranded DNA oligonucleotides functionalized with thiolated dTTP (4-thiothymidine-5'-triphosphate) were enzymatically generated by RCA reaction using the reaction scheme and templates (SEQ ID NOs: 3-13) described above. All of the oligonucleotides were encoded on a single pseudogene (SEQ ID NO: 16).

[0195] Incorporation of thiolated nucleotides into single-stranded oligonucleotides by phi29 DNA polymerase was highly successful at amounts of functionalized nucleotide that replaced the corresponding conventional nucleotide (dTTP) by up to 75%, i.e., a relative amount of 75% functionalized nucleotide (Figure 7).

[0196] The RCA products were digested with the type II restriction enzymes listed above; however, complete digestion of the functionalized RCA products required a 10-fold higher enzyme concentration than that used for RCA products containing only conventional nucleotides. When the conventional dTTP nucleotides were completely replaced with functionalized thiolated nucleotides, no single-stranded oligonucleotides were observed after treatment with type II restriction enzymes, but only a small accumulation of undigested RCA products was observed in the wells, suggesting that polymerase activity was also affected.

[0197] Example 7 Enzymatic generation of single-stranded oligonucleotides containing azidonucleotides. Single-stranded oligonucleotides 420 nucleotides in length (SEQ ID NO:2) containing increasing percentages of functionalized 2'-azido-dATP nucleotides replacing the corresponding conventional dATP nucleotides (Figure 9A) were enzymatically generated using phi29 DNA polymerase (Figure 9B) and Bst DNA polymerase (Figure 9C).

[0198] The high density of azide groups in the newly synthesized DNA strands allows for post-synthetic functionalization with alkyne molecules, either by Cu(I)-catalyzed Huisgen cycloaddition ("click" chemistry) or by strain-promoted [3+2] cycloaddition of azides with cycloalkynes, such as cyclooctyne or cyclononyne.

[0199] Two different polymerases with strand displacement activity were used in the amplification step: phi29 DNA polymerase or Bst DNA polymerase. Both polymerases are able to incorporate functionalized nucleotides into the amplification products, which were then digested and run on an agarose gel.

[0200] DNA products generated with phi29 DNA polymerase were visible in the gel at up to 75% of the modified nucleotides (Figure 9B), whereas Bst DNA polymerase products were visible at up to 100% (Figure 9C), although the Bst amplification buffer salts caused a smearing effect (even in the lane corresponding to 0% 2'-azido dATP). The functionalized nucleotides did not significantly affect the formation of hairpin structures that allowed successful incorporation and cleavage of the amplification products.

[0201] Example 8 Enzymatic generation of single-stranded oligonucleotides containing biotinylated nucleotides. A single-stranded oligonucleotide 420 nucleotides in length (SEQ ID NO:2) containing increasing percentages (25% to 100%) of functionalized biotin-16-aminoallyl-2'-dUTP replacing the corresponding conventional nucleotide dTTP (Figure 10A) was enzymatically generated using phi29 DNA polymerase (Figure 10B).

[0202] The incorporation of biotinylated nucleotides only slightly affected the DNA amplification reaction and, surprisingly, did not interfere with the formation of hairpin structures that allow cleavage of the RCA product, despite the potential for steric hindrance due to the large size of the functionalized nucleotides. The incorporation of multiple internal biotins into functionalized polynucleotides allows for conjugation with streptavidin-functionalized molecules.

[0203] Example 9 5. Enzymatic Creation of Single-Stranded Oligonucleotides Containing Modified Pyrimidines: 5-Bromo-2'-deoxyuridine-5'-triphosphate and 5-Propynyl-2'-deoxycytidine-5'-triphosphate Single-stranded oligonucleotides (SEQ ID NO:2) 420 nucleotides in length containing increasing percentages (25% to 100%) of unnatural 5-modified pyrimidines: 5-bromo-2'-deoxyuridine 5'-triphosphate (FIG. 11A) and 5-propynyl-2'-deoxycytidine-5'-triphosphate (FIG. 11B) replacing the corresponding conventional nucleotides dTTP and dCTP were enzymatically generated using phi29 DNA polymerase (FIG. 11C and FIG. 11D) and Bst DNA polymerase (FIG. 11E and FIG. 11F), respectively. Surprisingly, both functionalized nucleotides were successfully incorporated into the newly synthesized DNA sequence without affecting the formation of hairpin structures in the RCA products, thus allowing the cleavage reaction to occur.

[0204] Example 10 Enzymatic generation of single-stranded oligonucleotides containing 2'-O-methyl-ATP Single-stranded oligonucleotides (SEQ ID NO:2) of 420 nucleotides in length containing increasing percentages (25% to 100%) of 2'-O-methyl-ATP (Figure 12A) replacing the corresponding conventional nucleotide dATP were enzymatically generated using phi29 DNA polymerase (Figure 12B). Even with 100% of functionalized nucleotides, amplification products were still visible. This result contradicted previous studies that showed the inability of any known natural polymerase to efficiently accept these modified substrates (Romesberg, JACS 2004, 10.1021 / ja038525p).

[0205] In addition, no higher molecular weight undigested DNA bands were visible in the gel, indicating that the presence of functionalized nucleotides did not affect the formation of the hairpin structure and its digestion by the restriction enzyme, a surprising result considering the nuclease resistance conferred to the DNA molecule by the O-methyl group.

[0206] Example 11 Enzymatic generation of single-stranded oligonucleotides containing LNA-adenosine-5'-triphosphate A single-stranded oligonucleotide (SEQ ID NO:2) 420 nucleotides in length containing increasing percentages (25% to 100%) of LNA-adenosine-5'-triphosphate (Figure 13A) replacing the corresponding conventional nucleotide dATP was enzymatically generated using phi29 DNA polymerase (Figure 13B) and Bst DNA polymerase (Figure 13C).

[0207] Although LNA monomers structurally mimic RNA, even with 100% functionalized nucleotides, the efficiency of the polymerases, both phi29 DNA polymerase and Bst DNA polymerase, was not significantly affected. Moreover, the functionalized nucleotides did not interfere with the formation of hairpin structures that allow digestion of the amplification products.

Claims

1. 1. A method for making a single stranded functionalized oligonucleotide, the method comprising: (a) providing a circular DNA molecule comprising an oligonucleotide sequence flanked by a cleavage domain containing a sequence recognized by a cleavage enzyme, the cleavage domain comprising a sequence capable of forming a hairpin structure, the double-stranded portion of the hairpin structure comprising the sequence recognized by the cleavage enzyme; (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule of (a) as a template and one or more functionalized nucleotides (dNTPs); (c) enzymatically cleaving the product of the RCA reaction at the cleavage domain to release the single-stranded functionalized oligonucleotide; and (d) isolating or purifying the single-stranded functionalized oligonucleotide. A method comprising:

2. 2. The method of claim 1, wherein the circular DNA molecule is double-stranded, and the method comprises the additional step of cleaving one strand of the circular DNA molecule to prepare an RCA template before the RCA reaction is performed.

3. 3. The method of claim 1 or 2, wherein the functionalized dNTP is selected from the group consisting of a nucleotide containing an alkyne group, a fluorescently labeled nucleotide, a nucleotide containing a sterol group, a nucleotide containing a polyether group, a nucleotide containing a metal complex, a nucleotide containing a vinyl group, a nucleotide containing a thiol group, and a thione-oxidized nucleotide.

4. 4. The method of claim 1, wherein the functionalized dNTP is a nucleotide containing an alkyne group, an alkene group, an azide group, a halogen group, an O-methyl group, or a locked ribose sugar, preferably the functionalized dNTP is a nucleotide containing an alkyne group, a vinyl group, or an azide group.

5. 5. The method of claim 4, wherein the method further comprises conjugating a molecule or moiety to the oligonucleotide via the alkyne, vinyl or azide group.

6. 6. The method of claim 5, wherein the molecule or moiety is selected from the group consisting of fluorophores, sterols, polyethers, and metal complexes, and molecules containing thiol groups.

7. 7. The method of any one of claims 1 to 6, wherein the cleavage domain is immediately adjacent to the oligonucleotide sequence.

8. 8. The method according to any one of claims 1 to 7, wherein the cleavage enzyme is a type II restriction endonuclease, optionally a type IIS restriction endonuclease such as BseGI or BtsCI.

9. 9. The method of any one of claims 1 to 8, wherein the cleavage domains flanking the oligonucleotide sequences are identical.

10. 10. The method of claim 2, wherein the step of cleaving a single strand of the circular DNA molecule to prepare an RCA template comprises cleaving a single strand of the circular DNA molecule with a cleavage enzyme.

11. The method of claim 10, wherein the circular DNA molecule contains a sequence recognized by the cleavage enzyme.

12. 12. The method of claim 11, wherein the sequence recognized by the cleavage enzyme is between the cleavage domains that border the oligonucleotide sequence and is not within the oligonucleotide sequence.

13. 13. The method of any one of claims 10 to 12, wherein the cleavage enzyme is a nickase, and optionally the cleavage enzyme is Nb.BsrDI, Nt.BspQI, or a combination thereof.

14. 14. The method of any one of claims 1 to 13, wherein the RCA reaction uses phi29 DNA polymerase.

15. The method of any one of claims 1 to 13, wherein the RCA reaction uses Bst DNA polymerase.

16. 16. The method of any one of claims 1 to 15, wherein the circular DNA molecule comprises a plurality of oligonucleotide sequences, each of which is flanked by a cleavage domain.

17. The method of claim 16, wherein the oligonucleotide sequences are different.

18. Step (a) (i) cloning a linear DNA molecule comprising said oligonucleotide sequence flanked by cleavage domains into a DNA plasmid; (ii) amplifying the plasmid; (iii) excising the portion of the plasmid containing the DNA molecule that includes the oligonucleotide sequence flanked by cleavage domains; and (iv) circularizing the portion of the plasmid obtained in step (iii).

18. The method of any one of claims 1 to 17, comprising:

19. 20. The method of claim 18, wherein step (ii) comprises transfecting the DNA plasmid into a bacterium and propagating the bacterium.

20. 20. The method of claim 18 or 19, wherein the linear DNA molecule comprising the oligonucleotide sequence flanked by cleavage domains further comprises a 5' terminal region and a 3' terminal region each comprising a cleavage domain, and step (iii) comprises cleaving the cleavage domain within the terminal regions with a cleavage enzyme, optionally wherein the cleavage enzyme is BsmBI or Bsal.

21. 1. Use of a circular DNA molecule comprising an oligonucleotide sequence flanked by a cleavage domain in the generation of a single-stranded functionalized oligonucleotide, wherein the cleavage domain contains a sequence recognized by a cleavage enzyme, the cleavage domain comprises a sequence capable of forming a hairpin structure, and the double-stranded portion of the hairpin structure comprises the sequence recognized by the cleavage enzyme.

22. 22. The use according to claim 21, wherein the cleavage domain is as defined in any one of claims 7 to 9 and / or the DNA molecule is as defined in claim 16.

23. A kit for use in the method of any one of claims 1 to 20, comprising: (i) a circular DNA molecule comprising an oligonucleotide sequence flanked by a cleavage domain, the cleavage domain comprising or consisting of a sequence capable of forming a hairpin structure, the double-stranded portion of the hairpin structure comprising a sequence recognized by a cleavage enzyme; and (ii) optionally a functionalized dNTP as defined in claim 3 or 4; and (iii) one or more cleavage enzymes that cleave the cleavage domain of (i). Including the kit.

24. 24. The kit of claim 23, wherein the cleavage domain is as defined in any one of claims 7 to 9 and / or the DNA molecule is as defined in claim 16.

25. 21. The method of any one of claims 1 to 3 or 7 to 20, a method of making a pool of single-stranded functionalized oligonucleotides for use in single molecule fluorescence in situ hybridization (smFISH), wherein the functionalized nucleotides are fluorescently labeled nucleotides and each single-stranded functionalized oligonucleotide in the pool contains about 15 to 30, preferably about 20 to 25 nucleotides.

26. 21. The method of any one of claims 1 to 20, wherein the functionalized nucleotides are nucleotides that contain an azide group, an alkyne group, an alkene group, a nitrone group, a tetrazine group or a tetrazole group, or a combination thereof, and the method further comprises conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry, and wherein each single-stranded functionalized oligonucleotide in the pool contains about 15 to 30, preferably about 20 to 25 nucleotides.

27. 27. The method of claim 26, wherein the step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry is performed before the functionalized oligonucleotide is hybridized to a nucleic acid molecule comprising a target sequence.

28. 27. The method of claim 26, wherein the step of conjugating a fluorescent label to at least one functionalized nucleotide in each functionalized oligonucleotide by click chemistry is performed after the functionalized oligonucleotides are hybridized to a nucleic acid molecule comprising a target sequence.

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