Method for site-specific enzymatic labeling of nucleic acids in vitro by incorporation of non-natural nucleotides

Abstract

A method is provided for the site-specific enzymatic labeling of nucleic acids in vitro by incorporation of unnatural nucleotides. [Solution] Provided herein are unnatural nucleotide analogs that predominantly have hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated DNA replication and RNA polymerase-mediated RNA transcription. In this manner, the unnatural nucleobases can be site-specifically introduced into oligonucleotides (single- or double-stranded DNA or RNA), and the unnatural nucleobases can provide reactive linkers that can provide site-specific cleavage or undergo functionalization with cargo-bearing reagents by reaction with primary amino groups or by click chemistry with alkyne groups on the unnatural nucleobase linker.

Description

[Technical Field]

[0001] Government Support Statement This invention was made with government support under Grant No. GM060005 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61 / 863,649, filed August 8, 2013, which is incorporated by reference in its entirety. [Background technology]

[0003] Oligonucleotides and their uses have revolutionized biotechnology. However, oligonucleotides, including both DNA and RNA, each contain only the four natural nucleotides: adenosine (A), guanosine (G), cytosine (C), and thymine (T) in DNA and adenosine (A), guanosine (G), cytosine (C), and uridine (U) in RNA, which severely limits the potential functions and applications of oligonucleotides.

[0004] For example, the ability to sequence-specifically synthesize / amplify oligonucleotides (DNA or RNA) using polymerases by PCR or isothermal amplification systems (e.g., transcription with T7 RNA polymerase) has revolutionized biotechnology. In addition to all of the potential applications in nanotechnology, this has enabled a wide variety of new techniques, such as the in vitro evolution by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) of RNA and DNA aptamers and enzymes. For example, Oliphant AR, Brandl CJ & Struhl K (1989), Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 proteins, Mol. Cell Biol.,9:2944-2949;Tuerk C & Gold L (1990), Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science, 249:505-510; Ellington AD & Szostak JW (1990), In vitro selection of RNA molecules that bind specific ligands, Nature, 346:818-822.

[0005] Unfortunately, these applications are limited by the limited chemical / physical diversity present in the natural genetic alphabet (the four natural nucleotides A, C, G, and T in DNA and the four natural nucleotides A, C, G, and U in RNA). Accordingly, much interest has focused on technologies that allow the enzymatic synthesis / amplification of oligonucleotides that are site-specifically labeled with functional groups not present in the nucleotides of the natural genetic alphabet. Currently, available options for site-specific nucleic acid derivatization include solid-support-based chemical synthesis, combined chemical / enzymatic synthesis, and end-labeling procedures. End-labeling procedures are limited to oligonucleotide termini, and chemical synthesis is limited to short oligonucleotides (<200 nucleotides for DNA and <70 nucleotides for RNA). The problem is that enzymatic functionalization relies on enzymatic recognition of the modification of interest, which is not site-specific. Summary of the Invention [Problem to be solved by the invention]

[0006] The compositions and methods described herein are based on the in vitro expansion of the genetic alphabet, for example, using standard PCR or isothermal transcription methods to site-specifically incorporate unnatural nucleotides, as described herein, at any position in any DNA or RNA sequence, with a reactive linker suitable for reaction with a cargo reagent containing a complementary reactive group, or a linker having a cargo attached thereto.

[0007] In various embodiments, the linker is attached to the cargo at the nucleotide triphosphate stage, thereby allowing for the direct production of the desired site-specifically labeled oligonucleotide by an automated polynucleotide synthesis machine, such as a phosphoramidite polynucleotide synthesis machine.

[0008] In other embodiments, the linker contains a reactive center (e.g., a primary amine, an alkyne, a thiol, an aldehyde, or an azide), resulting in a reactive linker that allows for site-specific modification of a DNA or RNA oligonucleotide after its synthesis. This can be accomplished using a cargo reagent that contains a cargo (e.g., a molecule, a liposome, a nanoparticle, etc.) and a reactive group complementary to the reactive center of the reactive linker moiety. In some embodiments, the reactive center of the linker moiety is protected with a standard protecting group. Reaction of a nucleobase disclosed herein with a reactive linker (after deprotection, if necessary) and a cargo reagent that incorporates a reactive group complementary to the cargo into the reactive linker serves to provide a nucleobase bound to the cargo by a linked linker moiety.

[0009] The compositions of the present disclosure in various embodiments allow for the expansion of the limited repertoire of functionality of the four naturally occurring DNA and four naturally occurring RNA nucleotides, thereby including virtually any functionality desired for site-specific incorporation into DNA or RNA oligonucleotides, or into DNA or RNA analogs such as PNA or LNA. The cargo optionally includes functionality to alter molecular recognition (i.e., for aptamer generation), alter reactivity (including for catalysis), and / or allow for visualization and / or characterization studies (i.e., for diagnostic development). [Means for solving the problem]

[0010] Provided herein, in various embodiments, are compounds comprising a nucleobase analog of either of the following formulae β8a or β8b:

[0011] [ka] wherein each X is independently carbon or nitrogen; each R2 is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azido group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently sulfur, selenium, or oxygen; and the nucleobase analog is not 4TFP or 7TFP or a linker derivatization thereof.

[0012] Provided herein, in various embodiments, are compounds comprising a nucleobase analog of any of the following formulas:

[0013] [ka] wherein each X is independently carbon or nitrogen; each R1 is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a bonded linker to which a cargo is bonded; and each R2 is optional and, when present, independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azido group, a reactive linker comprising a cargo and a group of reactivity complementary to the reactive center. a reactive linker comprising a reactive center suitable for coupling to a cargo reagent, including a nucleobase analog, or a conjugated linker to which a cargo is attached, wherein each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently sulfur, selenium, or oxygen; and the nucleobase analog is not FIMO, MIMO, FEMO, PrMO, EMO, MEMO, IMO, MMO2, DMO, NMO, 5FM, 2OMe, TMO, FDMO, VMO, ZMO, CIMO, TfMO, CNMO, NaM, or QMO.

[0014] Throughout, wavy lines indicate points of attachment to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, or to an analog of a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, such as a locked ribose analog, a peptide group, etc. In some embodiments, the ribosyl, deoxyribosyl, or dideoxyribosyl moiety, or analog thereof, is in free form and is attached to a monophosphate, diphosphate, or triphosphate group, optionally includes an α-thiophosphate, β-thiophosphate, or γ-thiophosphate group, or is contained in RNA or DNA, or an RNA or DNA analog.

[0015] In some embodiments, when referring to either the ribosyl or deoxyribosyl moiety of an unnatural nucleobase X, dX, or (d)X provided herein, for example, dTPT3 or (d)TPT3 refers to a TPT3 nucleobase linked to one of the alternatives at the position indicated by the wavy line. Thus, the general term dX refers to a compound having a ribose or deoxyribose analog linked thereto as indicated by the wavy line. When specifically referring to a ribosyl nucleoside, the prefix "d" is dropped, i.e., TPT3 refers to the ribosyl form. When incorporated into a triphosphate polymerase substrate (i.e., TPT3TP, dTPT3TP), the nucleotide or linker-derivatized variants are considered to be incorporated into RNA or DNA oligonucleotides using RNA or DNA polymerases, respectively.

[0016] In some embodiments, the ribosyl, deoxyribosyl, or dideoxyribosyl analogs of the provided nucleoside analogs (e.g., β8a, β8b, α14a, α14b, α14c, α14d, α14e, α14f) contain a 2' functional group. Examples of functional groups include, but are not limited to, methoxy, halogen, -O-allyl, -O-methoxyethyl, primary amine, -O-dinitrophenol, -O-dinitrophenyl ether, alkyl, -O-alkyl, thiol, aminoethoxymethyl, aminopropoxymethyl, aminoethyl, cyanoethyl, and guanidinoethyl groups. In some embodiments, the ribosyl, deoxyribosyl, or dideoxyribosyl analogs contain a 4'-thio substitution (e.g., an oxygen in the sugar moiety is replaced with a sulfur).

[0017] In some embodiments, alkyl groups of nucleobase analogs include, but are not limited to, methyl, ethyl, propyl, and isopropyl groups. In some embodiments, halogen groups of nucleobase analogs include, but are not limited to, fluorine, chlorine, bromine, and iodine.

[0018] In some embodiments, the reactive linker of a nucleobase analog comprises a functional group including, but not limited to, alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, hydroxyl, carbonyl, aldehyde, haloformyl, carbonate, carboxylate, carboxyl, ester, methoxy, hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, methylenedioxy, orthocarbonate, carboxamide, primary amine, secondary amine, imide, azide, azo, cyanate, isocyanate, nitrate, nitrile, isonitrile, nitrosoxy, nitro, nitroso, pyridyl, sulfhydryl, sulfide, disulfide, sulfinyl, sulfo, thiocyanate, isothiocyanate, carbonothioyl, phosphino, phosphono, phosphate, borono, boronate, borino, borinate, and combinations thereof. In some embodiments, the reactive linker of the nucleobase analog comprises an amino group, an acetylene group, a thiol group, an aldehyde group, or an azide group.

[0019] In some embodiments, the ribosyl or deoxyribosyl moiety contains a triphosphate or α-thiotriphosphate group attached to its 5'-hydroxyl. In some embodiments, the ribosyl or deoxyribosyl moiety is incorporated into an RNA or DNA oligonucleotide strand, respectively, or the ribosyl or deoxyribosyl moiety, or an analog thereof, is incorporated into an RNA or DNA analog. In certain embodiments, the RNA or DNA analog is a peptide nucleic acid (PNA) or locked nucleic acid (LNA). In certain embodiments, the RNA or DNA analog is a bicyclic derivative. Bicyclic derivatives include, but are not limited to, 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), carbocyclic locked nucleic acid (CLNA), cyclohexene nucleic acid (CENA), and 2'-deoxy-2'-N,4'-C-ethylene-locked nucleic acid (AENA). In certain embodiments, the RNA or DNA analog is an acyclic derivative. In certain embodiments, the RNA or DNA analog is a non-locked nucleic acid (UNA). In certain embodiments, the RNA or DNA analog contains a pyranose ring instead of a ribose, hi certain embodiments, the RNA or DNA analog is an arabinonucleic acid (ANA) or a hexitol nucleic acid (HNA).

[0020] In some embodiments, the ribosyl or deoxyribosyl moiety, or analogs thereof, are substituted with protecting and activating groups suitable for use in an automated chemical oligonucleotide synthesis machine. One example of an automated chemical oligonucleotide synthesis machine is a phosphoramidite synthesis machine.

[0021] In some embodiments, at least one R2 of the nucleobase analog independently comprises a -C≡C-CH2NHR3 group, where R3 is hydrogen or an amino-protecting group. An example of an amino-protecting group is a dichloroacetyl group. In some embodiments, at least one R2 of the nucleobase analog independently comprises an acetylene group suitable for use in a click reaction with a cargo and a cargo reagent comprising an acetylene-reactive group. In some embodiments, at least one R2 of the nucleobase analog independently comprises a thiol group suitable for use in a reaction with a cargo and a cargo reagent comprising a thiol-reactive group. In some embodiments, at least one R2 of the nucleobase analog independently comprises an aldehyde group suitable for use in a reaction with a cargo and a cargo reagent comprising an aldehyde-reactive group. In some embodiments, at least one R2 of the nucleobase analog independently comprises an azide group suitable for use in a reaction with a cargo and a cargo reagent comprising an azide-reactive group. In some embodiments, at least one R2 of the nucleobase analog independently comprises -C≡C-(CH2)nC≡CH, where n is 1, 2, 3, 4, 5, or 6; or R2 is -C≡C-(CH2)n1-O(CH2)n2-C≡CH, where n1 and n2 are each independently 1, 2, or 3. In some embodiments, at least one R2 is independently a coupling linker attached to a cargo by reaction of an amino group with an amino-reactive group. An example of an amino-reactive group is an acylation group or an alkylation group, or an N-hydroxysuccinimide ester. In some embodiments, at least one R2 is independently a coupling linker attached to a cargo by reaction of an acetylene group with an acetylene-reactive group. An example of an acetylene-reactive group is an azide group. In certain embodiments, the acetylene group and the azide group are linked via a copper-catalyzed click reaction. In some embodiments, at least one R2 is independently a coupled linker attached to the cargo by reaction of a thiol with a thiol-reactive group. In some embodiments, at least one R2 is independently a coupled linker attached to the cargo by reaction of an aldehyde with an aldehyde-reactive group.In some embodiments, at least one R2 is independently a linker attached to a cargo by reaction of an azide with an azide-reactive group. An example of an azide-reactive group is a terminal alkyne or a strained cyclooctyne. In some embodiments, at least one R2 is independently hydrogen, and the compound comprises an α-thiotriphosphate group to which is linked a cargo reagent comprising a γ-bromo-α,β-unsaturated carbonyl, iodo, bromoacetyl, or aziridinyl sulfonamide group.

[0022] In some embodiments, the cargo of the nucleobase analog includes, but is not limited to, proteins, peptides, amino acids, oligonucleotides, small molecule pharmaceuticals, aliphatic groups, compounds containing photoreactive groups, compounds containing chemically reactive groups, compounds containing catalytic groups, compounds containing chloromethyl ketones, lipids, biotin, fluorescent compounds, fluorescence quenching compounds, liposomes, and nanoparticles.

[0023] Further, in various embodiments, the nucleobase analog TPT3

[0024] [ka] and derivatives and analogs thereof are provided.

[0025] Further, in various embodiments, the nucleobase analog FTPT3

[0026] [ka] and derivatives and analogs thereof are provided.

[0027] Further, in various embodiments, the nucleobase analog MMS

[0028] [ka] and derivatives and analogs thereof are provided.

[0029] Further, in various embodiments, the nucleobase analog DMS

[0030] [ka] and derivatives and analogs thereof are provided.

[0031] Further, in various embodiments, the present invention provides nucleobase analogues FEMS

[0032] [ka] and derivatives and analogs thereof are provided.

[0033] Further, in various embodiments, the present invention provides nucleobase analogs BrMS

[0034] [ka] and derivatives and analogs thereof are provided.

[0035] Further, in various embodiments, nucleobase analog IMSs are described herein.

[0036] [ka] and derivatives and analogs thereof are provided.

[0037] Provided herein, in some embodiments, are nucleobase pairs comprising a first nucleobase analog having either formula β9a or β9b, and a second nucleobase analog having either formula α15a or α15b,

[0038] [ka] wherein each X is independently carbon or nitrogen; each R1 is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R2 is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently oxygen, sulfur, or selenium; Acid-base pairs are dICS-dMMO2, dICS-2OMe, dSICS-dMMO2, dSICS-d2OMe, dSNICS-dMMO2, dSNICS-d2OMe, d4SICS-dMM O2,d4SICS-d2OMe,d5SICS-dFIMO,d5SICS-dMIMO,d5SICS-dFEMO,d5SICS-dPrMO,d5SICS-dEMO,d5SICS-d MEMO,d5SICS-dIMO,d5SICS-dMMO2,d5SICS-dDMO,d5SICS-dNMO,d5SICS-d5FM,d5SICS-d2OMe,d5SICS-d Not TMO, d5SICS-dFDMO, d5SICS-dVMO, d5SICS-dZMO, d5SICS-dCIMO, d5SICS-dTfMO, and d5SICS-dCNMO.

[0039] Provided herein, in some embodiments, is a nucleobase pair comprising a first nucleobase analog having the formula β9b and a second nucleobase analog having either the formula α15a or α15b,

[0040] [ka] wherein each X is independently carbon or nitrogen; each R is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R is optional and, when present, independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each Y is independently sulfur, oxygen, selenium, or a secondary amine; and each E is independently oxygen, sulfur, or selenium.

[0041] Provided herein, in some embodiments, are nucleobase pairs comprising a first nucleobase analog having either formula β9a or β9b, and a second nucleobase analog having either formula α16a or α16b,

[0042] [ka] wherein each X is independently carbon or nitrogen; each R is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently oxygen, sulfur, or selenium; and each E is independently sulfur or selenium.

[0043] The wavy line indicates the point of attachment to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, or to an analog of a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, such as a locked ribose analog, a peptide group, etc. In some embodiments, the ribosyl, deoxyribosyl, or dideoxyribosyl moiety, or analog thereof, is free and is attached to a monophosphate, diphosphate, or triphosphate group, optionally includes an α-thiophosphate, β-thiophosphate, or γ-thiophosphate group, or is contained in RNA or DNA, or an RNA or DNA analog.

[0044] In some embodiments, the alkynyl group is an ethynyl or propynyl group. In some embodiments, the alkyl group is a methyl, ethyl, propyl, or isopropyl group. In some embodiments, the halogen is fluorine, chlorine, bromine, or iodine.

[0045] In some embodiments, the ribosyl, deoxyribosyl, or dideoxyribosyl analog of the nucleobase pair comprises a 2' functional group. Exemplary functional groups include, but are not limited to, methoxy, halogen, -O-allyl, -O-methoxyethyl, primary amine, alkyl, -O-alkyl, thiol, -O-dinitrophenol, -O-dinitrophenyl ether, aminoethoxymethyl, aminopropoxymethyl, aminoethyl, cyanoethyl, and guanidinoethyl groups. In some embodiments, the ribosyl, deoxyribosyl, or dideoxyribosyl analog of the nucleobase pair comprises a 4'-thio substitution.

[0046] In some embodiments, the nucleobase pair comprises a nucleobase having the formula α15b. In some embodiments, the nucleobase pair comprises a nucleobase having the formula α15a. In some embodiments, the nucleobase pair comprises a nucleobase having the formula β9b. In some embodiments, the nucleobase pair comprises a nucleobase having the formula β9b, where each X is carbon, Y is sulfur, each R2 is hydrogen, and E is sulfur. In some embodiments, the nucleobase pair comprises a nucleobase having the formula α15b, where each X is carbon, each R2 is hydrogen, R1 is a methyl group, and E is oxygen. In some embodiments, the nucleobase pair comprises a first nucleobase analog having the formula β9a and a second nucleobase analog having the formula α16a. In some embodiments, the nucleobase pair comprises a first nucleobase analog having the formula β9a and a second nucleobase analog having the formula α16b. In some embodiments, the nucleobase pair comprises a first nucleobase analog having the formula β9b and a second nucleobase analog having the formula α16a. In some embodiments, the nucleobase pair comprises a first nucleobase analog having the formula β9b and a second nucleobase analog having the formula α16b.

[0047] Provided herein, in certain embodiments, is a nucleobase pair comprising a first nucleobase analog having the formula β9b and a second nucleobase analog having the formula β9b,

[0048] [ka] wherein each X is independently carbon or nitrogen; each R2 is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a linked linker to which a cargo is bonded; each Y is independently sulfur, oxygen, selenium, or a secondary amine; and each E is independently oxygen, sulfur, or selenium. In some embodiments, the nucleobase pairs are homo-nucleobase pairs.

[0049] Provided herein, in certain embodiments, is a nucleobase pair comprising a first nucleobase analog having the formula α16a and a second nucleobase analog having the formula α16a,

[0050] [ka] wherein each X is independently carbon or nitrogen; each R is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; and each E is independently sulfur or selenium. In some embodiments, the nucleobase pair is a homo-nucleobase pair.

[0051] Provided herein, in certain embodiments, is a nucleobase pair comprising a first nucleobase analog having the formula α16b and a second nucleobase analog having the formula α16b,

[0052] [ka] wherein each X is independently carbon or nitrogen; each R is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; and each E is independently sulfur or selenium. In some embodiments, the nucleobase pair is a homo-nucleobase pair.

[0053] Provided herein, in certain embodiments, is a method for producing a nucleobase analog comprising a first nucleobase analog having any of the formulas β9a, β9b, α15a, α15b, α16a, or α16b, and a nucleobase analog selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl, and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, 3-deazaguanine, 3-deazaadenine, and the like. ne), 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthine-9-yl(I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifluoromethyluracil and cytosine; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidines, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-Dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl derivatives of adenine and guanine, 6-azapyrimidine, 6-azo-uracil, 6-azo-cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine, 7-deaza-py ...pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrimidine, 6-azo-pyrim Aza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and guanines; N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase duplex formation stability, universal nucleic acids, hydrophobic nucleic acids, promisucuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines, phenoxazine cytidine ([5 ,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one, G-clamp, phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2, 3-d]pyrimidin-2-one, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid, wybutoxosine and a second nucleobase selected from the group consisting of uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine, and those in which the purine or pyrimidine base is replaced with a heterocycle.

[0054] Base pairs containing one or more unnatural nucleobases are exemplified by the dTPT3PA-dNaM unnatural base pair (i.e., the pair formed between dTPT3PA and dNaM; Figures 1A and 1B). Additionally, the orthogonal reactivity of the various reactive centers / linkers (i.e., phosphorothioates, amines, and alkynes) developed allows for the selective placement of various moieties on the same oligonucleotide (DNA or RNA). Another composition is further exemplified by the dTPT3PA-dMMO2pCO unnatural base pair, where, in various embodiments, the alkynyl group of dMMO2pCO is used to attach one functional group via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and the free amine, after deprotection of dTPT3PA, is used to attach a different functional group via N-hydroxysuccinimide (NHS) coupling.

[0055] While several other unnatural base pairs have been reported and derivatized with linkers, the linker-derivatized pairs described herein, dTPT3PA-dNaM, d5SICSCO-dNaM, and dTPT3PA-dMMO2pCO (Figure 2), are not only more efficient but also better at replicating within DNA and transcribing into RNA. In particular, dTPT3PA-dNaM replicates well and is therefore suitable for use in practicing the methods disclosed and claimed herein.

[0056] Provided herein are unnatural base pairs comprising dTPT3, and in some examples, dTPT3 is derivatized with a linker. Unnatural base pairs comprising dTPT3 or linker-derivatized dTPT3 (e.g., dTPT3PA) include, but are not limited to, dTPT3-MMS, dTPT3-DMS, dTPT3-FEMS, dTPT3-BrMS, dTPT3-IMS, dTPT3-dDMN, dTPT3-d4OMe, dTPT3-dIQ, dTPT3-d2MN, dTPT3-d3OMe, dTPT3-dQL, dTPT3-d2Np,dTPT3-dDM4,dTPT3-dDM,dTPT3-dBEN,dTPT3-d3FB,dTPT3-dMM1,dTPT3-dMMO1,dTPT3-dDM2,dTPT 3-dDM5,dTPT3-d2Py,dTPT3-d5MPy,dTPT3-dEPy,dTPT3-d3MPy,dTPT3-d34DMPy,dTPT3-d45DMPy,dTPT3-d4MPy, dTPT3-d35DMPy,dTPT3-dBP,dTPT3-dBTp,dTPT3-dBF,dTPT3-dIN,dTPT3-dTp,dTPT3-dB Tz,dTPT3-dMTp,dTPT3-dAM,dTPT3-dMAN,dTPT3-dDMMAN,dTPT3-dADM,dTPT3-dMMAN,dT PT3-dTOK588,dTPT3-dTOK576,dTPT3-dTOK587,dTPT3-dTOK586,dTPT3-dTOK580,dTPT3 -dPhMO,dTPT3-dPyMO1,dTPT3-PyMO2,dTPT3-dPMO1,dTPT3-dPMO2,dTPT3-dPMO3,dTPT3 -dFuMO1,dTPT3-dFuMO2,dTPT3-TpMO1,dTPT3-dTpMO2,dTPT3-dFIMO,dTPT3-dIMO,dTPT 3-dMIMO,dTPT3-dMEMO,dTPT3-dFEMO,dTPT3-dPrMO,dTPT3-dMMO2,dTPT3-d2OMe,dTPT3 -dDMO,dTPT3-dTMO,dTPT3-dNMO,dTPT3-dNOPy,dTPT3-d5FM,dTPT3-dNAM,dTPT3-dAMO1 ,dTPT3-dAPy,dTPT3-dAMO2,dTPT3-dMAPy,dTPT3-dAMO3,dTPT3-dDMAPy,dTPT3-dFDMO,Nucleobase analogs complementary to dTPT3 include dTPT3-dVMO, dTPT3-dQMO, dTPT3-dZMO, dTPT3-dCIMO, dTPT3-dTfMO, and dTPT3-CNMO, where the dTPT3 complementary base is either linker-derivatized or not (e.g., dMMO2pCO). dTPT3 is illustrated in Figure 9 as a β analog. An example of a linker-derivatized dTPT3 is illustrated in Figure 2, where in some instances, R is a reactive linker containing a reactive center suitable for binding to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dTPT3 include, but are not limited to, the α analogs or linker-derivatized α analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, dTPT3 or linker-derivatized dTPT3 is base paired with dTPT3 or linker-derivatized dTPT3 to form a homo-nucleobase pair. In some embodiments, dTPT3 or linker-derivatized dTPT3 is base paired with a β-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or a derivatized nucleobase thereof. In some embodiments, the linker moiety is protected with a protecting group (e.g., dTPT3PA). In some embodiments, the linker moiety is not protected with a protecting group (e.g., dTPT3A), and in some instances, the protecting group has been removed.

[0057] Provided herein are unnatural base pairs comprising dMMS, and in some examples, the dMMS is derivatized with a linker. Unnatural base pairs comprising dMMS or linker-derivatized dMMS (e.g., dMMSPA) include, but are not limited to, d7AI-dMMS, dM7AI-dMMS, dImPy-dMMS, dP7AI-dMMS, dPPP-dMMS, d8Q-dMMS, dICS-dMMS, dPICS-dMMS, dMICS-dMMS, d4MICS-dMMS, d5MICS-dMMS, dNICS-dMMS, dONICS-dMMS, d7OFP-dMMS, d7OTP-dMMS, d4OTP-dMMS, dPYR-dMMS, d4MP-dMMS, d3MP-dMMS, dPPYR- Nucleobase analogs complementary to dMMS include dMMS, dMOP-dMMS, d4MOP-dMMS, dSICS-dMMS, dSNICS-dMMS, d5SICS-dMMS, d4SICS-dMMS, dTPT1-dMMS, dTPT2-dMMS, dFPT1-dMMS, and dFTPT3-dMMS, where the dMMS complementary base is linker-derivatized or not (e.g., pFTPT3pA). dMMS is illustrated in Figure 11 as an α14a analog. In some embodiments, linker-derivatized dMMS contains a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dMMS include, but are not limited to, β analogs or linker-derivatized β analogs illustrated in Figures 9, 12, and 13. In some embodiments, dMMS or linker-derivatized dMMS is base paired with dMMS or linker-derivatized dMMS to form a homo-nucleobase pair. In some embodiments, dMMS or linker-derivatized dMMS is base paired with an alpha nucleobase, including but not limited to, any of the nucleobases illustrated in Figures 8, 10, 11, and 15, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0058] Provided herein are unnatural base pairs comprising dDMS, and in some examples, the dDMS is linker-derivatized. Unnatural base pairs comprising dDMS or linker-derivatized dDMS (e.g., dDMSPA) include, but are not limited to, d7AI-dDMS, dM7AI-dDMS, dImPy-dDMS, dP7AI-dDMS, dPPP-dDMS, d8Q-dDMS, dICS-dDMS, dPICS-dDMS, dMICS-dDMS, d4MICS-dDMS, d5MICS-dDMS, dNICS-dDMS, dONICS-dDMS, d7OFP-dDMS, d7OTP-dDMS, d4OTP-dDMS, dPYR-dDMS, d4MP-dDMS, d3MP-dDMS, and dPPY Nucleobase analogs complementary to dDMS include R-dDMS, dMOP-dDMS, d4MOP-dDMS, dSICS-dDMS, dSNICS-dDMS, d5SICS-dDMS, d4SICS-dDMS, dTPT1-dDMS, dTPT2-dDMS, dFPT1-dDMS, and dFTPT3-dDMS, where the dDMS complementary base is either linker-derivatized or not (e.g., pFTPT3pA). dDMS is illustrated in Figure 11 as an α14a analog. In some embodiments, the linker-derivatized dDMS comprises a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dDMS include, but are not limited to, β analogs or linker-derivatized β analogs illustrated in Figures 9, 12, and 13. In some embodiments, dDMS or linker-derivatized dDMS is base paired with dDMS or linker-derivatized dDMS to form a homo-nucleobase pair. In some embodiments, dMMS or linker-derivatized dMMS is base paired with an alpha nucleobase, including but not limited to, any of the nucleobases illustrated in Figures 8, 10, 11, and 15, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0059] Provided herein are unnatural base pairs comprising dFEMS, and in some instances, dFEMS is linker-derivatized. Unnatural base pairs comprising dFEMS or linker-derivatized dFEMS (e.g., dFEMSPA) include, but are not limited to, d7AI-dFEMS, dM7AI-dFEMS, dImPy-dFEMS, dP7AI-dFEMS, dPPP-dFEMS, d8Q-dFEMS, dICS-dFEMS, dPICS-dFEMS, dMICS-dFEMS, d4MICS-dFEMS, d5MICS-dFEMS, dNICS-dFEMS, dONICS-dFEMS, d7OFP-dFEMS, d7OTP-dFEMS, d4O These include TP-dFEMS, dPYR-dFEMS, d4MP-dFEMS, d3MP-dFEMS, dPPYR-dFEMS, dMOP-dFEMS, d4MOP-dFEMS, dSICS-dFEMS, dSNICS-dFEMS, d5SICS-dFEMS, d4SICS-dFEMS, dTPT1-dFEMS, dTPT2-dFEMS, dFPT1-dFEMS, and dFTPT3-dFEMS, where the dFEMS complementary base is either linker-derivatized or not (e.g., pFTPT3pA). dFEMS is illustrated in FIG. 11 as an α14a analog. In some embodiments, the linker-derivatized dFEMS comprises a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dFEMS include, but are not limited to, β-analogs or linker-derivatized β-analogs illustrated in Figures 9, 12, and 13. In some embodiments, dFEMS or linker-derivatized dFEMS is base paired with dFEMS or linker-derivatized dFEMS to form a homo-nucleobase pair. In some embodiments, dFEMS or linker-derivatized dFEMS is base paired with an α-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 8, 10, 11, and 15, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group.In some embodiments, the linker moiety is not protected with a protecting group, and in some instances the protecting group has been removed.

[0060] Provided herein are unnatural base pairs comprising dBrMS, and in some examples, the dBrMS is linker-derivatized. Unnatural base pairs comprising dBrMS or linker-derivatized dBrMS (e.g., dBrMSPA) include, but are not limited to, d7AI-dBrMS, dM7AI-dBrMS, dImPy-dBrMS, dP7AI-dBrMS, dPPP-dBrMS, d8Q-dBrMS, dICS-dBrMS, dPICS-dBrMS, dMICS-dBrMS, d4MICS-dBrMS, d5MICS-dBrMS, dNICS-dBrMS, dONICS-dBrMS, d7OFP-dBrMS, d7OTP-dBrMS, d Examples of suitable α14a analogs include 4OTP-dBrMS, dPYR-dBrMS, d4MP-dBrMS, d3MP-dBrMS, dPPYR-dBrMS, dMOP-dBrMS, d4MOP-dBrMS, dSICS-dBrMS, dSNICS-dBrMS, d5SICS-dBrMS, d4SICS-dBrMS, dTPT1-dBrMS, dTPT2-dBrMS, dFPT1-dBrMS, and dFTPT3-dBrMS, where the dBrMS complementary base is linker-derivatized or not (e.g., pFTPT3pA). dBrMS is illustrated in FIG. 11 as an α14a analog. In some embodiments, the linker-derivatized dBrMS comprises a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-attached conjugated linker. Nucleobase analogs complementary to dBrMS include, but are not limited to, β-analogs or linker-derivatized β-analogs illustrated in Figures 9, 12, and 13. In some embodiments, dBrMS or linker-derivatized dBrMS is base paired with dBrMS or linker-derivatized dBrMS to form a homo-nucleobase pair. In some embodiments, dBrMS or linker-derivatized dBrMS is base paired with an α-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 8, 10, 11, and 15, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group.In some embodiments, the linker moiety is not protected with a protecting group, and in some instances the protecting group has been removed.

[0061] Provided herein are unnatural base pairs comprising dIMS, and in some instances, the dIMS is linker-derivatized. Unnatural base pairs comprising dIMS or linker-derivatized dIMS (e.g., dIMSPA) include, but are not limited to, d7AI-dIMS, dM7AI-dIMS, dImPy-dIMS, dP7AI-dIMS, dPPP-dIMS, d8Q-dIMS, dICS-dIMS, dPICS-dIMS, dMICS-dIMS, d4MICS-dIMS, d5MICS-dIMS, dNICS-dIMS, dONICS-dIMS, d7OFP-dIMS, d7OTP-dIMS, d 4OTP-dIMS, dPYR-dIMS, d4MP-dIMS, d3MP-dIMS, dPPYR-dIMS, dMOP-dIMS, d4MOP-dIMS, dSICS-dIMS, dSNICS-dIMS, d5SICS-dIMS, d4SICS-dIMS, dTPT1-dIMS, dTPT2-dIMS, dFPT1-dIMS, dFTPT3-dIMS, where the dIMS complementary bases are either linker-derivatized or not (e.g., pFTPT3pA). dIMS are illustrated in Figure 11 as α14a analogs. In some embodiments, the linker-derivatized dIMS contain a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-attached coupled linker. Nucleobase analogs complementary to a dIMS include, but are not limited to, β-analogs or linker-derivatized β-analogs illustrated in Figures 9, 12, and 13. In some embodiments, a dIMS or linker-derivatized dIMS is base paired with a dIMS or linker-derivatized dIMS to form a homo-nucleobase pair. In some embodiments, a dIMS or linker-derivatized dIMS is base paired with an α-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 8, 10, 11, and 15, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0062] Provided herein are unnatural base pairs containing dICS, and in some examples, dICS is linker-derivatized. Unnatural base pairs containing dICS or linker-derivatized dICS (e.g., dICSPA) include, but are not limited to, dICS-dFIMO, dICS-dMIMO, dICS-dFEMO, dICS-dPrMO, dICS-dEMO, dICS-dMEMO, dICS-dIMO, dICS-dDMO, dICS-dNMO, dICS-d5FM, dICS-dTMO, dICS-dFDMO, dICS-dVMO, dICS-dZMO, dICS-dCIMO, dICS-dTfMO, dICS-dCNMO, dICS-dNAM, and dICS-dQMO, where the dICS complementary base is either linker-derivatized or not (e.g., dDMOpCO, dDMOpCC). dICS is illustrated as a β2 analog in Figure 9. In some embodiments, the linker-derivatized dICS comprises a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dICS include, but are not limited to, the α-analogs or linker-derivatized α-analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, dICS or linker-derivatized dICS is base-paired with dICS or linker-derivatized dICS to form a homo-nucleobase pair. In some embodiments, dICS or linker-derivatized dICS is base-paired with a β-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0063] Provided herein are unnatural base pairs comprising dPICS, and in some instances, the dPICS is linker-derivatized. Unnatural base pairs comprising dPICS or linker-derivatized dPICS (e.g., dPICSPA) include, but are not limited to, dPICS-dFIMO, dPICS-dMIMO, dPICS-dFEMO, dPICS-dPrMO, dPICS-dEMO, dPICS-dMEMO, dPICS-dIMO, dPICS-dMMO2, dPICS-dDMO, dPICS-dNMO, dPICS-d5FM, dPICS-d2OMe, dPICS-dTMO, dPICS-dFDMO, dPICS-dVMO, dPICS-dZMO, dPICS-dCIMO, dPICS-dTfMO, dPICS-dCNMO, dPICS-dNAM, dPICS-dQMO, wherein the dPICS complementary base is or is not linker-derivatized (e.g., dDMOpCO, dDMOpCC). dPICS is illustrated in Figure 9 as a β2 analog. In some embodiments, linker-derivatized dPICS comprises a functional group R, where R is a reactive linker comprising a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dPICS include, but are not limited to, the α analogs or linker-derivatized α analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, dPICS or linker-derivatized dPICS is base-paired with dPICS or linker-derivatized dPICS to form a homo-nucleobase pair. In some embodiments, dPICS or linker-derivatized dPICS is base-paired with a β nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0064] Provided herein are unnatural base pairs comprising dMICS, and in some examples, the dMICS is linker-derivatized. Unnatural base pairs comprising dMICS or linker-derivatized dMICS (e.g., dMICSPA) include, but are not limited to, dMICS-dFIMO, dMICS-dMIMO, dMICS-dFEMO, dMICS-dPrMO, dMICS-dEMO, dMICS-dMEMO, dMICS-dIMO, dMICS-dMMO2, dMICS-dDMO, dMICS-dNMO, dMICS-d5FM, dMICS-d2OMe, dMICS-dTMO, dMICS-dFDMO, dMICS-dVMO, dMICS-dZMO, dMICS-dCIMO, dMICS-dTfMO, dMICS-dCNMO, dMICS-dNAM, dMICS-dQMO, wherein the dMICS complementary base is or is not linker-derivatized (e.g., dDMOpCO, dDMOpCC). dMICS is illustrated in Figure 9 as a β2 analog. In some embodiments, the linker-derivatized dMICS comprises a functional group R, where R is a reactive linker comprising a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dMICS include, but are not limited to, the α analogs or linker-derivatized α analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, dMICS or linker-derivatized dMICS is base paired with dMICS or linker-derivatized dMICS to form a homo-nucleobase pair. In some embodiments, dMICS or linker-derivatized dMICS is base paired with a β nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0065] Provided herein are unnatural base pairs comprising d4MICS, and in some examples, d4MICS is derivatized with a linker. Unnatural base pairs comprising d4MICS or linker-derivatized d4MICS (e.g., d4MICSPA) include, but are not limited to, d4MICS-dFIMO, d4MICS-dMIMO, d4MICS-dFEMO, d4MICS-dPrMO, d4MICS-dEMO, d4MICS-dMEMO, d4MICS-dIMO, d4MICS-dMMO2, d4MICS-dDMO, d4MICS-dNMO, d4MI Examples of d4MICS-d5FM include CS-d5FM, d4MICS-d2OMe, d4MICS-dTMO, d4MICS-dFDMO, d4MICS-dVMO, d4MICS-dZMO, d4MICS-dCIMO, d4MICS-dTfMO, d4MICS-dCNMO, d4MICS-dNAM, and d4MICS-dQMO, where the d4MICS complementary bases are either linker-derivatized or not (e.g., dDMOPCO, dDMOPCC). d4MICS is illustrated as a β2 analog in Figure 9. In some embodiments, the linker-derivatized d4MICS comprises a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-attached conjugated linker. Nucleobase analogs complementary to d4MICS include, but are not limited to, α-analogs or linker-derivatized α-analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, d4MICS or linker-derivatized d4MICS is base paired with d4MICS or linker-derivatized d4MICS to form a homo-nucleobase pair. In some embodiments, d4MICS or linker-derivatized d4MICS is base paired with a β-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0066] Provided herein are unnatural base pairs comprising d5MICS, and in some examples, d5MICS is derivatized with a linker.Unnatural base pairs comprising d5MICS or linker-derivatized d5MICS (e.g., d5MICSPA) include, but are not limited to, d5MICS-dFIMO, d5MICS-dMIMO, d5MICS-dFEMO, d5MICS-dPrMO, d5MICS-dEMO, d5MICS-dMEMO, d5MICS-dIMO, d5MICS-dMMO2, d5MICS-dDMO, d5MICS-dNMO, d5MI Examples of d5MICS-d2OMe include CS-d5FM, d5MICS-d2OMe, d5MICS-dTMO, d5MICS-dFDMO, d5MICS-dVMO, d5MICS-dZMO, d5MICS-dCIMO, d5MICS-dTfMO, d5MICS-dCNMO, d5MICS-dNAM, and d5MICS-dQMO, where the d5MICS complementary bases are either linker-derivatized or not (e.g., dDMOPCO, dDMOPCC). d5MICS is illustrated as a β2 analog in Figure 9. In some embodiments, the linker-derivatized d5MICS comprises a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to d5MICS include, but are not limited to, α analogs or linker-derivatized α analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, d5MICS or linker-derivatized d5MICS is base paired with d5MICS or linker-derivatized d5MICS to form a homo-nucleobase pair. In some embodiments, d5MICS or linker-derivatized d5MICS is base paired with a β nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0067] Provided herein are unnatural base pairs comprising dNICS, and in some instances, the dNICS are linker-derivatized. Unnatural base pairs comprising dNICS or linker-derivatized dNICS (e.g., dNICSPA) include, but are not limited to, dNICS-dFIMO, dNICS-dMIMO, dNICS-dFEMO, dNICS-dPrMO, dNICS-dEMO, dNICS-dMEMO, dNICS-dIMO, dNICS-dDMO, dNICS-dNMO, dNICS-d5FM, dNICS-dTMO, dNICS-dFDMO, dNICS-dVMO, dNICS-dZMO, dNICS-dCIMO, dNICS-MMO2, dNICS-2OMe, dNICS-dTfMO, dNICS-dCNMO, dNICS-dNAM, dNICS-dQMO, wherein the dNICS complementary base is or is not linker-derivatized (e.g., dDMOpCO, dDMOpCC). dNICS is illustrated in Figure 9 as a β3 analog. In some embodiments, the linker-derivatized dNICS comprises a functional group R, where R is a reactive linker comprising a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dNICS include, but are not limited to, the α analogs or linker-derivatized α analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, the dNICS or linker-derivatized dNICS is base paired with the dNICS or linker-derivatized dNICS to form a homo-nucleobase pair. In some embodiments, the dNICS or linker-derivatized dNICS is base paired with a β nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0068] Provided herein are unnatural base pairs comprising dONICS, and in some examples, the dONICS is linker-derivatized. Unnatural base pairs comprising dONICS or linker-derivatized dONICS (e.g., dONICSPA) include, but are not limited to, dONICS-dFIMO, dONICS-dMIMO, dONICS-dFEMO, dONICS-dPrMO, dONICS-dEMO, dONICS-dMEMO, dONICS-dIMO, dONICS-dDMO, dONICS-dNMO, dONICS-d5FM, dONICS-dTMO, dONICS-dFDMO, dONICS-dVMO, dONICS-dZMO, dONICS-dCIMO, dONICS-MMO2, dONICS-2OMe, dONICS-dTfMO, dONICS-dCNMO, dONICS-dNAM, dONICS-dQMO, wherein the dONICS complementary base is or is not linker-derivatized (e.g., dDMOpCO, dDMOpCC). dONICS is illustrated in Figure 9 as a β3 analog. In some embodiments, linker-derivatized dONICS comprises a functional group R, where R is a reactive linker comprising a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dONICS include, but are not limited to, α analogs or linker-derivatized α analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, dONICS or linker-derivatized dONICS is base paired with dONICS or linker-derivatized dONICS to form a homo-nucleobase pair. In some embodiments, dONICS or linker-derivatized dONICS is base paired with a β nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0069] Provided herein are unnatural base pairs comprising dSICS, and in some instances, the dSICS ​​is linker-derivatized. Unnatural base pairs containing dSICS ​​or linker-derivatized dSICS ​​(e.g., dSICSPA) include, but are not limited to, dSICS-dFIMO, dSICS-dMIMO, dSICS-dFEMO, dSICS-dPrMO, dSICS-dEMO, dSICS-dMEMO, dSICS-dIMO, dSICS-dDMO, dSICS-dNMO, dSICS-d5FM, dSICS-dTMO, dSICS-dFDMO, dSICS-dVMO, dSICS-dZMO, dSICS-dCIMO, dSICS-dTfMO, dSICS-dCNMO, dSICS-dNAM, and dSICS-dQMO, where the dSICS ​​complementary base is or is not linker-derivatized (e.g., dDMOpCO, dDMOpCC). dSICS ​​is illustrated as a β5 analog in Figure 9. In some embodiments, the linker-derivatized dSICS ​​comprises a functional group R, where R is a reactive linker comprising a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dSICS ​​include, but are not limited to, the α-analogs or linker-derivatized α-analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, the dSICS ​​or linker-derivatized dSICS ​​is base paired with the dSICS ​​or linker-derivatized dSICS ​​to form a homo-nucleobase pair. In some embodiments, the dSICS ​​or linker-derivatized dSICS ​​is base paired with a β-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0070] Provided herein are unnatural base pairs comprising dSNICS, and in some examples, the dSNICS is linker-derivatized. Unnatural base pairs containing dSNICS or linker-derivatized dSNICS (e.g., dSNICSPA) include, but are not limited to, dSNICS-dFIMO, dSNICS-dMIMO, dSNICS-dFEMO, dSNICS-dPrMO, dSNICS-dEMO, dSNICS-dMEMO, dSNICS-dIMO, dSNICS-dDMO, dSNICS-dNMO, dSNICS-d5FM, dSNICS-dTMO, dSNICS-dFDMO, dSNICS-dVMO, dSNICS-dZMO, dSNICS-dCIMO, dSNICS-dTfMO, dSNICS-dCNMO, dSNICS-dNAM, and dSNICS-dQMO, where the dSNICS complementary base is or is not linker-derivatized (e.g., dDMOpCO, dDMOpCC). dSNICS is illustrated as a β5 analog in Figure 9. In some embodiments, the linker-derivatized dSNICS comprises a functional group R, where R is a reactive linker comprising a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to dSNICS include, but are not limited to, the α-analogs or linker-derivatized α-analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, the dSNICS or linker-derivatized dSNICS is base-paired with the dSNICS or linker-derivatized dSNICS to form a homo-nucleobase pair. In some embodiments, the dSNICS or linker-derivatized dSNICS is base-paired with a β-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0071] Provided herein are unnatural base pairs comprising d4SICS, and in some instances, the d4SICS is linker-derivatized. Unnatural base pairs comprising d4SICS or linker-derivatized d4SICS (e.g., d4SICSPA) include, but are not limited to, d4SICS-dFIMO, d4SICS-dMIMO, d4SICS-dFEMO, d4SICS-dPrMO, d4SICS-dEMO, d4SICS-dMEMO, d4SICS-dIMO, d4SICS-dDMO, d4SICS-dNMO, d4SICS-d5FM, d4SICS-dTMO, d4SICS-dFDMO, d4SICS-dVMO, d4SICS-dZMO, d4SICS-dCIMO, d4SICS-dTfMO, d4SICS-dCNMO, d4SICS-dNAM, d4SICS-dQMO, wherein the d4SICS complementary base is or is not linker-derivatized (e.g., dDMOPCO, dDMOPCC). d4SICS is illustrated in Figure 9 as a β5 analog. In some embodiments, linker-derivatized d4SICS comprises a functional group R, where R is a reactive linker comprising a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to d4SICS include, but are not limited to, the α analogs or linker-derivatized α analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, d4SICS or linker-derivatized d4SICS is base paired with d4SICS or linker-derivatized d4SICS to form a homo-nucleobase pair. In some embodiments, d4SICS or linker-derivatized d4SICS is base paired with a β nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0072] Provided herein are unnatural base pairs comprising d7OFP, and in some examples, the d7OFP is derivatized with a linker. Unnatural base pairs comprising d7OFP or linker-derivatized d7OFP (e.g., d7OFPPA) include, but are not limited to, d7OFP-dFIMO, d7OFP-dMIMO, d7OFP-dFEMO, d7OFP-dPrMO, d7OFP-dEMO, d7OFP-dMEMO, d7OFP-dIMO, d7OFP-dMMO2, d7OFP-dDMO, d7OFP-dNMO, d7OFP The d7OFP complementary bases may be linker-derivatized or non-linker-derivatized (e.g., dDMOPCO, dDMOPCC). d7OFP is illustrated as a β5 analog in Figure 9. In some embodiments, the linker-derivatized d7OFP comprises a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to d7OFP include, but are not limited to, the α-analogs or linker-derivatized α-analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, d7OFP or linker-derivatized d7OFP is base paired with d7OFP or linker-derivatized d7OFP to form a homo-nucleobase pair. In some embodiments, d7OFP or linker-derivatized d7OFP is base paired with a β-nucleobase, including, but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0073] Provided herein are unnatural base pairs comprising d7OTP, and in some examples, the d7OTP is linker-derivatized. Unnatural base pairs comprising d7OTP or linker-derivatized d7OTP (e.g., d7OTPPA) include, but are not limited to, d7OTP-dFIMO, d7OTP-dMIMO, d7OTP-dFEMO, d7OTP-dPrMO, d7OTP-dEMO, d7OTP-dMEMO, d7OTP-dIMO, d7OTP-dMMO2, d7OTP-dDMO, d7OTP-dNMO, and d7OTP-d5F. Nucleobase analogs complementary to d7OTP include M, d7OTP-d2OMe, d7OTP-dTMO, d7OTP-dFDMO, d7OTP-dVMO, d7OTP-dZMO, d7OTP-dCIMO, d7OTP-dTfMO, d7OTP-dCNMO, d7OTP-dNAM, and d7OTP-dQMO, where the d7OTP complementary base is linker-derivatized or not (e.g., dDMOPCO, dDMOPCC). d7OTP is illustrated as a β5 analog in Figure 9. In some embodiments, linker-derivatized d7OTP comprises a functional group R, where R is a reactive linker containing a reactive center suitable for coupling to a cargo reagent, or R is a cargo-linked conjugated linker. Nucleobase analogs complementary to d7OTP include, but are not limited to, the α analogs or linker-derivatized α analogs illustrated in Figures 8, 10, 11, and 15. In some embodiments, d7OTP or linker-derivatized d7OTP is base paired with d7OTP or linker-derivatized d7OTP to form a homo-nucleobase pair. In some embodiments, d7OTP or linker-derivatized d7OTP is base paired with a β-nucleobase, including but not limited to, any of the nucleobases illustrated in Figures 9, 12, and 13, or derivatized nucleobases thereof. In some embodiments, the linker moiety is protected with a protecting group. In some embodiments, the linker moiety is not protected with a protecting group, and in some instances, the protecting group has been removed.

[0074] Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase TPT3 and a second nucleobase MMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase TPT3 and a second nucleobase DMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase TPT3 and a second nucleobase FEMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase TPT3 and a second nucleobase BrMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase TPT3 and a second nucleobase IMS.

[0075] Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase FTPT3 and a second nucleobase MMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase FTPT3 and a second nucleobase DMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase FTPT3 and a second nucleobase FEMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase FTPT3 and a second nucleobase BrMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase FTPT3 and a second nucleobase IMS.

[0076] Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase 5SICS and a second nucleobase MMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase 5SICS and a second nucleobase DMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase 5SICS and a second nucleobase FEMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase 5SICS and a second nucleobase BrMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase 5SICS and a second nucleobase IMS.

[0077] Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase SICS and a second nucleobase MMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase SICS and a second nucleobase DMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase SICS and a second nucleobase FEMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase SICS and a second nucleobase BrMS. Further provided herein, in various embodiments, is a nucleobase pair comprising a first nucleobase SICS and a second nucleobase IMS.

[0078] Further, in various embodiments, double-stranded oligonucleotide duplexes are provided herein, in which a first oligonucleotide strand comprises an unnatural nucleobase (e.g., a nucleobase analog) disclosed herein, and a second, complementary oligonucleotide strand comprises a complementary base pairing at its complementary base pairing site. For example, for dTPT3, the second complementary oligonucleotide strand comprises dNaM, dDMO, or dMMO2, or a linker-derivatized analog, at the complementary base pairing site. Thus, the pairing interaction between the first oligonucleotide strand and the second oligonucleotide strand comprises a specific nucleobase pairing interaction between the unnatural nucleobase moiety provided herein and the complementary nucleobase, which may be a natural or unnatural nucleobase.

[0079] In some embodiments, double-stranded oligonucleotide duplexes are provided herein, wherein a first oligonucleotide strand comprises a compound having the formula β8a or β8b, and a second, complementary oligonucleotide strand comprises a complementary base-paired nucleobase at its complementary base-pairing site. In some embodiments, the complementary base-paired nucleobase is a compound having the formula α14a, α14b, α14c, α14d, α14e, or α14f. In some embodiments, complementary base-pairing nucleobases include, but are not limited to, cytosine, guanine, adenine, thymine, uracil, 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, 3-deazaguanine, 3-deazaadenine, 4-thio-uracil, 4-thio- Thymine, uracil-5-yl, hypoxanthine-9-yl(I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifluoromethyluracil and cytosine; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidines, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-Dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl derivatives of adenine and guanine, 6-azapyrimidine, 6-azo-uracil, 6-azo-cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine , 7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and guanines; N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d ]pyrimidin-2-one, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetate , wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine, as well as those in which the purine or pyrimidine base is replaced with a heterocycle.

[0080] In some embodiments, double-stranded oligonucleotide duplexes are provided herein, wherein a first oligonucleotide strand comprises a compound having the formula α14a, α14b, α14c, α14d, α14e, or α14f, and a second, complementary oligonucleotide strand comprises a complementary base-pairing nucleobase at its complementary base-pairing site. In some embodiments, the complementary base-pairing nucleobase is a compound having the formula β8a or β8b. In some embodiments, complementary base-pairing nucleobases include, but are not limited to, cytosine, guanine, adenine, thymine, uracil, 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, 3-deazaguanine, 3-deazaadenine, 4-thio-uracil, 4-thio- Thymine, uracil-5-yl, hypoxanthine-9-yl(I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifluoromethyluracil and cytosine; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidines, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-Dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl derivatives of adenine and guanine, 6-azapyrimidine, 6-azo-uracil, 6-azo-cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine , 7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and guanines; N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d ]pyrimidin-2-one, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetate , wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine, as well as those in which the purine or pyrimidine base is replaced with a heterocycle.

[0081] In some embodiments, at least one R2 of the nucleobases in the double-stranded oligonucleotide duplex is a linker linked to a cargo. In some embodiments, the cargo is a reporter group, protein, or compound containing catalytic functionality.

[0082] In some embodiments, a first oligonucleotide strand comprising a nucleobase analog disclosed herein is prepared by synthesis using a nucleobase comprising a reactive linker followed by attachment of a cargo reagent to the first oligonucleotide strand, or alternatively, a first oligonucleotide strand is prepared by synthesis using a nucleobase comprising an attached linker attached to a cargo.

[0083] In some embodiments, the double-stranded oligonucleotide duplex has a first strand that includes dTPT3 or a derivative thereof and a second strand that includes dNaM, dDMO, or dMMO2, or a derivative thereof, at the complementary base-pairing site.

[0084] Further provided herein, in various embodiments, are methods for performing site-specific functionalization of a double-stranded oligonucleotide duplex, the methods comprising incorporating into a first oligonucleotide strand an unnatural nucleobase comprising a reactive linker that includes a reactive center, wherein the nucleobase has any of the following formulae: α14a, α14b, α14c, α14d, α14e, α14f, β8a, β8b, β9a, β9b, α15a, α15b, α16a, or α16b; then incorporating into a second strand complementary to the first strand, the first strand and the second strand being complementary to the double-stranded oligonucleotide. The method includes synthesizing under conditions to form a duplex, wherein the second strand comprises a nucleobase complementary to the unnatural nucleobase at a site-specific complementary position as described herein, and then contacting the double-stranded oligonucleotide incorporating the unnatural nucleobase comprising a reactive linker moiety with a cargo reagent comprising a reactive group complementary to the cargo under conditions suitable for reaction of the reactive linker with the complementary reactive group to obtain a linked linker, thereby obtaining a double-stranded oligonucleotide functionalized with a cargo attached by a linked linker.

[0085] Further provided herein, in various embodiments, are methods for performing site-specific functionalization of double-stranded oligonucleotide duplexes, the methods comprising incorporating into a first oligonucleotide strand an unnatural nucleobase comprising a reactive linker containing a reactive center, wherein the nucleobase is selected from the group consisting of d5SICSCO, d5SICSCC, dDMOCO, dDMOCC, dMMO2pCO, dMMO2pCC, dTPT3, dTPT3A, dTPT3PA, dTPT3CO, and dTPT3CC, followed by synthesizing a second strand complementary to the first strand. wherein the second strand comprises a nucleobase complementary to the unnatural nucleobase at a site-specific complementary position described herein under conditions such that the first and second strands form a double-stranded oligonucleotide duplex; and then contacting the double-stranded oligonucleotide incorporating the unnatural nucleobase comprising a reactive linker moiety with a cargo reagent comprising a reactive group complementary to the cargo under conditions suitable for reaction of the reactive linker with the complementary reactive group to obtain a linked linker and a double-stranded oligonucleotide functionalized with the cargo attached by the linked linker.

[0086] In one embodiment, the linker is attached to the cargo after the corresponding 5' triphosphate is incorporated into the DNA or RNA oligonucleotide using a DNA or RNA polymerase (after deprotection, if necessary). In another embodiment, a second oligonucleotide complementary to the first strand is synthesized, the second strand containing an unnatural nucleotide at a position complementary to the unnatural nucleotide in the first strand, and then the resulting double-stranded oligonucleotide is reacted with a cargo-bearing reagent that selectively reacts with the reactive center of the reactive linker to obtain a cargo-bearing functionalized double-stranded oligonucleotide (e.g., DNA / DNA, DNA / RNA, or RNA / RNA).

[0087] Further provided herein, in various embodiments, are structures comprising the formula N1-Zx-N2, where N1 is a nucleotide or analog thereof or a terminal phosphate group, N2 is a nucleotide or analog thereof or a terminal hydroxyl group, Z is a compound having any of the formula α14a, α14b, α14c, α14d, α14e, α14f, β8a, β8b, β9a, β9b, α15a, α15b, α16a, or α16b, and x is an integer from 1 to 20. In some embodiments, the structure is an oligonucleotide. In some embodiments, the oligonucleotide is a ribonucleic acid or a deoxyribonucleic acid. In some embodiments, the oligonucleotide is an aptamer or a nucleic acid-based sensor. In some embodiments, the oligonucleotide is a molecular beacon. In some embodiments, the oligonucleotide is an RNA analog or a DNA analog.

[0088] Further provided herein, in various embodiments, is a method for identifying a nucleic acid aptamer comprising at least one compound described herein (e.g., α14a, α14b, α14c, α14d, α14e, α14f, β8a, β8b, β9a, β9b, α15a, α15b, α16a, α16b) having an enhanced desired property against a target molecule, the method comprising: a) preparing a candidate mixture of single-stranded nucleic acid aptamers, wherein each nucleic acid aptamer of the candidate mixture of aptamers comprises at least one compound described herein (e.g., α14a, α14b, α14c, α14d, α14e, α14f, β8a, β8b, β9a, β9b, α15a, α15b, α16a, α16b). The method includes: (a) selecting one or more nucleic acid aptamers from the candidate mixture; (b) selecting one or more nucleic acid aptamers from the candidate mixture; (c) selecting one or more nucleic acid aptamers having a desired property for the target molecule; and (d) amplifying the one or more nucleic acid aptamers in vitro to obtain one or more nucleic acid aptamers having an enhanced desired property for the target molecule. In some embodiments, the method further includes (e) repeating steps (c) and (d). In some embodiments, the single-stranded nucleic acid aptamer is selected from the group consisting of single-stranded DNA and single-stranded RNA. In some embodiments, the desired property is binding affinity for the target. In some embodiments, the desired property is target binding activity. In some embodiments, the desired property is catalytic activity. In some embodiments, the desired property is inhibitory activity, activating activity, or a modification of inhibitory or activating activity. In some embodiments, the desired property is structure-switching activity or a modification of structure-switching activity. In some embodiments, the desired property is cooperative activity. In some embodiments, the desired activity is enhanced cellular efficacy.

[0089] Further, in certain embodiments, the present specification provides an aptamer comprising a compound having any of the following formulas: α14a, α14b, α14c, α14d, α14e, α14f, β8a, β8b, β9a, β9b, α15a, α15b, α16a, α16b. [Brief explanation of the drawings]

[0090] [Figure 1] Pairings of dTPT3-dNaM, d5SICS-dNaM, d5SICS-dMMO2, and d5SICS-dDMO in DNA or RNA are shown. [Figure 2] Linker-derivatized nucleotides dTPT3R, d5SICSR, dMMO2R, dMMO2pR, dDMOR, dNaMpR, dNaMpR, dFEMO, and dEMO are shown, where R = 3-aminopropyn-1-yl (denoted as A, e.g., dTPT3A); R = dichloroacetyl-3-aminopropyn-1-yl (denoted as PA); R = 4-oxahepta-1,6-diyn-1-yl (denoted as CO); and R = hepta-1,6-diyn-1-yl (denoted as CC). [Figure 3] 1 shows an overview of phosphorothioate-based post-synthetic site-specific labeling strategies. [Figure 4] An overview of amino-based post-synthetic site-specific labeling strategies is provided. Linker-modified nucleotides can also be directly incorporated into template DNA using standard solid-phase synthesis of oligonucleotides and the corresponding phosphoramidites. [Figure 5] We provide an overview of click chemistry-based post-synthetic site-specific labeling strategies. Linker-modified nucleotides can also be directly incorporated into template DNA using standard solid-phase synthesis of oligonucleotides and the corresponding phosphoramidites. [Figure 6]Representative data illustrating post-amplification labeling of DNA analyzed by streptavidin (SA) gel shift are shown. The fast-migrating band corresponds to dsDNA, while the slow-migrating band corresponds to a 1:1 complex between dsDNA and streptavidin. (A) Labeling efficiency is 72% with d5SICSPA-dNaM and 80% with dTPT3PA-dNaM. (B) Labeling efficiencies are 6%, 84%, and 56% with d5SICSPA-dNaM at the 1st (primer 1: unnatural base pair at position 1), 9th (primer 2: unnatural base pair at position 11), and 11th (primer 3: unnatural base pair at position 9) positions. The corresponding labeling efficiencies with dTPT3PA-dNaM are 72%, 94%, and 81%. [Figure 7] 1 shows gel electrophoresis data confirming full-length transcription of RNA containing linker-derivatized analogs of 5SICS or MMO2. [Figure 8-1] The 12 groupings of alpha nucleobase analogs, alpha1-alpha12, are shown. [Figure 8-2] The 12 groupings of alpha nucleobase analogs, alpha1-alpha12, are shown. [Figure 9] Six groupings of β nucleobase analogs, β1-β6, are shown. [Figure 10]

[0023] Figure 1 shows two groupings of alpha nucleobase analogs, alpha 13 and alpha 14, each X is independently carbon or nitrogen, each R1 is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached, each R2 is optional and, when present, independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azido group, a cargo comprising a cargo and a group of reactivity complementary to the reactive center, A reactive linker comprising a reactive center suitable for bonding to a reagent, a coupled linker having a cargo attached thereto, wherein each R is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide group, a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, a coupled linker having a cargo attached thereto, wherein each Y is independently sulfur, oxygen, selenium, or secondary amine, and each E is independently sulfur, selenium, or oxygen. [Figure 11] 1 shows examples of α14a nucleobase analogs, including linker-derivatized nucleobase analogs, MMSpCO, and MMSPA. [Figure 12]

[0023] Figure 1 shows two groupings of β nucleobase analogs β7 and β8, each X is independently carbon or nitrogen; each R2 is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently sulfur, selenium, or oxygen; and R is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached. [Figure 13] 1 shows examples of linker-derivatized nucleobase analogs of β8. [Figure 14-1] The percentage of unnatural base pairs retained in the DNA after amplification during six rounds of screening is shown. [Figure 14-2] The percentage of unnatural base pairs retained in the DNA after amplification during six rounds of screening is shown. [Figure 14-3] The percentage of unnatural base pairs retained in the DNA after amplification during six rounds of screening is shown. [Figure 15]each R1 is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R2 is optional and, when present, is independently hydrogen, an alkyl, an alkenyl, an alkynyl, a methoxy, a methanethiol, a methaneseleno, a halogen, a cyano, an azide, a nitro group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently oxygen, sulfur, or selenium; and each E2 is independently sulfur or selenium. [Figure 16] each R1 is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R2 is optional and, when present, is independently hydrogen, an alkyl, an alkenyl, an alkynyl, a methoxy, a methanethiol, a methaneseleno, a halogen, a cyano, an azide, a nitro group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each Y is independently sulfur, oxygen, selenium, or a secondary amine; and each E is independently oxygen, sulfur, or selenium. DETAILED DESCRIPTION OF THE INVENTION

[0091] As used herein, the phrases "under conditions suitable to provide" or "under conditions suitable to obtain" in the context of synthetic methods refer to reaction conditions, such as time, temperature, solvent, and reactant concentration, that are within the ordinary skill of an experimenter to vary and result in a useful amount or yield of reaction product. The desired reaction product need not be the only reaction product, nor need the starting material be completely consumed, provided that the desired reaction product can be isolated or otherwise further utilized.

[0092] By "chemically feasible" is meant a compound or bonding arrangement in which the commonly understood rules of organic structure are not violated; for example, it is understood that structures within the definition of the claims that include, under certain circumstances, a non-naturally occurring pentavalent carbon atom are not claimed. In all of these embodiments, the structures disclosed herein are intended to include only "chemically feasible" structures; for example, structures depicted with variable atoms or groups, and any recited structures that are not chemically feasible are not intended to be disclosed or claimed herein.

[0093] An "analog" of a chemical structure, as used herein, refers to a chemical structure that retains substantial similarity to the parent structure, but may not be readily synthetically derived from the parent structure. In some embodiments, a nucleotide analog is an unnatural nucleotide. In some embodiments, a nucleoside analog is an unnatural nucleoside. Related chemical structures that are readily synthetically derived from the parent chemical structure are referred to as "derivatives."

[0094] Accordingly, "DNA analog" or "RNA analog," as used herein, refers to DNA- or RNA-like polymers, such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), phosphorothioates, etc., which are well known in the art. DNA and RNA analogs, like DNA and RNA, can be synthesized on automated synthesizers, for example, using phosphoramidite chemistry or other chemical approaches suitable for use on synthesizers.

[0095] DNA includes, but is not limited to, cDNA and genomic DNA. DNA may be attached by covalent or non-covalent means to other biomolecules, including, but not limited to, RNA and peptides. RNA includes coding RNA, such as messenger RNA (mRNA). In some embodiments, the RNA is rRNA, RNAi, snoRNA, microRNA, siRNA, snRNA, exRNA, piRNA, long ncRNA, or any combination or hybrid thereof. In some examples, the RNA is a component of a ribozyme. DNA and RNA can be in any form, including, but not limited to, linear, circular, supercoiled, single-stranded, and double-stranded.

[0096] The term "amino-protecting group" or "amino-protected," as used herein, refers to a group intended to protect an amino group against undesired reactions during synthetic procedures and which can be subsequently removed to reveal the amine. Commonly used amino-protecting groups are disclosed in Protective Groups in Organic Synthesis, Greene, TW; Wuts, PGM, John Wiley & Sons, New York, NY, (3rd Edition, 1999).Amino-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; alkoxy- or aryloxy-carbonyl groups (which form urethanes containing the protected amine). Benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl oxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitro These include alkoxycarbonyl or aryloxycarbonyl groups (forming urethanes with protected amines) such as cyclophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl; and silyl groups such as trimethylsilyl and the like.Amine-protecting groups also include cyclic amino-protecting groups, such as phthaloyl and dithiosuccinimidyl, which incorporate the amino nitrogen into a heterocycle. Common amino-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, Alloc, Teoc, benzyl, Fmoc, Boc, and Cbz. Further protecting groups include methyl carbamate, 9-fluorenylmethyl carbamate, 2,2,2-trichloroethyl carbamate, t-butyl carbamate, 2-(trimethylsilyl)ethyl carbamate, allyl carbamate, benzyl carbamate, m-nitrophenyl carbamate, trifluoroacetamide, benzylamine, allylamine, and tritylamine. Protecting groups also include formamide, acetamide, trifluoroacetamide, p-toluenesulfonyl, trifluoromethanesulfonyl, trimethylsilylethanesulfonamide, and tert-butylsulfonyl. It is well within the skill of one in the art to select and use an amino protecting group appropriate for the synthetic task at hand.

[0097] Analogs of DNA and RNA include PNA (peptide nucleic acid) and LNA (locked nucleic acid) analogs.

[0098] Peptide nucleic acids (PNAs) are synthetic DNA / RNA analogs in which a peptide-like backbone replaces the sugar-phosphate backbone of DNA or RNA. PNA oligomers exhibit high binding strength and great specificity when binding to complementary DNA, with PNA / DNA base mismatches being more destabilizing than similar mismatches in DNA / DNA duplexes. This binding strength and specificity also applies to PNA / RNA duplexes. PNAs are resistant to enzymatic degradation and are not readily recognized by either nucleases or proteases. PNAs are also stable over a wide pH range. Further Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). “Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide”, Science 254 (5037): 1497-500. doi:10.1126 / science.1962210. PMID 1962210; and, Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, and Nielsen PE (1993), “PNA Hybridizes to Complementary Oligonucleotides Obeying the Watson-Crick Hydrogen Bonding Rules”. Nature 365 (6446): 566-8. doi:10.1038 / 365566a0.PMID See 7692304.

[0099] Locked nucleic acids (LNA) are modified RNA nucleotides in which the ribose moiety of the LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in a 3'-endo (North) conformation, often found in A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in oligonucleotides whenever needed. Such oligomers can be chemically synthesized and are commercially available. The locked ribose conformation enhances base stacking and backbone preorganization. For example, Kaur, H; Arora, A; Wengel, J; Maiti, S (2006), “Thermodynamic, Counterion, and Hydration Effects for the Incorporation of Locked Nucleic Acid Nucleotides into DNA Duplexes”, Biochemistry 45 (23): 7347-55. doi:10.1021 / bi060307w. PMID 16752924; Owczarzy R.; You Y., Groth CL, Tataurov AV (2011), “Stability and mismatch discrimination of locked nucleic acid-DNA duplexes.”, Biochem. 50 (43): 9352-9367. doi:10.1021 / bi200904e. PMC 3201676. PMID 21928795; Alexei A. Koshkin; Sanjay K. Singh, Poul Nielsen, Vivek K.Rajwanshi, Ravindra Kumar, Michael Meldgaard, Carl Erik Olsen, Jesper Wengel (1998), “LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerization, and unprecedented nucleic acid recognition”, Tetrahedron 54(14):3607–3 doi:10.1016 / S0040-4020(98)00094-5; and, Satoshi Obika; Daishu Nanbu, Yoshiyuki Hari, Ken-ichiro Morio, Yasuko In, Toshimasa Ishida, Takeshi Imanishi (1997), “Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. 38(50):8735–8 doi:10.1016 / S0040-4039(97)10322-7.

[0100] Molecular beacons or molecular beacon probes are oligonucleotide hybridization probes that can detect the presence of specific nucleic acid sequences in a homogeneous solution. Molecular beacons are hairpin-shaped molecules containing an internally quenched fluorophore whose fluorescence is restored upon binding to the target nucleic acid sequence. For example, Tyagi S, Kramer FR (1996), “Molecular beacons: probes that fluoresce upon hybridization”, Nat Biotechnol. 14 (3): 303-8. PMID 9630890; Tapp I, Malmberg L, Rennel E, Wik M, Syvanen AC (2000 Apr), “Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5'-nuclease TaqMan assay and Molecular Beacon probes”, Biotechniques 28 (4): 732-8. PMID 10769752; and, Akimitsu Okamoto (2011), “ECHO probes: a concept of florescence control for practical nucleic acid sensing”, Chem. Soc. Rev. 40: 5815-5828.

[0101] In some embodiments, a nucleobase is generally the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring or modified, may bear no resemblance to natural bases, or may be synthesized, for example, by organic synthesis. In certain embodiments, a nucleobase includes any atom or group of atoms that can interact with a base of another nucleic acid, with or without hydrogen bonding. In certain embodiments, an unnatural nucleobase is not derived from a natural nucleobase. Note that an unnatural nucleobase does not necessarily have the essential properties, but is referred to as a nucleobase for simplicity. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase can be attached to either deoxyribose or ribose.

[0102] In some embodiments, a nucleoside is a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides with mimetic bases and / or sugars. Nucleosides also include nucleosides containing any type of substituent. A nucleoside can be a glycosidic compound formed by a glycosidic bond between a nucleobase and a reducing group on a sugar.

[0103] In some embodiments, a nucleotide is a compound in which the sugar moiety of the nucleoside forms an ester containing phosphate, more preferably a monophosphate, diphosphate, or triphosphate. The sugar moiety of such a nucleoside or nucleotide can be ribofuranosyl, 2'-deoxyribofuranosyl, or 2'-substituted ribofuranosyl having a substituent at the 2'-position. Similarly, the phosphate moiety can be a thiophosphate. That is, the sugar and phosphate moieties can be the same forms as those found in known nucleosides, nucleotides, or their derivatives. Ribonucleotides whose sugar moiety is ribofuranosyl can be used as members of RNA. Deoxyribonucleotides whose sugar moiety is deoxyribofuranosyl can be used as members of DNA. A nucleotide can be a nucleoside that further contains a phosphate linking group. A nucleotide can include a nucleoside containing a phosphate moiety.

[0104] A class of natural base pairs, exemplified by d5SICS-dNaM and d5SICS-dMMO2 (Figure 1), has been developed and shown by us to replicate (including using PCR) and be replicated and transcribed by a variety of natural polymerases with efficiency and fidelity approaching that of natural base pairs (Malyshev, DA; Seo, YJ; Ordoukhanian, P.; Romesberg, FE, PCR with an Expanded Genetic Alphabet. J. Am. Chem. Soc. 2009. 131 (41), 14620-14621; Seo, YJ; Matsuda, S.; Romesberg, FE, Transcription of an Expanded Genetic Alphabet. J. Am. Chem. Soc. 2009, 131 (14), 5046-5047; Lavergne T.; Degardin M.; Malyshev DA; Quach HT; Dhami (See also Seo, YJ, Malyshev, DA, Lavergne, T., Ordoukhanian, P., and Romesberg, FE, Expanding the scope of replicable unnatural DNA: Stepwise optimization of a predominantly hydrophobic base pair. J. Am. Chem. Soc. 2013, 135, 5408-5419; Seo, YJ, Malyshev, DA, Lavergne, T., Ordoukhanian, P., and Romesberg, FE, J. Am. Chem. Soc. 2011, 133, 19878.) These unnatural base pairs are formed between nucleotide analogs that have unnatural, predominantly hydrophobic nucleobases. The base pairs are shown in Figure 1, where each unnatural nucleotide is incorporated into the oligonucleotide at a complementary (i.e., paired) position, and the nucleobase is attached to the 1'-position of the ribosyl or 2'-deoxyribosyl moiety at the position indicated by the wavy line.The nucleobases themselves, as is often the case with fully natural nucleic acids, are incorporated into RNA or DNA by phosphate or phosphorothioate groups attached to the 3' and 5' hydroxyl groups of the ribosyl or deoxyribosyl groups, respectively. Pairing thus occurs as part of complementary bases, as is well known in the formation of double-stranded oligonucleotide duplex structures. In various embodiments, the unnatural nucleobase dTPT3 and its linker-derivatized variants are provided herein, which are contemplated to pair in a similar manner with the unnatural nucleobases dNaM, dMMO2, and dDMO (or their linker-derivatized variants).

[0105] We demonstrated that the unnatural nucleotides dTPT3 and dTPT3PA are efficiently incorporated into DNA by DNA polymerases opposite dNaM (Figure 2). Both dTPT3 and dTPT3PA (PA = dichloroacetyl-3-aminopropyn-1-yl) are expected to pair with dNaM, dDMO, or dMMO2, or their linker-derivatized analogs, as well as other linker-derivatized variants of dTPT3, including those with R = 3-aminopropyn-1-yl (dTPT3A), R = 4-oxahepat-1,6-diyn-1-yl (dTPTCO), or R = hepta-1,6-diyn-1-yl (dTPTCC) (Figure 3). The incorporation rates of dTPT3 and its linker-derivatized variants opposite dNAM approach those of natural base pairs. Additional unnatural base pairs identified with efficient incorporation rates include dTPT3-dFEMO, dTPT3-dFIMO, dTPT3-dIMO, dFTPT3-dNaM, dFTPT3-dFEMO, dFTPT3-dFIMO, and dFTPT3-dIMO.

[0106] Further, in various embodiments, the present invention relates to α analogs (e.g., any one of Figures 8, 10, 11, 15, and derivatives thereof); β analogs (e.g., any one of Figures 9, 12, 13, 16, and derivatives thereof); d5SICSCO, d5SICSCC, dDMOCO, dDMOCC, dMMO2pCO, dMMO2pCC, dTPT3, dTPT3PA, dTPT3A, dTPT3CO, dTPT3CC, and ribosyl forms thereof, and analogs thereof (see Figure 2); and in the form of nucleosides, nucleosides 5, triphosphates and the like. Provided are unnatural nucleotides with nucleobase analogs, including analogs (e.g., ribosyl and 2'-deoxyribosyl), nucleotides and their analogs (e.g., ribosyl and 2'-deoxyribosyl, phosphate and phosphorothioate), including nucleotide reagents used in RNA / DNA synthesis (DMT-protected phosphoramidites) and for enzymatic incorporation into oligonucleotides, such as by PCR or T7 RNA polymerase-mediated transcription, and for incorporation into nucleic acids (oligonucleotides), such as DNA and RNA. Compounds containing unnatural nucleobase analogs can also be incorporated into DNA or RNA analogs, such as PNA, LNA, and other similar polynucleotide analog polymers.Exemplary nucleobase analogs provided herein include β analogs comprising formula β8a and β8b as shown in FIG. 12, wherein each X is independently carbon or nitrogen; each R2 is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently sulfur, selenium, or oxygen; and R is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached. Examples of β8 analogs include dTPT3 and its linker-derivatized analogs.Exemplary nucleobase analogs provided herein include α14 analogs having formula α14a-α14f, as shown in FIG. 10, wherein each X is independently carbon or nitrogen; each R1 is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; and each R2 is optional and, when present, independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azido group, a group of reactivity complementary to the cargo and the reactive center. a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a group of reactivity complementary to the cargo, a coupled linker to which a cargo is attached, wherein each R is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide group; a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a group of reactivity complementary to the cargo and the reactive center, a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or secondary amine; and each E is independently sulfur, selenium, or oxygen. Examples of α14 analogs include dMMS, dDMS, dFEMS, dBrMS, dIMS, and linker-derivatized analogs thereof.

[0107] Further provided herein, in various embodiments, are unnatural base pairs comprising any one of the α analogs or derivatives thereof disclosed herein and any one of the β analogs or derivatives thereof disclosed herein. Derivatives include, but are not limited to, substitutions and additions of linker moieties. The linker moiety may be attached to the analog during synthesis or after incorporation of the nucleobase into the nucleic acid. Exemplary unnatural base pairs include, but are not limited to, dTPT3-dNaM, dTPT3-dFEMO, dTPT3-dFIMO, dTPT3-dIMO, dFTPT3-dNaM, dFTPT3-dFEMO, dFTPT3-dFIMO, and dFTPT3-dIMO. Unnatural base pairs include, but are not limited to, dTPT3-MMS, dTPT3-DMS, dTPT3-FEMS, dTPT3-BrMS, dTPT3-IMS, dTPT3-dDMN, dTPT3-d4OMe, dTPT3-dIQ, dTPT3-d2MN, dTPT3-d3OMe, dTPT3-dQL, dTPT3-d2Np, dTPT3-dDM4, dTPT3-dDM, dTPT3- dBEN, dTPT3-d3FB, dTPT3-dMM1, dTPT3-dMMO1, dTPT3-dDM2, dTPT3-dDM5, dTPT3-d2Py, dTPT3-d5MPy, dT PT3-dEPy, dTPT3-d3MPy, dTPT3-d34DMPy, dTPT3-d45DMPy, dTPT3-d4MPy, dTPT3-d35DMPy, dTPT3-dBP, dT PT3-dBTp, dTPT3-dBF, dTPT3-dIN, dTPT3-dTp, dTPT3-dBTz, dTPT3-dMTp, dTPT3-dAM, dTPT3-dMAN, dTPT 3-dDMMAN, dTPT3-dADM, dTPT3-dMMAN, dTPT3-dTOK588, dTPT3-dTOK576, dTPT3-dTOK587, dTPT3-dTOK586 , dTPT3-dTOK580, dTPT3-dPhMO, dTPT3-dPyMO1, dTPT3-PyMO2, dTPT3-dPMO1, dTPT3-dPMO2, dTPT3-dPMO3 , dTPT3-dFuMO1, dTPT3-dFuMO2, dTPT3-TpMO1, dTPT3-dTpMO2, dTPT3-dFIMO, dTPT3-dIMO, dTPT3-dMIMO,dTPT3-dMEMO、dTPT3-dFEMO、dTPT3-dPrMO、dTPT3-dMMO2、dTPT3-d2OMe、dTPT3-dDMO、dTPT3-dTMO、dTPT3-dNMO、dTPT3-dNOPy、dTPT3-d5FM、dTPT3-dNAM、dTPT3-dAMO1、dTPT3-dAPy、dTPT3-dAMO2、dTPT3-dMAPy、dTPT3-dAMO3、dTPT3-dDMAPy、dTPT3-dFDMO、dTPT3-dVMO、dTPT3-dQMO、dTPT3-dZMO、dTPT3-dCIMO、dTPT3-dTfMO、dTPT3-CNMO、d7AI-dMMS、dM7AI-dMMS、dImPy-dMMS、dP7AI-dMMS、dPPP-dMMS、d8Q-dMMS、dICS-dMMS、dPICS-dMMS、dMICS-dMMS、d4MICS-dMMS、d5MICS-dMMS、dNICS-dMMS、dONICS-dMMS、d7OFP-dMMS、d7OTP-dMMS、d4OTP-dMMS、dPYR-dMMS、d4MP-dMMS、d3MP-dMMS、dPPYR-dMMS、dMOP-dMMS、d4MOP-dMMS、dSICS-dMMS、dSNICS-dMMS、d5SICS-dMMS、d4SICS-dMMS、dTPT1-dMMS、dTPT2-dMMS、dFPT1-dMMS、dFTPT3-dMMS、d7AI-dDMS、dM7AI-dDMS、dImPy-dDMS、dP7AI-dDMS、dPPP-dDMS、d8Q-dDMS、dICS-dDMS、dPICS-dDMS、dMICS-dDMS、d4MICS-dDMS、d5MICS-dDMS、dNICS-dDMS、dONICS-dDMS、d7OFP-dDMS、d7OTP-dDMS、d4OTP-dDMS、dPYR-dDMS、d4MP-dDMS、d3MP-dDMS、dPPYR-dDMS、dMOP-dDMS、d4MOP-dDMS、dSICS-dDMS、dSNICS-dDMS、d5SICS-dDMS、d4SICS-dDMS、dTPT1-dDMS、dTPT2-dDMS、dFPT1-dDMS、dFTPT3-dDMS、d7AI-dFEMS、dM7AI-dFEMS、dImPy-dFEMS、dP7AI-dFEMS、dPPP-dFEMS、d8Q-dFEMS, dICS-dFEMS, dPICS-dFEMS, dMICS-dFEMS, d4MICS-dFEMS, d5MICS-dFEMS, dNICS-dFEMS, dONICS-dFEMS, d7OFP-dFEMS, d7OTP-dFEMS, d4OTP-dFEMS, dPYR-dFEMS, d4MP-dFEMS, d3MP-dFEMS, dPPYR-dFEMS, dMOP-dFEMS, d4MOP-dFEMS, dSICS-dFEMS, dSNICS-dFEMS, d5SICS-dFEMS, d4SICS-dFEMS, dTPT1-dFEMS, dTPT2-dFEMS, dFPT1-dFEMS, dFTPT3-dFEMS, d7AI-dBrMS, dM7AI-dBrMS, dImPy-dBrMS, dP7AI-dBrMS, dPPP-dBrMS, d8Q-dBrMS, dICS-dBrMS, dPICS-dBrMS, dMICS-dBrMS, d4MICS-dBrMS, d5MICS-dBrMS, dNICS-dBrMS, dONICS-dBrMS, d7OFP-dBrMS, d7OTP-dBrMS, d4OTP-dBrMS, dPYR-dBrMS, d4MP-dBrMS, d3MP-dBrMS, dPPYR-dBrMS, dMOP-dBrMS, d4MOP-dBrMS, dSICS-dBrMS, dSNICS-dBrMS, d5SICS-dBrMS, d4SICS-dBrMS, dTPT1-dBrMS, dTPT2-dBrMS, dFPT1-dBrMS, dFTPT3-dBrMS, d7AI-dIMS, dM7AI-dIMS, dImPy-dIMS, dP7AI-dIMS, dPPP-dIMS, d8Q-dIMS, dICS-dIMS, dPICS-dIMS, dMICS-dIMS, d4MICS-dIMS, d5MICS-dIMS, dNICS-dIMS, dONICS-dIMS, d7OFP-dIMS, d7OTP-dIMS, d4OTP-dIMS, dPYR-dIMS, d4MP-dIMS, d3MP-dIMS, dPPYR-dIMS, dMOP-dIMS, d4MOP-dIMS, dSICS-dIMS, dSNICS-dIMS, d5SICS-dIMS, d4SICS-dIMS, dTPT1-dIMS, dTPT2-dIMS, dFPT1-dIMS, and dFTPT3-dIMS,One or two of the unnatural nucleobases of the unnatural base pair may be derivatized with a linker. Exemplary unnatural base pairs of this disclosure further include any of the pairs described in Example 1. Exemplary β analogs include those presented in Figures 9, 12, and 13. Exemplary β nucleobase analogs include β analogs having formula β8a and β8b, as shown in FIG. 12, wherein each X is independently carbon or nitrogen; each R2 is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently sulfur, selenium, or oxygen; and R is optional and, when present, is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached. Examples of β analogs include dTPT3, d5SICS, dFTPT3, and derivatives or analogs thereof. Exemplary α analogs include those provided in Figures 8, 10, and 11. Exemplary α analogs include α analogs having formula α14a-α14f, as shown in Figure 10, wherein each X is independently carbon or nitrogen; each R is independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R is optional and, when present, independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azido group, a reactive linker comprising a reactive center suitable for bonding to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; each R is optional and, when present, independently hydrogen, alkyl, alkenyl,A reactive linker comprising an alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, or azide group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a reactive group complementary to the reactive center, a conjugated linker to which a cargo is attached, wherein each Y is independently sulfur, oxygen, selenium, or a secondary amine, and each E is independently sulfur, selenium, or oxygen. Examples of α analogs include dMMS, dDMS, dBrMS, dIMS, dFEMS, dNAM, dMMO2, dDMO, dEMO, dFEMO, and derivatives or analogs thereof. In some embodiments, the unnatural base pair comprises an α analog and a natural base. In some embodiments, the unnatural base pair comprises a β analog and a natural base. Further provided herein in some aspects are unnatural base pairs comprising the same two unnatural nucleoside analogs or derivatives thereof.

[0108] Unnatural base pairs, in various embodiments, comprise one unnatural nucleobase disclosed herein (e.g., an α-analog or derivative thereof, a β-analog or derivative thereof) and another unnatural nucleobase, including, but not limited to, 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl, and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2′-deoxyuridine, 2-amino-2′-deoxyadenosine, 3-deazaguanine, 3-deazaadenine, and the like. nine), 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthin-9-yl(I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifluoromethyluracil and cytosine; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidines, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-Dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl derivatives of adenine and guanine, 6-azapyrimidine, 6-azo-uracil, 6-azo-cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine , 7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and guanines; N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d ]pyrimidin-2-one, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetate , wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine, as well as those in which the purine or pyrimidine base is replaced with a heterocycle. Alpha analogs of unnatural base pairs include, but are not limited to, dMMS, dDMS, dBrMS, dIMS, dFEMS, dNAM, dMMO2, dDMO, dEMO, dFEMO, and derivatives or analogs thereof. Beta analogs of unnatural base pairs include, but are not limited to, dTPT3, d5SICS, and dFTPT3. ,

[0109] In some embodiments, the unnatural nucleobases and unnatural base pairs disclosed herein are efficiently incorporation and extension with natural polymerases. In some embodiments, the unnatural nucleobases and unnatural base pairs disclosed herein are efficiently incorporation and extension with modified polymerases. The effect of an unnatural nucleobase or unnatural nucleobase derivative on polymerase recognition is assessed, in exemplary embodiments, by determining the steady-state efficiency (e.g., the second-order rate constant kcat / KM) of a polymerase to synthesize an unnatural base pair by intercalating the unnatural nucleotide opposite its complementary base in the template and subsequently extending the resulting unnatural primer terminus by intercalating the correct natural nucleotide. To determine fidelity, the corresponding rates of synthesis and extension for mispairs involving natural nucleotides may also be measured. In some embodiments, polymerase modifications are not required to improve incorporation or extension rates. The embodiments and examples disclosed herein may be performed with any known polymerase. Polymerases include naturally occurring polymerases and any variants thereof, including, but not limited to, mutants, recombinants, fusions, genetically modified, chemically synthesized products, and analogs. Naturally occurring polymerases and their modified variants are not limited to polymerases that retain the ability to catalyze a polymerization reaction. In some examples, naturally occurring and / or modified variants retain the ability to catalyze a polymerization reaction. Mutant polymerases include polymerases in which one or more amino acids are replaced with other amino acids (naturally occurring or non-naturally occurring) and polymerases with one or more amino acid insertions or deletions. In some embodiments, a polymerase refers to a fusion protein comprising at least two moieties linked to each other, e.g., one moiety comprising a peptide capable of catalyzing the polymerization of nucleotides into nucleic acids is linked to another moiety comprising a second moiety, such as a reporter enzyme or a processivity-modifying domain. One exemplary embodiment of such a polymerase is T7 DNA polymerase, which comprises a nucleic acid polymerization domain and a thioredoxin-binding domain, where thioredoxin binding enhances the processivity of the polymerase. In the absence of thioredoxin binding, T7DNA polymerases are discrete polymerases with a processing capacity of one to a few bases. DNA polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases, and phage DNA polymerases. Bacterial DNA polymerases include Escherichia coli DNA polymerases I, II, and III, IV and V, the Klenow fragment of Escherichia coli DNA polymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase, and Sulfolobus solfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases include DNA polymerases α, β, γ, δ, ε, η, ζ, σ, λ, μ, ι, and κ, as well as Rev1 polymerase (terminal deoxycytidyl transferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerase, PZA DNA polymerase, phi-15 DNA polymerase, Cp1 DNA polymerase, Cp7 DNA polymerase, T7 DNA polymerase, and T4 polymerase. Archaeal DNA polymerases include Thermus aquaticus (Tag) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavus (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima (Tma) DNA polymerase, and Bacillus stearothermophilus(Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase; Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. 9°N-7 DNA polymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase (D.Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Pyrococcus islandicum DNA polymerases include thermostable and / or thermophilic DNA polymerases such as Thermococcus fumicolans DNA polymerase, Aeropyrum pernix DNA polymerase, and DNA polymerases isolated from the heterodimeric DNA polymerase DP1 / DP2. RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and K11 polymerase; eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and archaeal RNA polymerases.

[0110] In some embodiments, the polymerase has specificity for unnatural nucleotides comprising an α or β nucleobase analog that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity of the polymerase for natural nucleotides. In some embodiments, the polymerase has specificity for unnatural nucleotides comprising an α or β nucleobase analog and a modified sugar that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity of the polymerase for natural nucleotides and / or unnatural nucleotides that do not contain modified sugars. In some embodiments, the polymerase has specificity for unnatural nucleotides comprising linker-derivatized α or β nucleobase analogs that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.99% of the specificity of the polymerase for natural nucleotides and / or unnatural nucleotides that do not comprise a linker. In some embodiments, the unnatural nucleobase is dTPT3. In some embodiments, the unnatural nucleobase is dMMS. In some embodiments, the unnatural nucleobase is dDMS. In some embodiments, the unnatural nucleobase is dBrMS. In some embodiments, the unnatural nucleobase is IMS. In some embodiments, the unnatural nucleobase is dFEMS. In some embodiments, the unnatural nucleobase is MMSpCO. In some embodiments, the unnatural nucleobase is dMMSPA. In some embodiments, the unnatural nucleobase is dFTPT3. In some embodiments, the unnatural nucleobase is dTPTPA. In some embodiments, the unnatural nucleobase is dTPT3CO. In some embodiments, the unnatural nucleobase comprises formula α14a or a derivative or analog thereof. In some embodiments, the unnatural nucleobase comprises formula α14b or a derivative or analog thereof.In some embodiments, the unnatural nucleobase comprises formula α14c, or a derivative or analog thereof. In some embodiments, the unnatural nucleobase comprises formula α14d, or a derivative or analog thereof. In some embodiments, the unnatural nucleobase comprises formula α14e, or a derivative or analog thereof. In some embodiments, the unnatural nucleobase comprises formula α14f, or a derivative or analog thereof. In some embodiments, the unnatural nucleobase comprises formula β8a, or a derivative or analog thereof. In some embodiments, the unnatural nucleobase comprises formula β8b, or a derivative or analog thereof.

[0111] Polymerases can be characterized according to their fidelity when used with specific natural and / or unnatural nucleotides, or collections of natural and / or unnatural nucleotides, where the unnatural nucleotides include the α or β nucleobase analogs described herein. In various embodiments, fidelity generally refers to the accuracy with which a polymerase incorporates the correct nucleotide into a growing oligonucleotide when making a copy of an oligonucleotide template. When natural and unnatural nucleotides are present, for example, at equal concentrations, to compete for strand synthesis at the same site in the polymerase-strand-template nucleic acid binary complex, polymerase fidelity can be measured as the ratio of correct natural and unnatural nucleotide incorporation to incorrect natural and unnatural nucleotide incorporation. DNA polymerase fidelity can be calculated as the ratio of (kcat / KM) for natural and unnatural nucleotides to (kcat / KM) for incorrect natural and unnatural nucleotides, where kcat and KM are the Michaelis-Menden parameters of steady-state enzyme kinetics. In some embodiments, the polymerase, with or without proofreading activity, has a fidelity value of at least about 100, 1000, 10,000, 100,000, or 1x10. In some embodiments, the polymerase has a fidelity value for unnatural nucleotide incorporation of at least about 100, 1000, 10,000, 100,000, or 1x10. In some embodiments, the unnatural nucleotide is dTPT3TP or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide is dNAM or a derivative thereof. In some embodiments, the unnatural nucleotide is dNaMTP or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide is dTPT3 or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8a or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14a or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8a or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14b or a derivative thereof.In some embodiments, the unnatural nucleotide comprises β8a or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14c or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8a or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14d or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8a or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14e or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8a or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14f or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8b or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14a or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8b or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14b or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8b or a derivative thereof, and its corresponding nucleobase on the template oligonucleotide comprises α14c or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8b or a derivative thereof and its corresponding nucleobase on the template oligonucleotide comprises α14d or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8e or a derivative thereof and its corresponding nucleobase on the template oligonucleotide comprises α14e or a derivative thereof. In some embodiments, the unnatural nucleotide comprises β8f or a derivative thereof and its corresponding nucleobase on the template oligonucleotide comprises α14f or a derivative thereof.

[0112] In some embodiments, unnatural base pairs are synthesized / amplified with the efficiency and fidelity of natural base pairs. In various embodiments, unnatural base pairs include any α nucleobase analog or derivative thereof, and / or any β nucleobase analog or derivative thereof. Examples of β analogs include dTPT3, d5SICS, dFTPT3, and derivatives or analogs thereof. Examples of α analogs include dMMS, dDMS, dBrMS, dIMS, dFEMS, dNAM, dMMO2, dDMO, dEMO, dFEMO, and derivatives or analogs thereof. In some embodiments, unnatural base pairs are efficiently amplified with a fidelity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.85%, 99.9% or higher per doubling in a wide variety of sequence contexts, including GC and AT rich sequences, randomized sequences, and sequences containing multiple unnatural nucleobase pairs. For example, unnatural nucleobase pairs comprising one or more unnatural nucleobases have a synthesis efficiency and / or fidelity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar to the amplification efficiency and / or fidelity of natural base pairs. As another example, unnatural nucleobase pairs comprising one or more unnatural nucleobases have a synthesis efficiency and / or fidelity that is at most 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% or less efficient and / or accurate than natural base pairs. In some embodiments, unnatural nucleobase pairs are transcribed with excellent efficiency and selectivity in both strand configurations (e.g., dX must template a YTP insertion, and dY must template a XTP insertion). In some embodiments, incorporation of unnatural nucleotides does not reduce the rate of full-length transcription relative to the rate at which a fully natural sequence is transcribed.In some embodiments, incorporation of the unnatural nucleotide reduces the rate of full-length transcription by a factor of less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 relative to the rate at which the fully natural sequence is transcribed. In some embodiments, the unnatural base pair comprises dTPT3 or a derivative or analog thereof, and dNaM or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises dTPT3 or a derivative or analog thereof, and dNaM or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises dTPT3 or a derivative or analog thereof, and dNaM or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises dTPT3 or a derivative or analog thereof, and dNaM or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises dTPT3, or a derivative or analog thereof, and dNaM, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8a, or a derivative or analog thereof, and α14a, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8a, or a derivative or analog thereof, and α14b, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8a, or a derivative or analog thereof, and α14c, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8a, or a derivative or analog thereof, and α14d, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8a, or a derivative or analog thereof, and α14e, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8a, or a derivative or analog thereof, and α14f, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8b, or a derivative or analog thereof, and α14a, or a derivative or analog thereof.In some embodiments, the unnatural base pair comprises β8b, or a derivative or analog thereof, and α14b, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8b, or a derivative or analog thereof, and α14c, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8b, or a derivative or analog thereof, and α14d, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8b, or a derivative or analog thereof, and α14e, or a derivative or analog thereof. In some embodiments, the unnatural base pair comprises β8b, or a derivative or analog thereof, and α14f, or a derivative or analog thereof.

[0113] Further provided herein, in various embodiments, are unnatural base pairs comprising one or more unnatural nucleobases (e.g., an alpha nucleobase, a beta nucleobase, or an alpha nucleobase and a beta nucleobase), wherein one or two of the nucleobases comprise a linker, wherein the linker comprises a reactive center. Exemplary reactive centers include, but are not limited to, alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, hydroxyl, carbonyl, aldehyde, haloformyl, carbonate, carboxylate, carboxyl, ester, methoxy, hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, methylenedioxy, orthocarbonate, carboxamide, primary amine, secondary amine, imide, azide, azo, cyanate, isocyanate, nitrate, nitrile, isonitrile, nitrosoxy, nitro, nitroso, pyridyl, sulfhydryl, sulfide, disulfide, sulfinyl, sulfo, thiocyanate, isothiocyanate, carbonothioyl, phosphino, phosphono, phosphate, borono, boronate, borino, borinate, and combinations thereof. An example of a linker-derivatized nucleobase is TPT3R, shown in Figure 2, where the superscript R indicates the linker. In some embodiments, the linker is modified with a protecting group, e.g., TPT3PA, where the linker is a protected propargyl linker.

[0114] In some embodiments, the nucleobase analogs provided herein comprise an amino functional linker or a protected amino functional linker (e.g., dXPA). In certain embodiments, the amino functional linker is 3-aminopropyn-1-yl. In some embodiments, the nucleobase analogs provided herein comprise an alkyne azide ether linker for derivatization via click chemistry or a protected alkyne azide ether linker for derivatization via click chemistry. In certain embodiments, the alkyne azide ether linker is 4-oxahepat-1,6-diyn-1-yl. In some embodiments, the nucleobase analogs provided herein comprise an alkyne azide trimethylene linker for derivatization via click chemistry or a protected alkyne azide trimethylene linker for derivatization via click chemistry. In certain embodiments, the alkyne azide trimethylene linker is hepta-1,6-diyn-1-yl. In some embodiments, X is a β nucleoside analog having any of the formulas in Figures 9, 12, 13, and 16. In some embodiments, X is ICS, PICS, MICS, 4MICS, 5MICS, NICS, ONICS, SICS, SNICS, 5SICS, 4SICS, 7OFP, 7OTP, TPT2, TPT3, or FTPT3. In some embodiments, X is an α nucleoside analog having any of the formulas in Figures 8, 10, 11, and 15. In some embodiments, X is FIMO, MIMO, FEMO, PrMO, EMO, MEMO, IMO, MMO2, DMO, NMO, 5FM, 2OMe, TMO, FDMO, VMO, ZMO, CIMO, TfMO, CNMO, MMS, DMS, BrMS, IMS, FEMS, NAM, or QMO.

[0115] In some embodiments, the linker is a propynyl linker such as those used with natural nucleotides. These linkers contain a propargyl amine, with the amine serving as a reactive site for attaching other functionalities.

[0116] In various embodiments, the linker-derivatized nucleobase comprises a spacer. A typical spacer is acetamidohexanamide. The spacer may be hydrophilic. The spacer may connect the linker to a functional group. Spacers include, but are not limited to, spacer C3 (3-carbon spacer), spacer C6 (6-carbon spacer), photocleavable spacer, hexanediol spacer, spacer 9 (triethylene glycol spacer), spacer C12 (12-carbon spacer), spacer 18 (18-atom hexa-ethylene glycol spacer), and 1',2'-dideoxyribose spacer.

[0117] An unnatural nucleobase pair comprising one or two linker-derivatized nucleobases is amplified, in some instances, with an efficiency and fidelity similar to that of a natural base pair or a non-linker-derivatized unnatural base pair. For example, an unnatural nucleobase pair comprising one or two linker-derivatized unnatural nucleobases has a synthesis efficiency and / or fidelity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar to that of a natural base pair or a non-linker-derivatized unnatural base pair. As another example, an unnatural nucleobase pair comprising one or two linker-derivatized unnatural nucleobases has a synthesis efficiency and / or fidelity that is at most 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the synthesis efficiency and / or fidelity of a natural base pair or a non-linker-derivatized unnatural base pair. In some embodiments, the unnatural nucleobase pair comprises dTPT3PA. In some embodiments, the unnatural nucleobase pair comprises dTPT3CO. In some embodiments, the unnatural nucleobase pair comprises dMMSpCO. In some embodiments, the unnatural nucleobase pair comprises dMMSPA. In some embodiments, the unnatural nucleobase pair comprises dNaMR. In some embodiments, the unnatural nucleobase pair comprises dMMO2R. In some embodiments, the unnatural nucleobase pair comprises dDMOR. In some embodiments, the unnatural nucleobase pair comprises d5SICSR. In some embodiments, the unnatural nucleobase pair comprises dMMSR. In some embodiments, the unnatural nucleobase pair comprises dDMSR. In some embodiments, the unnatural nucleobase pair comprises dFEMSR. In some embodiments, the unnatural nucleobase pair comprises dBrMSR. In some embodiments, the unnatural nucleobase pair comprises dIMSR.

[0118] In some embodiments, a linker-derivatized unnatural nucleobase increases the insertion efficiency during oligonucleotide synthesis compared to the same unnatural nucleobase without a linker. In some embodiments, a linker-derivatized unnatural nucleobase decreases the insertion efficiency during oligonucleotide synthesis compared to the same unnatural nucleobase without a linker. In some examples, a linker-derivatized unnatural nucleobase has approximately the same insertion efficiency during oligonucleotide synthesis compared to the same unnatural nucleobase without a linker. In some embodiments, a protected linker-derivatized unnatural nucleobase increases the insertion efficiency during oligonucleotide synthesis compared to the same unnatural nucleobase without a protected linker. In some embodiments, a protected linker-derivatized unnatural nucleobase decreases the insertion efficiency during oligonucleotide synthesis compared to the same unnatural nucleobase without a protected linker. In some examples, a protected linker-derivatized unnatural nucleobase has approximately the same insertion efficiency during oligonucleotide synthesis compared to the same unnatural nucleobase without a protected linker.

[0119] Exemplary methods for analyzing unnatural base pair synthesis efficiency (insertion of an unnatural nucleobase opposite its partner in a template) and extension (continuous primer extension) are provided. In various embodiments, one or both of the nucleobases in an unnatural base pair may be a linker-derivatized unnatural nucleobase. One method uses a pre-steady-state assay. The assay is based on determining the amount of a primer (e.g., a 23-mer) extended by a polymerase (e.g., the Klenow fragment of E. coli DNA polymerase I) under a fixed set of conditions through the addition of an unnatural nucleotide opposite its complementary nucleotide in a template (e.g., a 45-mer). In this assay, the efficiency of unnatural base pair synthesis is characterized by measuring the percent incorporation (%inc) of the next correct unnatural triphosphate at a given concentration, e.g., using a ratio such as [24-mer + 25-mer] / [23-mer + 24-mer + 25-mer]. In this assay, the efficiency of extension is characterized by measuring the percent extension (%ext) at a given concentration of the next correct nucleotide and a saturating concentration of the unnatural nucleotide, e.g., using the ratio [25mer] / [24mer+25mer]. The results of a typical pre-steady-state assay are shown in Table 1, where the unnatural triphosphates are 5SICS, FPT1, TPT1, TPT2, TPT3, FTPT3, TPT3PA, or 5SICSPA. In some embodiments, the percent incorporation of the unnatural nucleobase is at least 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percent extension of the next correct nucleotide after insertion of the unnatural nucleobase is at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. In some embodiments, synthesis efficiency is increased by derivatization of the unnatural nucleobase.For example, by the addition of a linker, a protective linker, and / or a linker conjugated to a cargo molecule. As another example, derivatization includes atom substitution, addition, or deletion. In some embodiments, the percent elongation is increased by derivatizing an unnatural nucleobase. Derivatizing an unnatural nucleobase, in some instances, increases the efficiency of insertion of a nucleotide complementary to a base pair in the template by at least 1-2 orders of magnitude. This increase in efficiency can be due to an increase in kcat and a decrease in KM.

[0120] [Table 1]

[0121] Further provided herein is a means for assessing replication. In one method, a template nucleic acid duplex containing an unnatural base pair (e.g., dTPT3-dNaM or its analog) is amplified by PCR. In one example, a set of PCR reactions uses 48 cycles with OneTaq polymerase. In another example, a set of PCR reactions uses 20 amplification cycles with exonuclease-negative Taq. Efficiency is determined by monitoring the amplification level. Fidelity, generally defined as the extension of unnatural base pairs per doubling, is determined from the percentage of amplified DNA that retains unnatural base pairs. The percentage of amplified DNA that retains unnatural base pairs can be determined from the relative peak intensity of a sequencing chromatogram. In some embodiments, the fidelity of unnatural base pair replication is at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99%. Unnatural base pair replication may also proceed with little or no sequence bias, where little sequence bias reduces fidelity by less than 1%. Typical fidelities are described in Example 1 and shown in Tables 4, 5, and 6.

[0122] Further provided herein, in various embodiments, are oligonucleotides, including single-stranded and double-stranded (e.g., duplex) DNA and / or RNA, comprising one or more unnatural nucleobases described herein (e.g., any alpha nucleobase or analog or derivative thereof, and / or any beta nucleobase or analog or derivative thereof). The nucleobases may be any alpha or beta nucleobase, including those in Figures 2, 8, 9, 10, 11, 12, 13, 15, and 16. Double-stranded oligonucleotides include DNA-DNA duplexes, DNA-RNA hybrid duplexes, and RNA-RNA duplexes. In some embodiments, the oligonucleotides comprise linker-derivatized nucleobases.

[0123] In some embodiments, an oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or more unnatural nucleobases. In some embodiments, the percentage of unnatural nucleobases in the oligonucleotide is between about 0% and about 1%, between about 0% and about 2%, between about 0% and about 3%, between about 0% and about 4%, between about 0% and about 5%, between about 0% and about 10%, between about 1% and about 10%, between about 1% and about 15%, between about 1% and about 20%, between about 5% and about 10%, between about 5% and about 20%, between about 10% and about 30%, between about 1% and about 50%, or between about 1% and about 100%.

[0124] Examples of oligonucleotides containing one or more unnatural nucleobases include, but are not limited to, DNA aptamers and RNA aptamers. DNA and RNA aptamers include, but are not limited to, primers and molecular beacons. DNA aptamers may include barcodes.

[0125] In some embodiments, the oligonucleotide comprises dTPT3 or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises d5SICS or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises dNaM or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises dMMS or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises dDMS or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises dFEMS or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises dBrMS or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises dIMS or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises β8a or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises β8b or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises α14a or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises α14b or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises α14c or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises α14d or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises α14e or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises α14f or a derivative or analog thereof.

[0126] In some embodiments, the oligonucleotide comprises dTPT3 or a derivative or analog thereof, and dNaM or a derivative or analog thereof. In some embodiments, the oligonucleotide comprises a dTPT3-dNaM base pair. In some embodiments, the oligonucleotide comprises one or more base pairs selected from dTPT3-dFEMO, dTPT3-dFIMO, dTPT3-dIMO, dFTPT3-dNaM, dFTPT3-dFEMO, dFTPT3-dFIMO, and dFTPT3-dIMO. In some embodiments, the oligonucleotide is dTPT3-MMS, dTPT3-DMS, dTPT3-FEMS, dTPT3-BrMS, dTPT3-IMS, dTPT3-dDMN, dTPT3-d4OMe, dTPT3-dIQ, dTPT3-d2MN, dTPT3-d3OMe, dTPT3-dQL, dTPT3-d2Np, dTPT3-dDM4, dTPT3-dDM, dTPT3-dBEN, dTPT3 -d3FB, dTPT3-dMM1, dTPT3-dMMO1, dTPT3-dDM2, dTPT3-dDM5, dTPT3-d2Py, dTPT3-d5MPy, dTPT3-dEPy, dTPT3-d3MP y, dTPT3-d34DMPy, dTPT3-d45DMPy, dTPT3-d4MPy, dTPT3-d35DMPy, dTPT3-dBP, dTPT3-dBTp, dTPT3-dBF, dTPT3-dI N, dTPT3-dTp, dTPT3-dBTz, dTPT3-dMTp, dTPT3-dAM, dTPT3-dMAN, dTPT3-dDMMAN, dTPT3-dADM, dTPT3-dMMAN, dTPT 3-dTOK588, dTPT3-dTOK576, dTPT3-dTOK587, dTPT3-dTOK586, dTPT3-dTOK580, dTPT3-dPhMO, dTPT3-dPyMO1, dTPT 3-PyMO2, dTPT3-dPMO1, dTPT3-dPMO2, dTPT3-dPMO3, dTPT3-dFuMO1, dTPT3-dFuMO2, dTPT3-TpMO1, dTPT3-dTpMO2, dTPT3-dFIMO, dTPT3-dIMO, dTPT3-dMIMO, dTPT3-dMEMO, dTPT3-dFEMO, dTPT3-dPrMO, dTPT3-dMMO2, dTPT3-d2OMe,dTPT3-dDMO、dTPT3-dTMO、dTPT3-dNMO、dTPT3-dNOPy、dTPT3-d5FM、dTPT3-dNAM、dTPT3-dAMO1、dTPT3-dAPy、dTPT3-dAMO2、dTPT3-dMAPy、dTPT3-dAMO3、dTPT3-dDMAPy、dTPT3-dFDMO、dTPT3-dVMO、dTPT3-dQMO、dTPT3-dZMO、dTPT3-dCIMO、dTPT3-dTfMO、dTPT3-CNMO、d7AI-dMMS、dM7AI-dMMS、dImPy-dMMS、dP7AI-dMMS、dPPP-dMMS、d8Q-dMMS、dICS-dMMS、dPICS-dMMS、dMICS-dMMS、d4MICS-dMMS、d5MICS-dMMS、dNICS-dMMS、dONICS-dMMS、d7OFP-dMMS、d7OTP-dMMS、d4OTP-dMMS、dPYR-dMMS、d4MP-dMMS、d3MP-dMMS、dPPYR-dMMS、dMOP-dMMS、d4MOP-dMMS、dSICS-dMMS、dSNICS-dMMS、d5SICS-dMMS、d4SICS-dMMS、dTPT1-dMMS、dTPT2-dMMS、dFPT1-dMMS、dFTPT3-dMMS、d7AI-dDMS、dM7AI-dDMS、dImPy-dDMS、dP7AI-dDMS、dPPP-dDMS、d8Q-dDMS、dICS-dDMS、dPICS-dDMS、dMICS-dDMS、d4MICS-dDMS、d5MICS-dDMS、dNICS-dDMS、dONICS-dDMS、d7OFP-dDMS、d7OTP-dDMS、d4OTP-dDMS、dPYR-dDMS、d4MP-dDMS、d3MP-dDMS、dPPYR-dDMS、dMOP-dDMS、d4MOP-dDMS、dSICS-dDMS、dSNICS-dDMS、d5SICS-dDMS、d4SICS-dDMS、dTPT1-dDMS、dTPT2-dDMS、dFPT1-dDMS、dFTPT3-dDMS、d7AI-dFEMS、dM7AI-dFEMS、dImPy-dFEMS、dP7AI-dFEMS、dPPP-dFEMS、d8Q-dFEMS、dICS-dFEMS、dPICS-dFEMS、dMICS-dFEMS、d4MICS-dFEMS、d5MICS-dFEMS, dNICS-dFEMS, dONICS-dFEMS, d7OFP-dFEMS, d7OTP-dFEMS, d4OTP-dFEMS, dPYR-dFEMS, d4MP-dFEMS, d3MP-dFEMS, dPPYR-dFEMS, dMOP -dFEMS, d4MOP-dFEMS, dSICS-dFEMS, dSNICS-dFEMS, d5SICS-dFEMS, d4SICS-dFEMS, dTPT1-dFEMS, dTPT2-dFEMS, dFPT1-dFEMS, dFTPT3-dFEMS, d7AI -dBrMS, dM7AI-dBrMS, dImPy-dBrMS, dP7AI-dBrMS, dPPP-dBrMS, d8Q-dBrMS, dICS-dBrMS, dPICS-dBrMS, dMICS-dBrMS, d4MICS-dBrMS, d5MICS-dBrM S, dNICS-dBrMS, dONICS-dBrMS, d7OFP-dBrMS, d7OTP-dBrMS, d4OTP-dBrMS, dPYR-dBrMS, d4MP-dBrMS, d3MP-dBrMS, dPPYR-dBrMS, dMOP-dBrMS, d4MOP -dBrMS, dSICS-dBrMS, dSNICS-dBrMS, d5SICS-dBrMS, d4SICS-dBrMS, dTPT1-dBrMS, dTPT2-dBrMS, dFPT1-dBrMS, dFTPT3-dBrMS, d7AI-dIMS, dM7AI- dIMS, dImPy-dIMS, dP7AI-dIMS, dPPP-dIMS, d8Q-dIMS, dICS-dIMS, dPICS-dIMS, dMICS-dIMS, d4MICS-dIMS, d5MICS-dIMS, dNICS-dIMS, dONICS-dIM S, d7OFP-dIMS, d7OTP-dIMS, d4OTP-dIMS, dPYR-dIMS, d4MP-dIMS, d3MP-dIMS, dPPYR-dIMS, dMOP-dIMS, d4MOP-dIMS, dSICS-dIMS, dSNICS-dIMS, d5SICS-dIMS, d4SICS-dIMS, dTPT1-dIMS, dTPT2-dIMS, dFPT1-dIMS, dFTPT3-dIMS, and one or more unnatural nucleobases of the unnatural base pair may be derivatized with a linker.

[0127] Oligonucleotides comprising unnatural nucleobases disclosed herein include, but are not limited to, 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl, and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, 3-deazaguanine, 3-deazaadenine, 4-thio-uracil, 4-thio-thymine, uracil- 5-yl, hypoxanthine-9-yl(I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifluoromethyluracil and cytosine; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidines, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-Dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl derivatives of adenine and guanine, 6-azapyrimidine, 6-azo-uracil, 6-azo-cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine , 7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and guanines; N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d ]pyrimidin-2-one, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid, wybutoxosine butoxosine), pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine, and may further include one or more additional unnatural bases, including those in which the purine or pyrimidine base is replaced with a heterocycle.

[0128] Oligonucleotides containing unnatural nucleobases disclosed herein include, but are not limited to, modifications at the 2' position: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-Oalkyl, 2'-F, 2'-OCH3, 2'-O(CH2)2OCH3, where alkyl, alkenyl, and alkynyl are substituted or substituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, -O[(CH2)nO]mCH3, -O(CH2)nOCH3, -O(CH2)nNH2, -O(CH2)nCH3, -O (CH2)n-ONH2, and -O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; and / or modifications at the 5' position, including 5'-vinyl, 5'-methyl (R or S); modifications at the 4' position, including 4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of the oligonucleotide, or groups for improving the pharmacodynamic properties of the oligonucleotide, and any combination thereof.

[0129] In some embodiments, oligonucleotides comprising unnatural nucleobases disclosed herein further comprise an unnatural backbone, including, but not limited to, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkylphosphotriesters, C1-C10 phosphonates, 3'-alkylenephosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3'-aminophosphoramidates, aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.

[0130] Methods for determining the stability of oligonucleotide duplexes containing unnatural base pairs (with or without linkers) include thermodynamic analysis of circular dichroism (CD) measurements and UV melting experiments. In some embodiments, DNA duplex stability studies are used to facilitate the selection of appropriate unnatural nucleotide base pairs, unnatural nucleobases, or unnatural nucleobase derivatives or substitutions. Appropriately selected unnatural base pairs include those that increase oligonucleotide hybridization fidelity at other positions within the duplex. Appropriately selected unnatural base pairs include those that increase oligonucleotide duplex stability. Appropriately selected nucleobases may also be used to optimize oligonucleotides for biotechnological or therapeutic applications where high-fidelity hybridization and discrimination are critical. In some instances, unnatural base pairs are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more as stable as natural base pairs in oligonucleotide duplexes. In some instances, the Tm of a duplex containing one or more unnatural base pairs may be 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4.5°C, 4°C, 3.5°C, 3°C, 2.9°C, 2.8°C, 2.7°C, 2.6°C, 2.5°C, 2.4°C, 2.3°C, 2.2°C, 2.1°C, 2.2°C, 2.3°C, 2.4°C, 2.5°C, 2.6°C, 2.7°C, 2.8°C, 2.9°C, 2.8°C, 2.9°C, 2.8°C, 2.9°C, 2.8°C, 2.9°C, 2.8°C, 2.8°C, 2.9°C, 2.8°C, 2.8°C, 2.9°C, 2.8°C, 2.8°C, 2.8°C, 2.8°C, 2.8°C, 2.8°C, 2.8°C, 2.8°C, 2.8°C, 2.8°C, 2.9 ... , 1.9°C, 1.8°C, 1.7°C, 1.6°C, 1.5°C, 1.4°C, 1.3°C, 1.2°C, 1.1°C, 1°C, 0.9°C, 0.8°C, 0.7°C, 0.6°C, 0.5°C, 0.4°C, 0.3°C, 0.2°C, 0.1°C, and wherein one or more unnatural nucleobases are replaced with one or more natural nucleobases. In some embodiments, the presence of unnatural base pairs in an oligonucleotide duplex does not significantly perturb the structure of the duplex.

[0131] In some embodiments, oligonucleotides containing linker-derivatized nucleobases allow for site-specific modification of the DNA or RNA during or after enzymatic synthesis. The unnatural nucleotides disclosed herein (e.g., nucleotides containing unnatural α or β nucleobase analogs) are, in some instances, modified with linkers that allow for the attachment of various functional groups (e.g., cargo) without eliminating polymerase recognition. Site-specific functionalities include, but are not limited to, fluorophores, NMR handles for characterization (e.g., F19), IR probes (e.g., azide and cyano groups), biotin (e.g., for ease of identification and / or purification), affinity tags, liposomes, and nanoparticles. In one embodiment, the linker provides bioconjugation through cross-coupling (e.g., iodo groups). In one embodiment, the linker provides bioconjugation through click chemistry (e.g., azide and alkyne substituents). In one embodiment, oligonucleotides containing linker-derivatized nucleobases are useful as primers and / or molecular beacons.

[0132] Further provided herein, in various embodiments, is the use of any nucleoside analog (α or β) disclosed herein, or an analog or derivative thereof, in site-specific cleavage or functionalization of oligonucleotides. In some embodiments, the nucleoside analog comprises one or more linkers configured for site-specific modification. Examples of nucleotide analogs comprising a linker moiety include, but are not limited to, d5SICSCO, d5SICSCC, dDMOCO, dDMOCC, dMMO2pCO, dMMO2pCC, dTPT3, dTPT3A, dTPT3PA, dTPT3CO, dMMSpCO, dMMSPA, and dTPT3CC, or ribosyl forms or analogs thereof. Provided herein, in various embodiments, are compositions of matter for functionalized oligonucleotides, methods for preparing functionalized oligonucleotides, and methods for using functionalized oligonucleotides. Various embodiments provide for dTPT3, dTPT3PA, dTPT3A, dTPT3CO, and dTPT3CC, or other linker-derivatized analogs of dTPT3, to be incorporated into oligonucleotides, and further reaction and derivatization of such unnatural nucleobase analogs incorporated into oligonucleotides with various reagents for selective reaction with the unnatural nucleobase analogs in oligonucleotides, while naturally occurring nucleobases (A, T, G, C, U) are unreactive to any suitable extent with such reagents. The dTPT3-based family of unnatural nucleotides with linkers is particularly important, as it has been found to be more efficiently replicated by DNA polymerases than base pairs containing d5SICS or its linker-derivatized variants. This greatly facilitates many of the potential uses. In some embodiments, the percent incorporation of the non-natural nucleotide, including the linker, into the oligonucleotide is at least 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.In some embodiments, the percent extension of the next exact nucleotide into the oligonucleotide (wherein the next exact nucleotide results from the incorporation of the non-natural nucleotide, including the linker) is at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. In some embodiments, the addition of the site-specific functionality reduces the percent incorporation of the unnatural nucleotide into the oligonucleotide by no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the fidelity of the linker-derivatized unnatural nucleotide is at least 98%, 98.1%, 98.2%, 98.3, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99%. Accordingly, in various embodiments, provided herein are methods of using linker-derivatized unnatural nucleotides to generate DNA or RNA that is site-specifically modified with another molecule of interest. In some embodiments, site-specific inclusion of various functionalities occurs before or after amplification. In some embodiments, site-specific functionalization is used for SELEX applications.

[0133] A typical strategy for generating site-specifically modified DNA or RNA with another molecule of interest is called the phosphorothioate strategy (Figure 3), which relies on the site-specific incorporation of phosphorothioate groups into DNA or RNA via the ribo- or deoxyribo-α-thiotriphosphate of one of the unnatural nucleosides shown in Figures 1 or 2. After incorporation into DNA or RNA, the phosphorothioate may be used to link reagents bearing γ-bromo-α,β-unsaturated carbonyl moieties, iodo(or bromo)acetyl moieties, or aziridinyl sulfonamide moieties to generate site-specifically functionalized DNA or RNA. Alternatively, after incorporation into DNA or RNA, phosphorothioates may be used to site-specifically cleave DNA or RNA using iodine in alkaline solution or iodoethanol. Thus, the phosphorothioate strategy results in site-specific modification of the nucleic acid backbone, providing a method for site-specific cleavage of oligonucleotide chains.

[0134] Another strategy for generating DNA or RNA site-specifically modified with another molecule or target, called the linker strategy (Figures 4 and 5), utilizes derivatization of unnatural nucleobases with linkers (Figure 2) that can be used to attach the target functional group either by polymerization (by PCR or T7 RNA polymerase-mediated polymerization using an appropriate functionalized nucleobase triphosphate reagent that is incorporated into the DNA or RNA strand being synthesized) or by reaction of the unnatural nucleobase linker after incorporation into an oligonucleotide strand with an appropriate functionalization reagent, e.g., an NHS-containing reagent that further contains the desired functional group; NHS reacts with the free amino group of an amino-functionalized unnatural nucleobase, such as d5SICSA, dMMO2A, or dTPT3A. Figure 4 further illustrates the amino-functionalized linker strategy using d5SICSA and dMMO2PA, which allows for site-specific dual labeling of duplex DNA.

[0135] For example, functionalization can be achieved after incorporation of unnatural nucleobases into oligonucleotides using linkers bearing primary amino groups (e.g., dTPT3A). More specifically, functionalization can be carried out by reaction of the primary amino (e.g., propargylamino group) with a reagent bearing a cargo containing an N-hydroxysuccinimide (NHS) ester (Figure 4). Analogs developed for this application include d5SICSA, d5SICSPA, dMMO2A, dMMO2PA, dTPT3PA, and dTPT3A (recall that "A" refers to the nucleotide containing the propargylamine, and "PA" refers to the linker with the same protecting group; see Figure 2 and its caption). The use of dTPT3PA bearing a protected primary propargylamino group and dTPT3A bearing a protected primary propargylamino group for sequence-specific functionalization of oligonucleotides is disclosed and claimed herein.

[0136] Site-specific functionalization of oligonucleotides can also be achieved using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (i.e., a "click chemistry" linker strategy; Figure 5), and for these uses, d5SICSCO, d5SICSCC, dDMOCO, dDMOCC, dMMO2pCO, dMMO2pCC, dTPT3CO, and dTPT3CC (Figure 2) may be used. In each case, the ribosyl triphosphate of the unnatural nucleobase can be used for transcription to generate site-specifically labeled RNA, and the deoxyribosyl triphosphate of the unnatural nucleobase can be used, for example, to generate site-specifically labeled DNA. Disclosed and claimed herein are unnatural nucleobases containing an acetylene (alkynyl) linker group suitable for use in CuAAC conjugates, d5SICSCO, d5SICSCC, dDMOCO, dDMOCC, dMMO2pCO, dMMO2pCC, dTPT3CO, and dTPT3CC, methods for their preparation, and methods for their use in preparing site-specifically labeled oligonucleotides.

[0137] Demonstration of a general phosphorothioate strategy (Figure 3). To demonstrate the feasibility of our system, we prepared the α-thiotriphosphate of an unnatural nucleotide, d5SICS (d5SICS-αS), and incorporated it into DNA opposite its cognate unnatural nucleotide, dNaM, using standard PCR. The amplification efficiency and fidelity of d(5SICS-αS)TP incorporation were greater than 99%, virtually identical to the results obtained with d5SICS. To functionalize this unnatural base pair, we reacted the site-specifically incorporated phosphorothioate bond with iodoacetyl-PEG2-biotin to label the DNA duplex with a biotin functionality. To characterize this site-specific adduct, we incubated it in the presence of streptavidin and then quantified functionalization by gel shift assay. We were able to convert 60-70% of the phosphorothioate bond to a functionalized derivative. This is a typical efficiency (70%) for labeling protocols previously reported in the literature (Fidanza, J.A.; Ozaki, H.; McLaughlin, L.W., Site-specific labeling of DNA sequences containing phosphorothioate diesters. J. Am. Chem. Soc. 2002, 114 (14), 5509-5517.). These conjugates exhibited high stability under conditions typical of thermal denaturation of DNA duplexes, i.e., overnight at 50°C within the pH range of 6.0-8.3 (<10% degradation), as well as at 95°C for 3 minutes at pH 8.3 (<5% degradation). We believe that the phosphorothioate strategy can be used equally well with other unnatural base pairs, including d5SICS-dMMO2 and d5SICS-dNaM.

[0138] Provided herein, in various embodiments, is a phosphorothioate strategy that uses the unnatural base pairs dTPT3-dNaM, dTPT3-dMMO2, or dTPT3-dDMO, or linker-derivatized variants thereof.

[0139] The phosphorothioate and linker-based strategies are not mutually exclusive and, when combined, allow for simultaneous modification of a given site with up to three functional groups: one attached to the first nucleobase of the nucleobase pair, a second attached to the second nucleobase of the nucleobase pair, and a third attached to the backbone adjacent 5' to the unnatural nucleotide.

[0140] Demonstration of the primary amine-based linker strategy (Figure 4). To further demonstrate the feasibility of our system, we synthesized and characterized amino- and protected amino-linker-derivatized variants of d5SICS and dMMO2 (Figure 2). When paired in DNA opposite their cognate non-natural partners, we showed that each was successfully amplified by PCR and transcribed into RNA. Coupling of DNA containing dMMO2A or d5SICSA, prepared by PCR amplification with NHS-ester biotin, proceeded with 55% and 70% efficiency, respectively.

[0141] We showed that ribonucleotide triphosphates of 5SICSPA, 5SICSA, MMO2PA, or MMO2A were transcribed into RNA by T7 RNA polymerase with high efficiency and fidelity (Fig. 7 ).

[0142] Provided herein, in various embodiments, is site-specific modification of DNA or RNA using dTPT3L-dNaM, dTPT3L-dMMO2, or dTPT3L(d)-DMO (R is a linker, e.g., R=H for dTPT3, R=3-aminopropyn-1-yl for dTPT3A, R=dichloroacetyl-3-aminopropyn-1-yl for dTPT3PA, R=4-oxahepato-1,6-diyn-1-yl for dTPT3CO, and R=hepta-1,6-diyn-1-yl for dTPT3CC).

[0143] Demonstration of a general linker strategy using alkynes (Figure 5). To further demonstrate the feasibility of our system, we synthesized and characterized alkynyl-functionalized variants of d5SICS, dDMO, and dMMO2, including d5SICSCO, d5SICSCC, dDMOCO, dDMOCC, dMMO2pCO, and dMMO2pCC (Figure 2). Each of these alkyne-functionalized unnatural nucleotides should be efficiently PCR amplified when present in DNA. Once amplified, DNA containing, for example, d5SICSCO-dNaM base pairs can also be efficiently site-specifically modified with azide groups bearing small molecules or one or more proteins using click chemistry, e.g., copper-catalyzed click reactions. Furthermore, we demonstrated the utility of dEMO and dFEMO (Figure 2) for incorporation into oligonucleotides and the use of these functionalized oligonucleotides in click chemistry reactions that use azides to site-specifically functionalize oligonucleotides.

[0144] Demonstration of a linker strategy using dTPT3PA (Figure 6). Attachment of a linker to the d5SICS and dMMO2 scaffolds significantly reduces the efficiency with which unnatural nucleotides can be enzymatically incorporated into DNA, which is expected to limit their practical application. However, we have discovered that the dTPT3 scaffold is much more tolerant of linker addition (Figure 6). For example, dTPT3PATP is incorporated into the primer opposite dNaM in the template by DNA polymerase with virtually the same efficiency and fidelity as natural base pairs. Accordingly, various embodiments provide the use of unnatural nucleobases based on the dTPT3 scaffold, including dTPT3PA (protected amino-functional linker), dTPT3A (amino-functional linker), dTPT3CO (alkyne-azide ether linker for click chemistry derivatization), and dTPT3CC (alkyne-azide trimethylene linker for click chemistry derivatization), in the synthesis of site-specifically functionalized oligonucleotides.

[0145] Scheme 1 illustrates examples of dTPT3 containing various linkers that can be used to site-specifically modify DNA or RNA.

[0146] [ka]

[0147] For clarity, only the nucleobase portion of the dTPT3 scaffold is shown, but it is understood that these are used as nucleotides. Functionalization reactions can be performed before or after incorporation of the unnatural nucleobase into the oligonucleotide. Scheme 1 (top reaction) illustrates the use of dTPT3A, which contains a linker bearing a primary amine that is acylated using an activated ester to form an amide, with the R group containing a cargo. The middle reaction in Scheme 1 illustrates the use of dTPT3CO, which contains an alkynyl-bearing linker that is reacted with an azide to generate a triazole via click chemistry (dTPT3CC can also be used), with the R group of the resulting triazole containing a cargo. The bottom reaction illustrates the most common case of a dTPT3 scaffold derivative with a linker group R1 containing a reactive moiety that can selectively form a covalent bond with an R2 group containing a reactive moiety complementary to the reactive moiety of the linker, e.g., thiol-maleimide, hydrazine-aldehyde, etc.

[0148] In one embodiment, a linker containing an azide reactive group serves to attach to an aklyne-containing cargo via a click reaction. In one embodiment, a linker containing a thiol group can form a reversible disulfide bond or an irreversible bond with a variety of cargo-accepting groups, including but not limited to, maleimide, bromide, iodide, sulfonyl derivatives, activated esters, and isothiocyanate derivatives. In one embodiment, a linker containing an azide group is reactive to a cargo molecule containing a phosphine group.

[0149] In one embodiment, an oligonucleotide comprising one or more linker-derivatized unnatural nucleobases is configured for use as a molecular beacon, in which the fluorophore is a cargo molecule attached to the reactive center of the linker-derivatized unnatural nucleobase. Exemplary fluorophore cargo molecules include, but are not limited to, 6-FAM, fluorescein, Cy3™, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, Cy5™), TAMRA, MAX, TET™, ROX (carboxy-X-rhodamine), TYE™ 563, hexachlorofluorescein, TEX615, TYE™ 665, TYE705, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 750, IRDye® 800CW, ATTO™ 488, ATTO™ 532, Includes ATTO™ 550, ATTO™ 565, ATTO™ Rho101, ATTO™ 590, ATTO™ 633, ATTO™ 647N, Rhodamine Green™-X, Rhodamine Red™-X, 5-TAMRA™, Texas Red®-X, Lightcycler® 640, and Dy 750.

[0150] In some embodiments, unnatural base pairs enable the site-specific incorporation of various functionalities into DNA for use in SELEX (synthetic evolution of nucleic acids) applications, including the generation of DNA and / or RNA aptamers. DNA and RNA aptamers have a variety of targets, including nucleic acids, small molecules, peptides, carbohydrates, and cells. SELEX involves forming a library of nucleic acid molecules, contacting the library with a target molecule to select nucleic acid molecules that bind to the target molecule, and amplifying library members that bind to the target molecule. Selection and amplification are continued multiple times until sufficient aptamers are recovered. In one aspect, the aptamer comprises any of the unnatural bases disclosed herein. In some embodiments, a SELEX experiment in which library components include unnatural nucleobases generates aptamers with greater affinity for a target molecule in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds or fewer of selection than a library that does not contain unnatural nucleobases. In some embodiments, aptamers containing one or more unnatural nucleobases have greater affinity for a target molecule than aptamers containing only natural nucleobases. The addition of one or more unnatural nucleobases to a SELEX library increases the chemical and structural diversity of the resulting DNA or RNA aptamers. In some embodiments, the unnatural aptamer has at least nanomolar affinity for its target molecule. In some embodiments, the unnatural aptamer has at least picomolar affinity for its target molecule. For example, the unnatural aptamer has an affinity for its target molecule of 1-1,000 pM. In some embodiments, the unnatural aptamer has at least femtomolar affinity for its target molecule. For example, the unnatural aptamer has an affinity for its target molecule of 1-1,000 fM. Unnatural aptamers selected using SELEX can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more unnatural nucleobases. In some embodiments, the unnatural aptamer comprises dTPT3 or a derivative or analog thereof. In some embodiments, the unnatural aptamer comprises a nucleobase having the formula α14a, or a derivative or analog thereof.In some embodiments, the unnatural aptamer comprises a nucleobase having formula α14b, or a derivative or analog thereof. In some embodiments, the unnatural aptamer comprises a nucleobase having formula α14c, or a derivative or analog thereof. In some embodiments, the unnatural aptamer comprises a nucleobase having formula α14d, or a derivative or analog thereof. In some embodiments, the unnatural aptamer comprises a nucleobase having formula α14e, or a derivative or analog thereof. In some embodiments, the unnatural aptamer comprises a nucleobase having formula α14f, or a derivative or analog thereof. In some embodiments, the unnatural aptamer comprises a nucleobase having formula β8a, or a derivative or analog thereof. In some embodiments, the unnatural aptamer comprises a nucleobase having formula β8b, or a derivative or analog thereof.

[0151] The various combinations of components described above with respect to exemplary reaction mixtures and reaction methods can be provided in kit form. Such kits can include individual components that are separated from one another, e.g., carried in separate containers or packaging. Kits can include one or more subcombinations of components described herein, but where one or more subcombinations are separate from the other components of the kit. The subcombinations can be combined to form reaction mixtures described herein (or combined to carry out the reactions described herein). In certain embodiments, the subcombinations of components provided in individual containers or packaging are insufficient to carry out the reactions described herein.

[0152] However, the kit as a whole may include various containers or packaging, the contents of which can be combined to carry out the reactions described herein.

[0153] The kit may include suitable packaging materials for containing the contents of the kit. The packaging materials can be constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials used herein may include, for example, those commonly used in commercially available kits sold for use with nucleic acid sequencing systems. Typical packaging materials include, but are not limited to, glass, plastic, paper, foil, etc., which can hold the components described herein within certain limits.

[0154] The packaging material may include a label indicating a specific use of the components. The use of the kit indicated by the label may be one or more of the methods described herein as being suitable for the particular combination of components in the kit. For example, the label may indicate that the kit is useful for a method of conjugating a cargo molecule to a linker moiety of an unnatural nucleobase in an oligonucleotide.

[0155] Instructions for use of the packaged reagents or components may also be included in the kit, and typically include a clear statement of reaction parameters such as the relative amounts of kit components and sample to be mixed, maintenance periods for the reagent / sample mixture, temperature, buffer conditions, etc.

[0156] It will be understood that not all components required for a particular reaction need be present in a particular kit. Rather, one or more additional components may be obtained from other sources. Instructions provided with the kit may identify the additional components provided and where to obtain them.

[0157] In one embodiment, the kit provides one or more unnatural nucleobases or derivatives thereof and reagents configured for site-specific functionalization using the one or more unnatural nucleobases or derivatives thereof. [Example]

[0158] Currently, the free nucleosides and phosphoramidites of d5SICS and dNaM are commercially available from Berry and Associates (Dexter, MI).

[0159] Example 1. PCR-based screening to identify unnatural base pairs The α6 triphosphates were prepared from previously reported nucleosides (Kubelka, T., Slavetinska, L., Eigner, V., and Hocek, M. Synthesis of 2,6-disubstituted pyridin-3-yl C-2'-deoxyribonucleosides through chemoselective transformations of bromo-chloropyridine C-nucleosides. Org. Biomol. Chem., 11, 4702-4718) following the efficient synthesis of nucleoside 5'-O-(1-thiotriphosphate), 5'-triphosphate, and 2',3'-cyclophosphorotoate using Ludwig, J., and Eckstein, F. Rapid and 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one. The purity of all other triphosphates was confirmed by MALDI-TOF and UV-VIS. Taq and OneTaq DNA polymerases were purchased from New England Biolabs (Ipswich, MA). Deoxyribonucleotide triphosphate mix was purchased from Fermentas (Glen Burnie, MD). SYBR Green I nucleic acid gel stain (10,000x) was purchased from Life Technologies (Carlsbad, CA).The synthesis of the DNA templates used for rounds 1–5 of screening, D8 (Malyshev, D.A., Dhami, K., Quach, H.T., Lavergne, T., Ordoukhanian, P., Torkamani, A., and Romesberg, F.E. Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet. Proc. Natl. Acad. Sci. USA, 109, 12005–12010), and D6 (Malyshev, D.A., Seo, Y.J., Ordoukhanian, P., and Romesberg, F.E. PCR with an expanded genetic alphabet. J. Am. Chem. Soc., 131, 14620–14621), used for all other amplifications, was previously described. Sanger sequencing was performed as previously described (Malyshev, D.A., Dhami, K., Quach, H.T., Lavergne, T., Ordoukhanian, P., Torkamani, A. and Romesberg, F.E. Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet. Proc. Natl. Acad. Sci. USA, 109, 12005-12010).Raw Sanger sequencing traces were used to determine percent retention of unnatural base pairs, which was converted to fidelity per doubling as described (Malyshev, D.A., Dhami, K., Quach, H.T., Lavergne, T., Ordoukhanian, P., Torkamani, A. and Romesberg, FE. Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet. Proc. Natl. Acad. Sci. USA, 109, 12005-12010; Malyshev, D.A., Seo, Y.J., Ordoukhanian, P. and Romesberg, FE. PCR with an expanded genetic alphabet. J. Am. Chem. Soc., 131, 14620-14621).

[0160] All PCR amplifications were performed in a total volume of 25 μL using the following conditions: 1x OneTaq reaction buffer, 0.5x Sybr Green I, MgSO4 adjusted to 4.0 mM, 0.2 mM of each dNTP, 50 μM of each unnatural triphosphate, 1 mM Primer 1 and Primer 2 (see Table 2), and 0.02 U / μl DNA polymerase. Other conditions specific to each screening round are listed in Table 3. Amplified products were purified using DNA Clean and Concentrator-5 spin columns from Zymo Research (Irvine, CA). After purification, PCR products were sequenced on a 3730 DNA Analyzer (Applied Biosystems) to determine retention of unnatural base pairs as described below. Fidelity was characterized from unnatural base pair (UBP) retention as determined by sequencing with Primer1 on a 3730 DNA Analyzer (Applied Biosystems).

[0161] [Table 2]

[0162] [Table 3]

[0163] Specific PCR analysis conditions. PCR using the most promising UBPs was performed under the conditions described in Table 3. The PCR products were further purified on a 2% agarose gel, followed by single-band excision and subsequent cleanup using the Zymo Research Zymoclean Gel DNA Recovery Kit. After elution with 20 μl of water, DNA concentration was measured using fluorescent dye binding (Quant-iT dsDNA HS Assay kit, Life Technologies). UBP retention and therefore amplification fidelity were determined by sequencing the amplified products generated in triplicate with both Primer 1 and Primer 2. Amplification of DNA containing the α6 analog pair was performed with OneTaq polymerase under the following thermocycling conditions: initial denaturation at 96°C for 1 minute; 16 cycles of 96°C for 10 seconds, 60°C for 15 seconds, and 68°C for 1 minute. Fidelity was determined by sequencing the amplified products in Primer 1 in triplicate. Amplification of DNA containing the UBP formed between dTPT3 and d2MN or dDM2 was performed using OneTaq or Taq polymerase for 16 cycles under the following thermocycling conditions: 1) OneTaq: initial denaturation at 96°C for 1 min, 96°C for 10 s, 60°C for 15 s, and 68°C for 1 min; or 2) Taq: initial denaturation at 96°C for 1 min, 96°C for 5 s, 60°C for 5 s, and 68°C for 10 s. Fidelity was determined by sequencing the amplification products in Primer 1 in triplicate.

[0164] Results. To screen for well-represented UBPs, unnatural deoxynucleoside triphosphates were grouped for analysis into dMMO2 / dNaM- or d5SICS / dTPT3-like analogs, although the distinction was not entirely clear in all cases. In total, 80 dMMO2 / dNaM analogs were grouped into 12 "α groups" (α1-α12; Figure 8), and 31 d5SICS / dTPT3 analogs were grouped into six "β groups" (β1-β6; Figure 9). Note that the group designations used herein should not be confused with the anomeric designations (all tested analogs are β-glycosides). Additionally, to increase the structure-activity relationship (SAR) content of the screen, a substituted pyridyl nucleobase (Kubelka, T., Slavetinska, L., Eigner, V., and Hocekm, M. Synthesis of 2,6-disubstituted pyridin-3-yl C-2'-deoxyribonucleosides through chemoselective transformations of bromo-chloropyridine C-nucleosides. Org. Biomol. Chem., 11, 4702-4718) was phosphorylated to include group α6. For screening, a 134-mer single-stranded DNA template containing a centrally positioned dNaM (referred to as D8) was PCR-amplified in the presence of natural triphosphates (200 μM each), all pairwise combinations of α and β triphosphate bases shown in Figures 8 and 9 (50 μM each), and 0.02 U / μL DNA polymerase. During the first round of PCR, dNaM templates the incorporation of the α analog, which is then replaced by the β analog when the original strand is copied in the second round, and the resulting UBP is amplified in subsequent rounds. The amplification products of each reaction were analyzed by Sanger sequencing. The presence of unnatural nucleotides results in an abrupt termination reaction in the sequencing chromatogram, and the level of UBP retention can be quantified by the amount of readthrough. The percentage of UBP retained in DNA after amplification during each round of screening is shown in Figure 14.

[0165] The first round of screening used 0.1 nanograms of template and 16 cycles of amplification under relatively permissive conditions, including OneTaq polymerase and a 1-minute extension time. In this example, OneTaq is considered permissive because it is a mixture of Taq (a family A polymerase) and DeepVent (a family B polymerase), the latter of which has exonuclease proofreading that allows excision of incorrectly incorporated triphosphates. Under these conditions, only pairs containing groups β5 or β6 showed high retention.

[0166] The combinations of β5 or β6 and α groups that showed the highest retention were carried forward to a second round of screening, which divided them into smaller groups (denoted a, b, or c; Figures 8 and 9). High retention rates (≥97%) were observed for β5a with α2c, α9a, α9c, α10a, α10c, α12b, or α12c; β5b with α9a, α9b, α10cm, or α12b; and β6b with α10c. Moderate retention rates (84-96%) were observed for β5a and α1a, α1b, α6a, α9b, α10b, or α12a; β5b and α1a, α1b, α2c, α6a, α9c, α10a, α12a, or α12c; β6a and α1b or α10c; and β6b and α1a, α6a, α9a-c, α10a, α10b, or α12a-c.

[0167] For the third round of screening, α analogs were analyzed in groups of one to three compounds, and group β6a was subdivided into its two triphosphate constructs, dTPT1TP and dFPT1TP. The highest retention rates (≥90%) were observed for β5a with α1a, α2cII, α9a-c, α10aI, α10aII, α10c, α12b, or dTfMOTP; β5b with α9a, α9c, or α10c; dFPT1TP with α10aI; and β6b with α1a, α9a-c, α10aI, α10aII, α10c, α12b, dNMOTP, dTfMOTP, or dCNMOTP. Only slightly lower retention rates (80-89%) were observed for β5a and α2cI, α12a, dNMOTP, dQMOTP, or dTOK587TP; β5b and α1a, α2cII, α10aI, α10aII, α12b, or dTOK587TP; dFPT1TP and α10c; and β6b and α12a, dQMOTP, dFuMO1TP, or dTOK587TP.

[0168] For the fourth round of screening, all α derivatives in Figure 9 were analyzed as individual triphosphates, with the exception of α9b and α9c, which remained grouped. The highest retention (≥91%) was observed for β5a and α9b, α9c, dFIMOTP, dIMOTP, dFEMOTP, dMMO2TP, d2OMeTP, ​​dDMOTP, d5FMTP, dNaMTP, dVMOTP, dZMOTP, dClMOTP, dTfMOTP, dQMOTP, d2MNTP, dDM2TP, or dTOK587TP; β5b and α9b, α9c, dFIMOTP, dIMOTP, dFEMOTP, dNaMTP, dZMOTP, dClMOTP, dQMOTP, dMM1TP , dDM2TP, or dTOK587TP; the β6 analog dFPT1TP and the α analogs d2OMeTP or dNaMTP; and β6b and α9b, α9c, dFIMOTP, dIMOTP, dFEMOTP, dMMO2TP, dDMOTP, dTMOTP, dNMOTP, d5FMTP, dNaMTP, dVMOTP, dZMOTP, dClMOTP, dTfMOTP, dQMOTP, dCNMOTP, d2MNTP, dTOK587TP, or dFuMO2TP.

[0169] To increase the stringency of the screening, the fifth round was performed with Taq polymerase instead of OneTaq, because it lacks exonuclease proofreading and therefore increases the sensitivity of synthesis to mismatches. This round further separated all remaining α and β groups into individual triphosphates. The highest retention (≥90%) was observed for dSICSTP and dNaMTP; dSNICSTP and dNaMTP; dTPT2TP and dFDMOTP; dTPT3TP and dFIMOTP, dIMOTP, or dNaMTP; and dFTPT3TP and dFIMOTP, dIMOTP, dFEMOTP, dNMOTP, dNaMTP, dClMOTP, dTfMOTP, or dCNMOTP.

[0170] To better distinguish between UBPs, we advanced the 62 most promising candidate UBPs to a sixth round of screening, in which the template concentration was reduced 10-fold (to 10 pg), allowing for greater amplification and thus better discrimination. The template was then changed to D6 (Malyshev, DA, Seo, YJ, Ordoukhanian, P., and Romesberg, FE PCR with an expanded genetic alphabet. J. Am. Chem. Soc., 131, 14620-14621), in which the three nucleotides flanking one side of the unnatural nucleotide were randomized among the natural nucleotides. Furthermore, the denaturation and annealing steps were each reduced to 5 s, and the extension time was reduced to 10 s. Under these conditions, we explored amplification using OneTaq or Taq alone. Results using OneTaq showed the highest retention (>95%) for dSICSTP and dNaMTP; dSNICSTP and dFEMOTP; dTPT3TP and dFIMOTP, dIMOTP, dFEMOTP, dZMOTP, or dNaMTP; and dFTPT3TP and dIMOTP or dFEMOTP. Moderate retention (86%-94%) was observed for dSICSTP, dFEMOTP, or dDM2TP; d5SICSTP and dNaMTP; dSNICSTP or dIMOTP; dTPT2TP and dNaMTP; dTPT3TP and dNMOTP, dClMOTP, dQMOTP, dCNMOTP, or d2MNTP; and dFTPT3TP and dFIMOTP, dNaMTP, dZMOTP, dClMOTP, dTfMOTP, or dCNMOTP. Retention during Taq-mediated amplification was generally reduced compared to retention using OneTaq, but the general trends were similar. The highest retention (>96%) was observed for dTPT3TP and dFIMOTP or dIMOTP, and for dFTPT3TP and dFIMOTP. Slightly lower retention (89%-94%) was observed for dTPT3TP and dFEMOTP, dNaMTP, or dCNMOTP; and for dFTPT3TP and dIMOTP, dFEMOTP, dNaMTP, dClMOTP, dCNMOTP, or d2MNTP.

[0171] Amplification with the most promising combinations of triphosphates, dTPT3TP or dFTPT3TP, and dFIMOTP, dIMOTP, dFEMOTP, or dNaMTP, was then performed for 52 cycles, using Taq and a 10-s extension time to explore particularly stringent conditions, or OneTaq and a 30-s extension time to explore more practical conditions (Table 4). Both amplified strands were sequenced in triplicate to determine UBP retention with high accuracy. With Taq, dTPT3-dNaM, dTPT3-dFIMO, dFTPT3-dNaM, and dFTPT3-dFIMO showed the highest retention, while pairs containing dIMO and dFEMO showed somewhat less retention. With OneTaq, dTPT3-dNaM and dFTPT3-dNaM showed the highest retention, closely followed by dFTPT3-dFIMO and dTPT3-dFIMO.

[0172] [Table 4]

[0173] The screening data suggested that some pairs formed between dTPT3 and previously untested pyridine-based derivatives of α6 replicated reasonably well. Therefore, we tested the amplification of DNA containing these UBPs in triplicate using OneTaq with 16 amplification cycles including a 1-minute extension time (Table 5). Pairs formed between dTPT3 and dTOK580, dTOK582, or dTOK586 replicated poorly. However, pairs formed between dTPT3 and dTOK588, dTOK581, dTOK576, and dTOK587 were amplified with retention rates of 62%, 65%, 85%, and 94%, respectively.

[0174] [Table 5]

[0175] Finally, the screening data suggested that pairs formed between dTPT3 and d2MN or dDM2 were not replicated reasonably well, even though both d2MN and dDM2 possess putative essential ortho H-binding receptors. Therefore, these pairs were further tested by 16-cycle amplification with OneTaq or Taq alone, including either a 1-minute or 10-second extension time (Table 6). Only weak retention was observed with Taq alone. However, retention rates were superior for both pairs with OneTaq. The retention rates for the dTPT3-dDM2 pair were 58% and 69% with 1-minute and 10-second extension times, respectively. Remarkably, dTPT3-d2MN was amplified with 96% and 94% retention rates with 1-minute and 10-second extension times, respectively.

[0176] [Table 6]

[0177] Discussion. A PCR-based screen was performed to identify the most promising UBPs. To increase the SAR content of the screen, seven novel α-derivatives based on a pyridyl scaffold containing various substituents at the ortho and para positions relative to the glycosidic bond were included.

[0178] Structure-activity relationship data. Even under permissive conditions, with exonuclease proofreading and a 1-minute extension time, only the mixed grouping of α analogs with β analogs showed significant levels of retention, demonstrating that efficient replication requires pairing of α scaffolds with β scaffolds. However, the only dβ groups that showed high retention were β5 and β6. This reveals the privileged status of the d5SICS / dTPT3-like scaffold over all others tested. The preferential contribution to high retention by group β5 was found to be attributable to pairs containing dSICS ​​and, to a lesser extent, dSNICS, rather than pairs containing d5SICS. For example, under all conditions, dSICS-dNaM replicated better than d5SICS-dNaM. d5SICS results from the optimization of dSICS ​​for pairing with dMMO2; apparently, the increased volume of dNaM makes the added methyl group detrimental. Furthermore, dSNICS-dNaM replicated as well (with OneTaq) or better (with Taq) than d5SICS-dNaM, suggesting that the 6-aza substituent optimizes UBP synthesis by facilitating the insertion of unnatural triphosphates opposite dNaM or by increasing the efficiency with which unnatural nucleotides template dNaMTP insertion. Finally, dSNICS-dFEMO replicated better than d5SICS-dNaM as well, but only in the presence of proofreading, suggesting that although triphosphate insertion may be less efficient, increased elongation efficiency leads to an overall increase in fidelity. The preferential contribution to high fidelity by group β6 was provided by dTPT3 and dFTPT3. In general, both pair well with dNaM, dFEMO, dFIMO, or dIMO. dTPT3 paired particularly well with dFIMO and dIMO, suggesting that the para-iodo substituent mediates a favorable interaction and that it also pairs well with dFEMO, especially dNaM, in the presence of exonuclease activity. dFTPT3 paired well with either dIMO or dFEMO in the presence of exonuclease activity, as well as with dFIMO and dNaM in its absence.

[0179] Although nitrogen substituents of pyridine-based α analogs (group α6) were generally unfavorable for replication, more detailed analysis of UBPs formed with dTPT3 revealed several trends. Methyl, chloro, or amino substituents at the ortho position to the C-glycosidic bond did not generate sufficient replication pairs, likely due to insufficient extension after incorporation of the unnatural triphosphate. The ortho-methoxy substituent of dTOK581 results in superior replication, likely due to its ability to both hydrophobically pack the template during UBP synthesis and accept H-bonds, including those from polymerase-based H-bond donors, during extension. Data further revealed that the methylsulfanyl ortho-substituents of dTOK588 and dTOK576, and especially dTOK587, result in superior replication. This improvement is likely due to an optimal compromise between hydrophobic packing ability and the ability to accept H-bonds from the polymerase at the primer terminus. Additionally, the para-substituents in this series of derivatives can contribute to efficient replication, with bromo-substituents being the most efficient, followed by secondary methylsulfanyl groups, and finally simple methyl groups. When dTOK587 was paired with dTPT3 with its combination of ortho-methylsulfanyl and para-bromo-substituents, the resulting UBP replicated with 99.3% fidelity (calculated from retention levels) using OneTaq with a 1-minute extension time, slightly better than d5SICS-dMMO2 under similar conditions. Clearly, similar ortho-methylsulfanyl and para-bromo-substituents must be tested on more efficiently replicating α-like scaffolds, such as dFIMO and dNaM.

[0180] Replication of pairs formed between dTPT3 and d2MN or dDM2 merits discussion. DNA containing such pairs is not amplified by Taq alone but is fully amplified by OneTaq. This result was unexpected because neither d2MN nor dDM2 possesses the ortho H-bond acceptor hypothesized to be essential for extension of the nascent (natural or unnatural) primer end. Specifically, when a nucleotide is located at the end of the growing primer, the H-bond acceptor is positioned in the developing minor groove to accept an H-bond from the polymerase, and this H-bond is thought to be necessary for proper terminal alignment. When amplified with OneTaq at a 1-minute extension time, dTPT3-d2MN is replicated with 99.5% fidelity, which only decreases to 99.1% when the extension time is reduced to 10 s. The absence of amplification in the absence of proofreading, coupled with the slight decrease observed in the presence of proofreading as extension times decreased, suggests that the surprisingly high fidelity or amplification of DNA containing dTPT3-d2MN results from the selective extension of UBPs at mispairs. This suggests that the absence of an ortho H-bond acceptor is more detrimental to the extension of mispairs than to the extension of UBPs.

[0181] Efforts to Expand the Genetic Alphabet. Overall, the data confirm that dTPT3-dNaM is the most promising UBP tested. However, pairs formed between dTPT3 and dFEMO, dFIMO, or dIMO, or between dFTPT3 and dNaM, dFEMO, dFIMO, or dIMO, are similarly promising. In addition to the most promising UBPs listed above, it is noteworthy that a notable number of additional novel pairs replicate with only slightly reduced fidelity or replicate with high fidelity when amplification is performed under less stringent conditions (Table 7). In addition to the most efficiently replicated UBPs, where different physicochemical properties are expected to confer nucleotide constructs with various pharmacokinetically similar properties, these pairs offer diverse physicochemical properties to various scaffolds for further optimization efforts.

[0182] [Table 7]

[0183] Example 2. General procedure for triphosphate synthesis Proton sponge (1.3 equiv.) and free nucleoside derivative (1.0 equiv.) were dissolved in dry trimethyl phosphate (40 equiv.) and cooled to -15 °C under a nitrogen atmosphere. Freshly distilled POCl3 (1.3 equiv.) was added dropwise, and the resulting mixture was stirred at -10 °C for 2 h. Tributylamine (6.0 equiv.) and a solution of tributylammonium pyrophosphate (5.0 equiv.) in dimethylformamide (0.5 M) were added. The reaction was slowly warmed to 0 °C over 30 min and then quenched by the addition of 0.5 M aqueous Et3NH2CO3 (TEAB) pH 7.5 (2 volume equiv.). The mixture was diluted 2-fold with HO, and the products were separated on a DEAE Sephadex column (GE Healthcare) with an elution gradient of 0 to 1.2 M TEAB, evaporated, and co-evaporated with HO (3x). Further purification was performed by reverse-phase (C18) HPLC (0-35% CH3CN in 0.1 M TEAB, pH 7.5) (10%-31% yields).

[0184] [ka] Scheme S1. (a) Piperidine, Py, 100 °C, 12 h, then reflux for 1 h; (b) SOCl2, DMF, CHCl3, reflux for 3 h; (c) NaN3, 1,4-dioxane, H2O, 5 °C, 0.5 h; (d) diphenyl ether, 230 °C, 1 h

[0185] Nucleobase analogs 4a, 4b, 4c, and 4d were synthesized based on literature methods 1 and 2, as shown in Scheme S1. Briefly, aldehydes (1a–d) were condensed with malonic acid in piperidine as the solvent and pyridine as the catalyst at 100 °C for 12 h, followed by refluxing for 1 h, to give the corresponding acrylic acid intermediates (2a–d). Chlorination of these acids with thionyl chloride in chloroform in the presence of DMF gave acyl chlorides, which were not purified but could be used directly in the preparation of azides (3a–d). Compounds 3a–d were prepared in a biphasic mixture of 1,4-dioxane and water at 5 °C using sodium azide. The crude mixture of 3a–d in CHCl3 solution was added portionwise to diphenyl ether and heated to 230 °C to give the isocyanates, which underwent intramolecular cyclization to the fused 6-5 bicyclic systems 4a–d.

[0186] [ka] Schematic S2. (a) N,O-bis(TMS) acetamide, SnCl4 (1 M in CHCl2), CHCl2, 3 h, 45%; (b) Lawesson's reagent, toluene, reflux, overnight, 58%; (c) 30% NaOMe, MeOH, room temperature, 1 h, 85%; (d) proton sponge, POCl3, Bu3N, Bu3NPPi, (MeO)3P, DMF, -20 °C, 31%.

[0187] Compound 5a. To a solution of 4a (54 mg, 0.33 mmol) in CHCl (8 mL) at room temperature under a nitrogen atmosphere, bis(trimethylsilyl)acetamide (83 mg, 0.39 mmol) was added. After stirring for 40 min, 3,5-bis(toluoyl)-2-deoxyribosyl chloride (196 mg, 0.39 mmol) was added. The reaction mixture was cooled to 0 °C, and SnCl was added dropwise (1.0 M in CHCl, 160 μL, 0.16 mmol). The solution was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc, quenched with saturated aqueous NaHCO, extracted with EtOAc, dried, filtered, and evaporated. The crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 5a as a white foam (77 mg, 0.15 mmol, 45%). 1H NMR (500MHz, CDCl3) δ7.97-6.82(m, 11H, Ar-H), 6.44(d,J=7.5Hz, 1H, H-1'), 5.63(d,J=6.5Hz, 1H, H-3'), 4.76-4.68(m,2H,H-5'a, 5'b), 4.59(d,J =2.5Hz, H-4'), 2.89(dd, J=1.5, 0.5Hz, 1H, H-2'a), 2.59(s, 3H, Ar-CH3), 2.43(s, 3H, Ar-CH3), 2.43(s, 3H, Ar-CH3), 2.36-2.30(m, 1H, H-2'b).13C NMR (125MHz, CDCl3) δ166.6, 166.5, 158.5, 147.2, 144.8, 144.6, 140.0, 131.1, 130.3, 130.0, 1 29.9, 129.7, 127.1, 126.9, 125.6, 122.8, 102.7, 85.9, 83.3, 75.6, 64.8, 39.6, 22.1, 16.1.HRMS (ESI+) m / z calcd for C29H28NO6S (M+H+) 518.1632, found 518.1621

[0188] Compound 6a. Compound 5a (27 mg, 0.052 mmol) was dried by co-evaporation with anhydrous toluene. The residue was dissolved in anhydrous toluene (1 mL). Lawesson's reagent (41.5 mg, 0.10 mmol) was added, and the mixture was heated at reflux overnight. After filtration over cotton, the filtrate was concentrated, and the crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 6a (16 mg, 0.03 mmol, 58%) as a yellow foam. 1H NMR (500 MHz, CDCl3) δ 8.00-7.89 (m,4H, Ar-H), 7.71 (m,1H, Ar-H), 7.49-7.48 (m,1H, H-1'), 7.29-7.21 (m,4H, Ar-H), 7.65-7.62 (m,1H, Ar-H), 6.90 (d,J= 7.5 Hz, 1H, Ar-H), 5.64-5.62 (m,1H, H-4'), 4.85-4.74 (m,2H, H-5'a), 4.68-4.67 (m,1H, H-5'b), 3.38-3.34 (m,1H, H-3'), 2.26 (s,3H), 2.44 (s,3H), 2.41 (s,3H), 2.28-2.22 (m,1H).13C NMR (125 MHz, CDCl3) δ 174.6, 166.6, 144.9, 144.8, 142.9, 142.7, 142.3, 130.3, 130.0, 129.7, 127.9, 127.0, 126.9, 126.8, 108.3, 100.0, 91.4, 84.0, 74.9, 64.5, 39.3, 22.2, 22.1, 16.3.HRMS (ESI+) m / z calcd for C29H28NO5S2 (M+H+) 534.1403, found 534.1404.

[0189] Compound 7a. To a solution of 6a (20 mg, 0.037 mmol) in methanol (1.0 mL), 30% NaOMe (8.66 mg, 0.16 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and monitored by TLC. The reaction mixture was then concentrated, and the crude product was subjected to silica gel column chromatography (MeOH / CHCl) to give compound 7a (9.2 mg, 0.031 mmol, 85%) as a yellow foam. 1H NMR (500 MHz, CD3OD)oe 8.36 (d,J= 4 Hz, 1H, Ar-H), 7.58 (d,J= 1Hz, 1H, Ar-H), 7.35 (t,J= 4 Hz, 1H, H-1'), 7.22 (d,J= 8 Hz, 1H, Ar-H), 4.07-4.06 (m,1H, H-4'), 4.07 (d,J= 4 Hz, 1H, H-3'), 3.80 (dd,J= 24, 4 Hz, 2H, H-5'a, b), 2.79-2.76 (m,1H, H-2'a), 2.13-2.08 (m,1H, H-2'b).13C NMR (125 MHz, CD3OD)oe 173.60, 143.29, 142.26, 142.23, 129.35, 126.11, 108.01, 91.14, 88.44, 70.37, 61.35, 41.59, 14.81.HRMS (ESI+) m / z calcd for C13H16NO3S2 (M+H+) 298.0566, found 298.0569.

[0190] Compound 8a (dTPT1TP). Compound 8a (11.2 mg, 20.8 μmol, 31%) was synthesized using the general procedure for the synthesis of the triphosphate salt described above, starting from 7a (20 mg, 67.3 μmol). P NMR (162 MHz, DO) δ: -10.3 (d, J = 19.8 Hz, yP), -10.9 (d, J = 20.1 Hz, aP), -22.8 (t, J = 19.4 Hz, fP). MS (MALDI-TOF, matrix: 9-aminoacridine) (m / z): [MH]-calculated for C H NO P S , 536.3, found 536.7.

[0191] [ka] Scheme S3. (a) N,O-bis(TMS) acetamide, SnCl4 (1 M in CHCl2), CHCl2, 3 h, 41%; (b) Lawesson's reagent, toluene, reflux, overnight, 52%; (c) 30% NaOMe, MeOH, room temperature, 1 h, 85%; (d) proton sponge, POCl3, Bu3N, Bu3NPPi, (MeO)3P, DMF, -20 °C, 21%.

[0192] Compound 5b. To a solution of 4b (100 mg, 0.67 mmol) in CHCl (8 mL) at room temperature under a nitrogen atmosphere, bis(trimethylsilyl)acetamide (165 mg, 0.81 mmol) was added. After stirring for 40 min, 3,5-bis(toluoyl)-2-deoxyribosyl chloride (292 mg, 0.81 mmol) was added. The reaction mixture was cooled to 0 °C, and SnCl (1.0 M in CHCl, 200 μL, 0.2 mmol) was added dropwise. The solution was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc, quenched with saturated aqueous NaHCO, extracted with EtOAc, dried, filtered, and evaporated. The crude product was subjected to silica gel column chromatography (hexane / EtOAc) to afford compound 5b (137 mg, 0.27 mmol, 41%) as a white foam.1H NMR (500 MHz, CDCl3)oe 7.99 (d,J= 8.1 Hz, 2H, Ar-H), 7.93 (d,J= 8.1 Hz, 2H, Ar-H), 7.55 (d,J= 7.7 Hz, 1H, Ar-H), 7.33 - 7.28 (m,2H, Ar-H), 7.27 - 7.20 (m,2H,Ar-H), 6.82 (dd,J= 8.3, 5.6 Hz, 1H, Ar-H), 6.57 (d,J= 0.9 Hz, 1H, Ar-H), 6.41 (d,J= 7.7 Hz, 1H, H-1’), 5.68 - 5.61 (m,1H, H-4’), 4.75 (dd,J= 12.1, 3.4 Hz, 2H, H-5’a, b), 4.62 (q, J = 3.1 Hz, 1H, H￢3’), 2.94 (ddd,J= 14.3, 5.6, 1.7 Hz, 1H, H-2’a), 2.48 - 2.39 (s, 3x3H, Ar-CH3), 2.36 - 2.26 (m,1H, H-2’b).13C NMR (125 MHz, CDCl3)oe 166.6, 166.5, 159.2, 159.1, 154.5, 144.8, 144.6, 130.2, 130.0, 129.7, 127.5, 127.1, 126.9, 117.5, 103.3, 96.6, 86.0, 83.2, 75.5, 64.7, 39.8, 22.1, 14.1.HRMS (ESI+) m / z calcd for C29H28NO7 (M+H+) 502.1860, found 502.1885.。

[0193] Compound 6b. Compound 5b (29 mg, 0.056 mmol) was dried by co-evaporation with anhydrous toluene three times. The residue was dissolved in anhydrous toluene (1 mL). Lawesson's reagent (41.5 mg, 0.10 mmol) was added, and the mixture was heated at reflux overnight. After filtration through cotton, the filtrate was concentrated, and the crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 6b as a yellow foam (15 mg, 0.029 mmol, 52%). 1H NMR (500 MHz, CDCl3)oe 8.10-7.89 (m,5H, Ar-H), 7.52-7.48 (m,1H, H￢1'),7.29-7.22 (m,4H, Ar-H), 6.8 (d,J= 1Hz, 1H, Ar-H), 6.73 (d,J= 7.5 Hz, 1H,Ar-H), 5.65-5.62 (m,1H, H-4'), 4.84-4.74 (m,2H,H-5'a, b), 4.67-4.65 (m,1H, H-3'), 3.36-3.32 (m,1H, H-2'a), 2.44 (s, 3H, Ar-CH3), 2.43 (s,3H, s, 3H, Ar-CH3), 2.41 (s,3H, s, 3H, Ar-CH3), 2.27-2.21 (m,1H, H-2'b).13C NMR (125 MHz, CDCl3)oe 166.6, 156.9, 153.9, 144.8, 130.3, 130.0, 129.8, 129.7, 127.9, 106.4, 96.0,83.9, 56.6, 39.5, 22.1, 12.6.HRMS (ESI+) m / z calcd for C29H28NO6S (M+H+) 518.1632, found 518.1638.

[0194] Compound 7b. To a solution of 6b (20 mg, 0.039 mmol) in methanol (1.0 mL) was added dropwise 30% NaOMe (8.66 mg, 0.16 mmol). The reaction mixture was stirred at room temperature for 1 hour and monitored by TLC. The reaction mixture was then concentrated, and the crude product was subjected to silica gel column chromatography (MeOH / CHCl) to give compound 7b as a yellow foam (9.3 mg, 0.033 mmol, 85%). 1H NMR (500 MHz, CD3OD)oe 8.57 (d,J= 5 Hz, 1H, Ar-H), 7.42 (t,J= 4 Hz, 1H, H-1'), 7.13 (d,J= 7.5 Hz, 1H, Ar-H), 6.80 (s,1H, Ar-H), 4.50-4.47 (m,1H, H-4'), 4.12 (d,J= 3.5 Hz, 1H, H-3'), 3.95 (dd,J= 30, 3 Hz, 2H, H-5'a, b), 2.81-2.77 (m,1H, H-2'a), 2.50 (s,3H, Ar-CH3), 2.18-2.14 (m,1H, H-2'b).13C NMR (125 MHz, CD3OD) oe 172.9, 157.2, 154.5, 132.7, 131.3, 105.4, 100.8, 90.9, 88.5, 70.3, 61.3, 41.8, 12.7.HRMS (ESI+) m / z calcd for C13H16NO4S (M+H+) 282.0795, found 282.0790.

[0195] Compound 8b (dFPT1TP). Compound 8b (3.7 mg, 7.1 μmol, 10%) was synthesized using the general procedure for the synthesis of the triphosphate salt described above, starting from 7b (20 mg, 71.2 μmol). P NMR (162 MHz, DO) δ: -10.4 (d, J = 20.0 Hz, yP), -10.9 (d, J = 19.4 Hz, aP), -22.8 (t, J = 20.0 Hz, fP). MS (MALDI-TOF, matrix: 9-aminoacridine) (m / z): [MH]-calculated for C H NO P S, 520.3, found 520.1.

[0196] [ka] Scheme S4. (a) N,O-bis(TMS)acetamide, SnCl4 (1 M in CHCl2), CHCl2, 3 h, 40%; (b) Lawesson's reagent, toluene, reflux, overnight, 31%; (c) 30% NaOMe, MeOH, room temperature, 1 h, 81%; (d) proton sponge, POCl3, Bu3N, Bu3NPPi, (MeO)3P, DMF, -20 °C, 15%.

[0197] Compound 5c. To a solution of 4c (46 mg, 0.28 mmol) in CHCl (8 mL) at room temperature under a nitrogen atmosphere, bis(trimethylsilyl)acetamide (66 mg, 0.33 mmol) was added. After stirring for 40 min, 3,5-bis(toluoyl)-2-deoxyribosyl chloride (120 mg, 0.33 mmol) was added. The reaction mixture was cooled to 0 °C, and SnCl was added dropwise (1.0 M in CHCl, 140 μL, 0.14 mmol). The solution was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc, quenched with saturated aqueous NaHCO, extracted with EtOAc, dried, filtered, and evaporated. The crude product was subjected to silica gel column chromatography (hexane / EtOAc) to afford compound 5c as a white foam (58 mg, 0.11 mmol, 40%). 1H NMR (500 MHz, CDCl3)oe 7.98-7.90 (m,4H, Ar-H), 7.53 (d,J= 7.4 Hz, 1H, Ar-H), 7.27- 7.21 (m,4H, Ar-H), 6.83-6.82 (m,2H,Ar-H), 6.44 (d,J= 7.5 Hz, 1H, H￢1'), 5.63(d,J= 6.5 Hz, 1H, H-4'), 4.76 - 4.60 (m,2H, H-5'a, b), 4.59 (d,J= 2.5 Hz, 1H, H-3'), 2.89 (dd,J= 13, 5.5 Hz, H-2'a), 2.59 (s,3H, 2.43 (s,3H, Ar-CH3), 2.40 (s,3H, Ar-CH3), 2.37-2.30 (m,1H, H-2'b).13C NMR (125 MHz, CDCl3)oe 166.5, 158.0, 149.9, 146.3, 144.8, 144.6, 130.3, 130.0, 129.7, 128.8, 127.3, 122.8, 103.7, 100.0, 85.8, 83.2, 75.5, 64.8, 39.5, 22.1, 16.7.HRMS (ESI+) m / z calcd for C29H28NO6S (M+H+) 518.1632, found 518.1631.

[0198] Compound 6c. Compound 5c (50 mg, 0.097 mmol) was dried by co-evaporation with anhydrous toluene three times. The residue was dissolved in anhydrous toluene (1.5 mL). Lawesson's reagent (83 mg, 0.20 mmol) was added, and the mixture was heated at reflux overnight. After filtration through cotton, the filtrate was concentrated, and the crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 6c as a yellow foam (16 mg, 0.03 mmol, 31%). 1H NMR (500 MHz, CDCl3)oe 8.13-7.97 (m,5H, Ar-H), 7.52-7.49 (m,1H, H-1'), 7.37-7.29 (m,4H, Ar-H), 6.99 (d,J= 1Hz, 1H, Ar-H), 6.91 (d,J= 7.5 Hz, 1H, Ar-H), 5.73-5.71 (m,1H, H-4'), 4.91-4.82 (m,2H, H-5'a, b), 4.76-4.74 (m,1H, H-3'), 3.41-3.37 (m,1H, H-2'a), 2.68 (s,3H, Ar-CH3), 2.52 (s,3H, Ar-CH3), 2.49 (s,3H, Ar-CH3), 2.39-2.34 (m,1H, H-2'b).13C NMR (125 MHz, CDCl3)oe 172.1, 166.6, 154.0, 144.9, 144.7, 140.4, 130.3, 130.0, 129.7, 129.6, 127.0, 126.8, 122.7, 109.0, 91.2, 83.9, 75.0, 64.5, 39.4, 22.2, 22.1, 17.0.HRMS (ESI+) m / z calcd for C29H28NO5S2 (M+H+) 534.1403, found 534.1406.

[0199] Compound 7c. To a solution of 6c (20 mg, 0.037 mmol) in methanol (1.0 mL), 30% NaOMe (8.66 mg, 0.16 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and monitored by TLC. The reaction mixture was then concentrated, and the crude product was subjected to silica gel column chromatography (MeOH / CHCl) to give compound 7c as a yellow foam (8.9 mg, 0.03 mmol, 81%). 1H NMR (500 MHz, CD3OD)oe 8.48, (d,J= 7.5 Hz, 1H, Ar-H), 7.42 (t,J= 5 Hz, 1H, H-1'), 7.20 (d,J= 5 Hz, 1 H, Ar-H), 7.12 (s,1H, Ar-H), 4.51-4.48 (m,1H, H-4'), 4.13 (d,J= 5 Hz, 1H, H-3'), 3.95 (dd,J= 30, 5 Hz, 2H, H-5'a, b), 2.81-2.78 (m,1H, H-2'a), 2.67 (s,3H, Ar-CH3), 2.21-2.16 (m,1H, H-2'b).13C NMR (125 MHz, CD3OD)oe 171.1, 154.0, 144.1.141.1, 131.1, 122.7, 108.8, 90.9, 88.5, 70.5, 61.4, 41.7, 15.4.HRMS (ESI+) m / z calcd for C13H16NO3S2 (M+H+) 298.0566, found 298.0566.

[0200] Compound 8c. Compound 8c (10.8 mg, 20.2 μmol, 30%) was synthesized using the general procedure for the synthesis of the triphosphate salt described above starting from 7c (20 mg, 67.3 μmol). 31P NMR (162 MHz, DO) δ: -10.8 (d, J = 19.8 Hz, yP), -11.5 (d, J = 20.1 Hz, aP), -23.3 (t, J = 20.1 Hz, f3-P). MS (MALDI-TOF, matrix: 9-aminoacridine) (m / z): [MH]-calcd for C13H17NO12P3S2, 536.3, found, 536.1

[0201] [ka] Scheme S5. (a) N,O-bis(TMS) acetamide, SnCl4 (1 M in CHCl2), CHCl2, 3 h, 39%; (b) Lawesson's reagent, toluene, reflux, overnight, 33%; (c) 30% NaOMe, MeOH, room temperature, 1 h, 82%; (d) proton sponge, POCl3, Bu3N, Bu3NPPi, (MeO)3P, DMF, -20 °C, 30%.

[0202] Compound 5d. To a solution of 4d (200 mg, 1.32 mmol) in CHCl (8 mL) at room temperature under a nitrogen atmosphere, bis(trimethylsilyl)acetamide (298 mg, 1.46 mmol) was added. After stirring for 40 min, 3,5-bis(toluoyl)-2-deoxyribosyl chloride (563 mg, 1.46 mmol) was added. The reaction mixture was cooled to 0 °C, and SnCl was added dropwise (1.0 M in CHCl, 660 μL, 0.66 mmol). The solution was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc, quenched with saturated aqueous NaHCO, extracted with EtOAc, dried, filtered, and evaporated. The crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 5d (260 mg, 0.52 mmol, 39%) as a white foam. 1H NMR (500 MHz, CDCl3)oe 7.98-7.90 (m,4H, Ar-H), 7.70 (d,J= 6 Hz, 1H, Ar-H), 7.55 (d,J= 9.5 Hz, 1H, Ar-H), 7.28-7.16 (m,5H, Ar-H), 6.84-6.85 (m,1H, Ar-H), 6.57 (d,J= 9.5 Hz, H-1'), 5.66-5.64 (m,1H, H-4'), 4.75-4.72 (m,2H, H-5'a, b), 4.61 (m,1H, H-3'), 2.95-2.90 (m,1H, H-2'a), 2.43 (s,3H, Ar-CH3), 2.40 (s,3H, Ar-CH3), 2.39-2.31(m,1H, H-2'b).13C NMR (125 MHz, CDCl3)oe 166.2, 166.1, 158.1, 145.1, 144.4, 144.2, 133.8, 129.9, 129.6, 129.3, 129.1, 126.9, 126.5, 124.2, 103.5, 85.5, 82.9, 75.1, 64.4, 39.2, 21.7.HRMS (ESI+) m / z calcd for C20H20Cl2N2O5S (M+H+) 504.1475, found 504.1480.

[0203] Compound 6d. Compound 5d (50 mg, 0.1 mmol) was dried by co-evaporation with anhydrous toluene three times. The residue was dissolved in anhydrous toluene (1 mL). Lawesson's reagent (48 mg, 0.12 mmol) was added, and the mixture was heated at reflux overnight. After filtration through cotton, the filtrate was concentrated, and the crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 6d (17 mg, 0.033 mmol, 33%) as a yellow foam. 1H NMR (500 MHz, CDCl3)oe 8.14-7.82 (m, 7H, Ar-H), 7.51 (dd,J= 7.5, 6.0 Hz, 1H, H-1'), 7.32-7.23 (m,5H, Ar-H), 6.99 (d,J= 7.2 Hz, 1H, Ar-H), 74-5.73 (m,1H, H-4'), 4.92-4.83 (m,2H, H-5'a, b), 4.78-4.77 (m,1H, H-3'), 3.43-3.40 (m,1H, H-2'a), 2.51 (s,3H, Ar-CH3), 2.48 (s,3H, Ar-CH3), 2.39-2.36 (m,1H, H-2'b).13C NMR (125 MHz, CDCl3)oe 173.5, 166.6, 144.9, 144.8, 139.5, 138.0, 134.5, 130.3, 130.0, 129.7, 129.5, 126.8, 124.7, 109.5, 91.4, 84.0, 75.0, 64.5, 39.4, 22.2, 22.1.HRMS (ESI+) m / z calcd for C28H26NO5S2 (M+H+) 520.1247, found 520.1241.

[0204] Compound 7d. To a solution of 6d (20 mg, 0.039 mmol) in methanol (1.0 mL), 30% NaOMe (8.66 mg, 0.16 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and monitored by TLC. The reaction mixture was then concentrated, and the crude product was subjected to silica gel column chromatography (MeOH / CHCl) to give compound 7d (9.0 mg, 0.032 mmol, 82%) as a yellow foam. 1H NMR (500 MHz, CD3OD)oe 8.48 (d,J= 5 Hz, 1H, Ar-H), 8.01 (d,J= 5 Hz, 1H, Ar-H), 7.40-7.38 (m,2H, Ar-H), 7.29 (d,J= 10 Hz, 1H, H￢1'), 4.47-4.46 (m,1H, H-4'), 4.10 (m,1H, H-3'), 3.94-3.88 (m,2H, H-5'a ,b), 2.77-2.76 (m,1H, H-2'a), 2.19-2.14 (m,1H, H-2'b).13C NMR (125 MHz, CD3OD)oe 171.2, 144.7, 139.6, 137.6, 130.5, 124.2, 108.8, 90.7, 88.2, 70.1, 61.0, 41.3.HRMS (ESI+) m / z calcd for C12H14NO3S2 (M+H+) 284.041, found 284.0410.

[0205] Compound 8d (dTPT3TP). Compound 8d (5.7 mg, 10.9 μmol, 31%) was synthesized using the general procedure for the synthesis of the triphosphate salt described above, starting from 7d (10 mg, 35.3 μmol). P NMR (162 MHz, DO) δ: -9.3 (d, J = 19.5 Hz, yP), -10.8 (d, J = 19.8 Hz, aP), -22.4 (t, J = 20.0 Hz, fP). MS (MALDI-TOF, matrix: 9-aminoacridine) (m / z): [MH]-calculated for C12H15NO12P3S2-, 521.9, found 521.9.

[0206] [ka] Scheme S6. (a) i. Selectfluor, MeOH / CH3CN, reflux, 3 h; ii. TfOH-CH2Cl2 (1:1 v / v), 1 h, 85%; (b) Lawesson's reagent, toluene, reflux, overnight, 32%; (c) 30% NaOMe, MeOH, room temperature, 1 h, 85%; (d) proton sponge, POCl3, Bu3N, Bu3NPPi, (MeO)3P, DMF, -20 °C, 10%.

[0207] Compound 9. Compound 5d (55 mg, 0.11 mmol) was dissolved in 1.0 mL of MeOH-CHCN (1:1 v / v), Selectfluor (42 mg, 0.12 mmol) was added, and the mixture was heated at reflux for 3 h. The solvent was then evaporated, the residue was dissolved in 20 mL of EtOAc, and the organic phase was washed three times with water. The organic solvent was then evaporated, and the solid residue was dried by co-evaporation with anhydrous toluene three times. The residue was dissolved in 1 mL of TfOH-CHCl (1:1 v / v), and the mixture was stirred at room temperature for 1 h. The mixture was then concentrated, and the crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 9 (49 mg, 0.093 mmol, 85%) as a white solid. 1H NMR (500 MHz, CDCl3)oe 7.98-7.92 (m,4H, Ar-H), 7.75 (d,J= 5 Hz, 1H, Ar-H), 7.52 (d,J= 7.5 Hz, 1H, Ar-H), 7.32-7.21 (m,5H, Ar-H), 6.82-6.78 (m,1H, H-1'), 5.64-5.61 (m,1H, H-4'), 4.80-4.59 (m,2H, H-5'a, b), 4.62-4.59 (m,1H, H-3'), 2.93- 2.87 (m,1H, H-2'a), 2.43 (s,3H, Ar-CH3), 2.39 (s,3H, Ar-CH3), 2.34-2.27 (m,1H, H-2'b).13C NMR (125 MHz, CDCl3)oe 166.2, 166.1, 156.4, 144.5, 144.3, 137.7, 137.5, 134.6, 129.9, 129.6, 129.3, 126.6, 126.4, 120.2, 112.1, 111.7, 85.5, 83.1, 75.0, 64.2, 39.1, 21.8, 21.7.19F NMR (376 MHz, CDCl3)oe-151.5.HRMS (ESI+) m / z calcd for C28H25FNO6S (M+H+) 522.1381, found 522.1380.

[0208] Compound 10. Compound 9 (20 mg, 0.038 mmol) was dried by co-evaporation with anhydrous toluene three times. The residue was dissolved in anhydrous toluene (1 mL). Lawesson's reagent (18.5 mg, 0.046 mmol) was added, and the mixture was heated at reflux overnight. After filtration through cotton, the filtrate was concentrated, and the crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 10 (6.5 mg, 0.012 mmol, 32%) as a yellow foam. 1H NMR (500 MHz, CDCl3)oe 8.11-7.85 (m, 6H, Ar-H), 7.40-7.39(m,2H, Ar-H, H-1'), 7.28-7.21 (m,4H, Ar-H), 5.64-5.63 (m,1H, H-4'), 4.83 13C NMR (125 MHz, CDCl3)oe 170.9, 166.6, 166.5, 144.9, 144.8, 138.6, 130.3, 130.0, 129.9, 129.7, 129.7, 126.9, 126.7, 120.5, 116.3, 116.0, 100.0, 91.6, 84.3, 74.7, 64.3, 39.2, 22.2, 22.1.19F NMR (376 MHz, CDCl3)oe-142.9. HRMS (ESI+) m / z calcd for C28H25FNO5S2 (M+H+) 538.1153, found 538.1155.

[0209] Compound 11. To a solution of 10 (10 mg, 0.019 mmol) in methanol (1.5 mL), 30% NaOMe (4.33 mg, 0.08 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 h and monitored by TLC. The reaction mixture was then concentrated, and the crude product was subjected to silica gel column chromatography (MeOH / CHCl) to give compound 11 (4.9 mg, 0.016 mmol, 85%) as a yellow foam. 1H NMR (500 MHz, CD3OD)oe 8.68 (d,J= 5 Hz, 1H, Ar-H), 8.12 (d,J= 5 Hz, 1H, Ar-H), 7.52 (d,J= 5 Hz, 1H, Ar-H), 7.28 (t,J= 6.5 Hz, 1H, H-1'), 4.48 (m,1H, H-4'), 4.10 (m,1H, H-3'), 3.94 (dd,J= 35, 3 Hz, 2H, H-5'a, b), 2.78-2.75 (m,1H, H-2'a), 2.24-2.19 (m,1H, H-2'b).13C NMR (125 MHz, CD3OD)oe 170.2, 150.2, 148.3, 139.0, 131.7, 131.6, 119.8, 117.8, 117.4, 91.5, 88.7, 70.1, 61.0, 41.5.19F NMR (376 MHz, CD3OD)oe-145.3.HRMS (ESI+) m / z calcd for C12H13FNO3S2 (M+H+) 302.0315, found 302.0314.

[0210] Compound 12 (dFTPT3TP). Compound 12 (2.0 mg, 3.7 μmol, 22%) was synthesized using the general procedure for the synthesis of the triphosphate salt described above, starting from 11 (5 mg, 16.6 μmol). P NMR (162 MHz, DO) oe -10.9 (d, J = 20.0 Hz, yP), -11.6 (d, J = 21.1 Hz, aP), -23.3 (t, J = 23.1 Hz, fP). F NMR (376 MHz, DO) oe -138.5 (s). MS (MALDI-TOF, matrix: 9-aminoacridine) (m / z): [MH]- calculated for C H F NO P S -, 539.9, found, 540.1.

[0211] [ka] Scheme S7. (a) ICl, CHCl, 0°C to room temperature, overnight, 63%; (b) Lawesson's reagent, toluene, reflux, overnight, 27%; (c) 2,2-dichloro-N-prop-2-yn-1-ylacetamide, (PPh)Pd, CuI, EtN, DMF, room temperature, overnight, 91%; (d) 30% NaOMe, MeOH, room temperature, 1 h, 74%; (e) proton sponge, POCl, BuN, BuNPPi, (MeO)P, DMF, -20°C, 25%.

[0212] Compound 13. To a solution of 5d (73 mg, 0.145 mmol) in CHCl (1 mL) at 0 °C under a nitrogen atmosphere, iodine monochloride (1.0 M in CHCl, 0.15 mL, 0.15 mmol) was added dropwise. The resulting mixture was stirred overnight at room temperature. The reaction mixture was quenched with saturated aqueous NaHCO and saturated aqueous NaSO, extracted with CHCl, dried, filtered, and evaporated. The crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 13 (57 mg, 0.091 mmol, 63%) as a white foam. 1H NMR (500 MHz, CDCl3) δ 7.98-7.93 (m,4H, Ar-H), 7.83 (s,1H, Ar-H), 7.72 (d,J= 5 Hz, 1H, Ar-H), 7.28-7.17 (m,5H, Ar-H), 6.78-6.75 (m,1H, H-1'), 5.65-5.63 (m,1H, H-4'), 4.76 (m,2H, H-5'a, b), 4.63-4.62 (m,1H, H-3'), 2.95-2.91 (m,1H, H-2'a), 2.43 (s,3H, Ar-CH3), 2.35 (s,3H, Ar-CH3), 2.34-2.29 (m,1H, H-2'b).13C NMR (125 MHz, CDCl3) δ 166.6, 166.5, 157.6, 147.4, 144.9, 144.6, 133.7, 132.7, 130.3, 130.1, 129.8, 129.7, 129.4, 128.5, 127.0, 126.8, 86.1, 83.8, 75.7, 64.7, 39.9, 22.2, 22.1.HRMS (ESI+) m / z calcd for C28H25INO5S (M+H+) 630.0442, found 630.0440.

[0213] Compound 14. Compound 13 (30 mg, 0.048 mmol) was dried by co-evaporation with anhydrous toluene three times. The residue was dissolved in anhydrous toluene (1 mL), Lawesson's reagent (23 mg, 0.057 mmol) was added, and the mixture was heated at reflux overnight. After filtration through cotton, the filtrate was concentrated, and the crude product was subjected to silica gel column chromatography (hexane / EtOAc) to give compound 14 (8.4 mg, 0.013 mmol, 27%) as a yellow foam. 1H NMR (500 MHz, CDCl3) δ 8.31 (s,1H, Ar-H), 7.99-7.82 (m,5H, Ar-H), 7.39-7.36 (m,1H, H-1'), 7.29-7.20 (m,5H, Ar-H), 5.65-5.64 (m,1H, H-4'), 4.83-4.81 (m,2H, H-5'a, b), 4.71-4.70 (m,1H, H-3'), 3.35 (dd,J= 15, 5.5 Hz, 1H, H-2'a), 2.44 (s,3H, Ar-CH3), 2.39 (s,3H, Ar-CH3), 2.27-2.21 (m,1H, H-2'b).13C NMR (125 MHz, CDCl3) δ 172.9, 166.6, 144.9, 144.7, 144.6, 141.8, 137.8, 135.0, 130.3, 130.2, 129.8, 129.7, 128.6, 126.9, 126.7, 91.7, 84.5, 75.3, 64.6, 39.5, 22.2, 22.1.HRMS (ESI+) m / z calcd for C28H25INO5S2 (M+H+) 646.0213, found 646.0219.

[0214] Compound 15. To a solution of 14 (10 mg, 0.015 mmol) in DMF (2 mL) under a nitrogen atmosphere, (PPh3)4Pd (1.7 mg, 0.0015 mmol), CuI (0.57 mg, 0.011 mmol), and Et3N (5 μL, 0.030 mmol) were added. The reaction mixture was degassed, and a solution of Cl2CHCONHCH2CCH (3.8 mg, 0.0225 mmol) in DMF (0.5 mL) was added. The reaction mixture was stirred overnight at room temperature and monitored by TLC. The reaction mixture was diluted with EtOAc, quenched with saturated aqueous NaHCO3, extracted with EtOAc, dried, filtered, and evaporated. The crude product was subjected to silica gel column chromatography (MeOH / CHCl2) to give compound 15 (9.2 mg, 0.0135 mmol, 91%) as a yellow foam. 1H NMR (500 MHz, CDCl3) δ 8.26 (s,1H, Ar-H), 7.99-7.82 (m,5H, Ar-H), 7.40-7.37 (m,2H, Ar-H, H-1'), 7.29-7.21 (m, 4 H, Ar-H), 6.71 (br, 1H, NH),6.95 (s,1H, CHCl2), 5.65-5.64 (m,1H, H-4'), 4.85-4.79 (m,2H, H-5'a, b), 4.73 (m,1H, H-3'), 4.26-4.11 (m,2H, NHCH2), 3.38-3.34 (m,1H, H-2'a), 2.44 (s,3H, Ar-CH3), 2.40 (s,3H, Ar-CH3), 2.31-2.25 (m,1H, H-2'b).13C NMR (125 MHz, CDCl3)oe 173.3, 166.6, 164.1, 144.8, 139.0, 138.3, 133.4, 130.3, 130.1, 129.8, 129.7, 126.9, 124.3, 104.8, 91.8, 88.3, ​​84.5, 78.8, 75.2, 66.4, 64.7, 39.6, 31.3, 22.2. (ESI+) m / z calcd for C33H29Cl2N2O6S2 (M+H+) 683.0839, found 683.0854.

[0215] Compound 16. To a solution of 15 (9.2 mg, 0.0135 mmol) in methanol (1.0 mL) was added dropwise 30% NaOMe (2.92 mg, 0.32 mmol). The reaction mixture was stirred at room temperature for 1 hour and monitored by TLC. The reaction mixture was concentrated, and the crude product was subjected to silica gel column chromatography (MeOH / CHCl) to give compound 16 (4.5 mg, 0.01 mmol, 74%) as a yellow foam. 1H NMR (500 MHz, CD3OD)oe 8.69 (s,1H, Ar-H), 8.06 (d,J= 5 Hz, 1H, Ar-H), 7.53 (d,J= 5 Hz, 1H, Ar-H), 7.30 (t,J= 5 Hz, 1H, H-1'), 6.33 (s,1H, CHCl2 ), 4.47-4.46 (m,1H, H-4'), 4.36 (s, 2H, NHCH2), 4.11-4.08 (m,1H, H-3'), 3.97 (dd,J= 12, 3Hz, 2H, H-5'a, b), 2.79-2.74 (m,1H, H-2'a), 2.21-2.16 (m,1H, H-2'b).13C NMR (125 MHz, CD3OD)oe 172.9, 138.4, 134.3, 123.9, ,122.8, 104.7, 100.0, 91.2, 88.6, 77.1, 70.3, 66.4, 61.1, 41.7, 30.2.HRMS (ESI+) m / z calcd for C17H17Cl2N2O4S2 (M+H+) 447.0001, found 447.0020.

[0216] Compound 17 (dTPT3PATP). Compound 17 (2.2 mg, 3.1 μmol, 28%) was synthesized using the general procedure for the synthesis of the triphosphate salt described above, starting from 16 (5 mg, 11.2 μmol). P NMR (162 MHz, DO) δ: -10.85 (d, J = 19.9 Hz, yP), -11.63 (d, J = 20.0 Hz, aP), -23.07 (s), -23.26 (t, J = 19.7 Hz, f3-P). MS (MALDI-TOF, matrix: 9-aminoacridine) (m / z): [MH]-calculated for C17H18Cl2N2O13P3S2-, 684.9, found, 685.0.

[0217] Example 3. General procedure for PCR amplification to determine fidelity. Materials. Taq and OneTaq DNA polymerases were purchased from New England Biolabs (Ipswich, MA). dNTP mix was purchased from Fermentas (Glen Burnie, MD). SYBR Green I nucleic acid gel stain (10,000x) was purchased from Life Technologies (Carlsbad, CA).

[0218] DNA Oligonucleotides. Complete oligonucleotide sequences are provided in Table 8. Complete natural primers were purchased from Intergrated DNA Technologies (Coralville, Iowa). Reagents and solvents for the synthesis of non-natural primers 1-3 were obtained from Glen Research (Sterling, VA) and / or Applied Biosystems (Foster City, CA). Oligonucleotides were prepared using standard automated DNA synthesis with extra-light natural phosphoramidites (Glen Research) and dNaM phosphoramidites (Berry & Associates, Inc.) on a controlled pore glass support (0.20 μmol, 1000A, Glen Research) and an ABI Expedite 8905 synthesizer. After automated synthesis, the oligonucleotides were cleaved from the support, deprotected by incubation in concentrated aqueous ammonia overnight at room temperature, purified by DMT purification (glen-pak™ cartridges, Glen Research), and desalted on Sephadex G-25 (NAP-25 columns, GE Healthcare). The concentration of the single-stranded oligonucleotides was determined by UV absorption at 260 nm.

[0219] PCR analysis. PCR amplification was performed in a total volume of 25 μL and under conditions specific to each analysis, as described in Table 9. After amplification, a 5 μL aliquot was analyzed on a 6% non-denaturing PAGE gel run with a 50 bp ladder (Life Technologies) to confirm the size of the amplified product. The remaining solution was purified by a spin column (DNA Clean and Concentrator-5; Zymo Research, Irvine, CA) and then by a 4% agarose gel, recovered with a Zymoclean Gel DNA Recovery Kit (Zymo Research), quantified by fluorescent dye binding (Quant-iT dsDNA HS Assay kit, Life Technologies), and sequenced on a 3730 DNA Analyzer (Applied Biosystems). Fidelity was determined as the average % retention of unnatural base pairs per doubling, as described below.

[0220] Fidelity Determination. Percent retention of unnatural base pairs (F) was measured using raw sequencing data and normalized to fidelity per doubling. Briefly, the presence of unnatural nucleotides causes an abrupt termination of the sequencing profile, and mutation to natural nucleotides results in "readthrough." The degree of "readthrough" is thus inversely correlated with unnatural base pair retention. To use sequencing data as a quantitative measure of PCR fidelity, we performed calibration experiments ranging from 50-100% retention of unnatural base pairs. Therefore, low retention (<50%) and high "readthrough" lead to inaccurate quantification.

[0221] High retention rates (>50%) were quantified by adjusting the start and end points in the Sequencing Analysis software (Applied Biosystems) and determining the individual average signal intensities of each pathway (A, C, G, and T) for peaks within these defined points (35-45 nucleotides in length) before (section L) and after S36 (section R). The R / L ratios were normalized using a sequencing calibration plot to account for both noise in the sequencing chromatogram and readthrough in the control sample. The normalized R / L ratio (R / L norm) corresponds to the proportion of natural sequences in the pool. Finally, F was calculated as 1-(R / L) norm, and the retention rate of unnatural base pairs per doubling (fidelity, f) was calculated as 1 / (Flog2A), where A is the amplification and log2A is the number of doublings. Sequencing errors were minimized by sequencing each sample in triplicate in each direction before and after PCR amplification. The corresponding data are provided in Table 10. Under standard PCR conditions, DNA containing dTPT3-dNaM was amplified by OneTaq with greater than 99.98% fidelity and only 4-fold less efficiency than DNA containing only natural base pairs. This fidelity corresponds to an error rate of 10-4 per nucleotide, which overlaps with the error rates of 10-4 to 10-7 for fully natural DNA using commonly used PCR systems. Using Taq polymerase, the efficiency is only 2.5-fold lower than natural base pairs, with a fidelity of 99.7%. This fidelity corresponds to an error rate of 10-3, which is similar to the error rate observed in Taq-mediated amplification of natural DNA.

[0222] [Table 8]

[0223] [Table 9]

[0224] [Table 10]

[0225] Example 4: Site-specific labeling of TPT3: Analysis by streptavidin gel shift assay. A 134-mer DNA containing a centrally located dTPT3PA-dNaM or d5SICSPA-dNaM was synthesized. The DNA templates were amplified by PCR under the conditions described in Table 9. Upon completion, NaOH (1 M, 12.5 μL) was added directly to the PCR sample to a final concentration of 0.2 M and incubated at room temperature for 5 hours. After the addition of NaOAc (3 M, pH 5.5, 7.5 μL) and 200 μL of cold ethanol, the sample was mixed and incubated overnight on ice, and the DNA was precipitated by centrifugation at 10,000 rfu for 30 minutes at 4°C. The supernatant was removed, and the pellet was carefully washed with 80% ethanol. The sample was resuspended in 50 μL of annealing buffer (50 mM Na phosphate, pH 7.5, 100 mM NaCl, 1 mM EDTA), heated to 95°C, and cooled to room temperature for 30 minutes. NHS-PEG4-biotin (Thermo Scientific) solution in annealing buffer (40 mM, 50 μL) was mixed with the DNA sample and incubated overnight at room temperature. The sample was purified using a spin column (DNA Clean and Concentrator-5 (Zymo Research)) and eluted in 10 μL of elution buffer. Half of the sample (5 μL) was mixed with 1 μg of streptavidin (Promega) in annealing buffer, incubated at 37°C for 30 minutes, mixed with 5x non-denaturing loading solution (Qiagen), and loaded onto a 6% non-denaturing PAGE. The other half was mixed with 5x non-denaturing loading solution and loaded directly onto the gel as a control. After running the gel at 110V for 30 minutes, the gel was immersed in 1x Sybr Gold Nucleic Acid Stain (Life Technologies) for 30 minutes and visualized using a Molecular Imager Gel Doc XR+ equipped with a 520DF30 filter (Bio-Rad). A diagram of the labeling strategy described is provided below.

[0226] [ka]

[0227] Example 5. General procedure for transcription of unnatural base pairs. To characterize the transcription of unnatural base pairs formed by dTPT3 and dNaM, or their analogs or derivatives (the derivatives contain a linker moiety), ribonucleotides and deoxyribonucleotides were synthesized and converted to the corresponding triphosphates or deoxyphosphoramidites, and the deoxyphosphoramidites were incorporated into DNA templates using an automated DNA synthesizer. Transcription experiments were performed using 100 nM DNA substrate, 1x Takara buffer (40 mM Tris-HCl, pH 8.0, 8 mM MgCl2, 2 mM spermidine), DEPC-treated, nuclease-free sterile water (Fisher), T7 polymerase (50 units), 20 μM of each natural NTP, α-32P-ATP (2.5 μCi, MP Biomedicals), and 5 μM TPT3TP or 5 μM NamTP. After 2 h of incubation at 37 °C, the reaction is quenched by adding 10 μL of gel loading solution (10 M urea, 0.05% bromophenol blue), and the reaction mixture is loaded onto a 20% polyacrylamide-7 M urea gel, electrophoresed, and analyzed by phosphorimaging. Transcription efficiency is determined by measuring the amount of full-length product formed (at low conversion rates) as a function of time.

[0228] Example 6. General procedure for thermodynamic analysis of DNA duplexes containing unnatural base pairs. UV melting experiments are performed using a Cary 300Bio UV-visible spectrophotometer. The absorbance of the sample (3 μL of oligonucleotide containing unnatural base pairs, 10 mM PIPES buffer, pH 7.0, 100 mM NaCl, 10 mM MgCl2) is monitored at 260 nm from 21 °C to 80 °C at a heating rate of 0.5 °C per minute. The melting temperature is determined by induction using the Cary Win UV thermal application software.

[0229] Thermodynamic parameters were determined by van't Hoff analysis: Tm-1 = R[ln([Ct] / 4)]ΔH + ΔS° / ΔH°, where ΔH° and ΔS° are the standard enthalpy and entropy changes, respectively, determined from UV experiments, R is the universal gas constant, and [Ct] is the total oligonucleotide chain concentration. The change in the number of water molecules associated with the melting process (Δnw) is obtained from the dependence of Tm on water activity (aw) according to the equation Δnw = (-ΔH / R)[δ(Tm-1) / δ(lnaw)]. The slope of the plot of the reciprocal temperature of melting (K-1) against the logarithm of water activity for various concentrations of ethylene glycol (0, 2, 5, 7, 10, and 15% by weight) is obtained as the value of δ(Tm-1) / δ(lnaw).

[0230] CD experiments are performed using an Aviv Model 61DS spectropolarimeter equipped with a Peltier thermoelectric temperature regulation unit (3 µM oligonucleotide concentration, 10 mM PIPES buffer, pH 7.0, 100 mM NaCl, 10 mM MgCl). Data are collected using a 1 cm pathlength quartz cuvette with a scan from 360 to 220 nm, a 3 s time constant, and a wavelength step size of 0.5 nm at 25 °C.

[0231] Example 7. In vitro selection using unnatural nucleobases An oligonucleotide library containing unnatural nucleic acids is generated. Samples of the library are subjected to sequential binding and elution with a target molecule (e.g., a protein). The pool of bound nucleic acids is amplified by PCR and subjected to another round of selection for binding to the target molecule. This selection process is repeated several times. To increase the selection pressure during the final rounds of selection, the target molecule is enriched and / or the incubation time is reduced. The surviving nucleic acids are sequenced as potential aptamers. The binding affinity of the potential aptamers is determined using flow cytometry.

[0232] Example 8. General procedure for DNA click reaction. To 14 μL of DNA solution (0.2 pmol) in DMSO, 1 μL of azido-PEG(3+3)-SS-biotin (20 mM in HO) was added, followed by 2 μL of the ligand (BimC4A)3 (4 mM in HO), 1 μL of sodium ascorbate (100 mM in HO), and 1 μL of 5x PBS buffer. The mixture was then vortexed, and the final component, 1 μL of freshly prepared CuSO4 solution (4 mM in HO), was added. The solution was shaken at 37°C for 2 h, after which the resulting product DNA was purified (DNA Clean & Concentrator-5 kit, Zymo Research Corp.). The purified sample was used directly in gel mobility assays (see below).

[0233] Example 9. General procedure for post-amplification DNA labeling (from Seo et al., JACS 2011, 133, 19878). For enzymatic post-synthesis labeling, dsDNA containing free amino groups was incubated with 10 mM EZ-Link sulfo-NHS-SS-biotin or EZ-Link NHS-PEG4-biotin (Thermo Scientific) in phosphate labeling buffer (50 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1 mM EDTA) for 1 h at room temperature, followed by purification using a Qiagen PCR purification kit. With dichloroacetyl-protected amine derivatives such as dTPT3PA or d5SICSPA, the amines first required deprotection, which was achieved by overnight incubation in concentrated aqueous ammonia at room temperature. The ammonia was removed via a SpeedVac concentrator (water aspirator, followed by an oil vacuum pump). To cleave the disulfide-containing linker (i.e., SS-biotin or SS-PEG4-biotin), the dsDNA was treated with DTT (30 mM final concentration) for 1 h at 37°C. For backbone labeling, dsDNA containing backbone phosphorothioates was incubated overnight with 25 mM EZ-Link iodoacetyl-PEG2-biotin (Thermo Scientific) in phosphate labeling buffer at 50°C, and the product was purified with a Qiagen PCR purification kit. All reactions manipulating the attached biotin moiety were quantified by streptavidin gel shift assay.

[0234] Gel mobility assay. DNA samples (10–50 nanograms) were mixed with 1 μg of streptavidin (Promega) in phosphate labeling buffer (50 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1 mM EDTA) and incubated at 37°C for 30 min, mixed with 5x non-denaturing loading solution (Qiagen), and loaded onto a 6% non-denaturing PAGE gel. Gels were run for 25–40 min at 150 V, stained with 1x Sybr Gold nucleic acid stain (Life Technologies) in TBE for 30 min, and visualized using a Molecular Imager Gel DocXR+ equipped with a 520DF30 filter (Bio-Rad). Intense bands corresponding to dsDNA (~150 bp) and a 1:1 complex between dsDNA and streptavidin (~400 bp) were evident. Weaker bands corresponding to higher order (lower mobility) complexes of DNA and streptavidin or, in some cases, non-biotinylated single-stranded DNA resulting from incomplete annealing after PCR were also evident.

[0235] All patents and publications referenced herein are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

[0236] The terms and expressions that have been used are used as terms of description rather than of limitation, and it is not intended to use such terms or expressions to exclude all equivalents of the features shown and described, or portions thereof, but it is understood that various modifications are possible within the scope of the claimed embodiments. Thus, while the present embodiments have been disclosed in terms of particularly preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be employed by those skilled in the art, and such modifications and variations are deemed to be within the scope of the present disclosure as defined by the appended claims.

Claims

1. A compound comprising a nucleobase analog of any of the following formulas: 【Chemistry 1】 During the ceremony, each X is independently carbon or nitrogen; R ２ are each optional and, when present, independently represent hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azido group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently sulfur or selenium; The wavy line indicates the point of attachment to the ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof, which is in free form, attached to a monophosphate, diphosphate, or triphosphate, and optionally includes an α-thiophosphate, β-thiophosphate, or γ-thiophosphate group, or is contained in an RNA or DNA, or an RNA or DNA analog.

2. A compound comprising a nucleobase analog of any of the following formulas: 【Chemistry 2】 During the ceremony, each X is independently carbon or nitrogen; R １ are each independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of complementary reactivity to the reactive center, or a coupled linker having a cargo attached thereto; R ２ are each optional and, when present, independently represent hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azido group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently sulfur or selenium; The wavy line indicates the point of attachment to the ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof, which is in free form, bound to a monophosphate, diphosphate, or triphosphate, and optionally includes an α-thiophosphate, β-thiophosphate, or γ-thiophosphate group, or is contained in an RNA or DNA or RNA or DNA analog.

3. The compound of claim 1 , wherein the reactive center comprises an amino group, an acetylene group, a thiol group, an aldehyde group, or an azide group.

4. The compound of claim 2 , wherein the reactive center comprises an amino group, an acetylene group, a thiol group, an aldehyde group, or an azide group.

5. 2. The compound of claim 1, wherein the ribosyl or deoxyribosyl moiety comprises a triphosphate or α-thiotriphosphate group attached to its 5'-hydroxyl.

6. The compound of claim 2, wherein the ribosyl or deoxyribosyl moiety contains a triphosphate or α-thiotriphosphate group attached to its 5'-hydroxyl.

7. 2. The compound of claim 1, wherein the ribosyl or deoxyribosyl moiety is incorporated into an RNA or DNA oligonucleotide strand, respectively, or the ribosyl or deoxyribosyl moiety or analog thereof is incorporated into an RNA or DNA analog.

8. The compound of claim 2, wherein the ribosyl or deoxyribosyl moiety is incorporated into an RNA or DNA oligonucleotide strand, respectively, or the ribosyl or deoxyribosyl moiety or analog thereof is incorporated into an RNA or DNA analog.

9. At least one R ２ are independently -C≡C-CH ２ NHR ３ group, wherein R ３ is hydrogen or an amino protecting group; an acetylene group suitable for use in a click reaction between a cargo and a cargo reagent comprising an acetylene-reactive group; a thiol group suitable for use in a reaction between a cargo and a cargo reagent comprising a thiol-reactive group; an aldehyde group suitable for use in a reaction between a cargo and a cargo reagent comprising an aldehyde-reactive group; an azide group suitable for use in a reaction between a cargo and a cargo reagent comprising an azide-reactive group; and -C≡C-(CH ２ ) ｎ -C≡CH, -C≡C-CH ２ NHR ３ group, and —C≡C—(CH ２ ) ｎ１ -O(CH ２ ) ｎ２ 10. The compound of claim 1 comprising a group selected from the group consisting of: -C≡CH.

10. At least one R ２ are independently -C≡C-CH ２ NHR ３ group, wherein R ３ is hydrogen or an amino protecting group; an acetylene group suitable for use in a click reaction between a cargo and a cargo reagent comprising an acetylene-reactive group; a thiol group suitable for use in a reaction between a cargo and a cargo reagent comprising a thiol-reactive group; an aldehyde group suitable for use in a reaction between a cargo and a cargo reagent comprising an aldehyde-reactive group; an azide group suitable for use in a reaction between a cargo and a cargo reagent comprising an azide-reactive group; and -C≡C-(CH ２ ) ｎ -C≡CH, -C≡C-CH ２ NHR ３ group, and —C≡C—(CH ２ ) ｎ１ -O(CH ２ ) ｎ２ The compound of claim 2 comprising a group selected from the group consisting of -C≡CH.

11. A nucleobase pair comprising a first nucleobase analogue having the formula β9b and a second nucleobase analogue having either the formula α15a or α15b, 【Transformation 3】 During the ceremony, each X is independently carbon or nitrogen; R １ are each independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of complementary reactivity to the reactive center, or a coupled linker having a cargo attached thereto; R ２ are each optional and, when present, independently represent hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently oxygen, sulfur, or selenium; The wavy line indicates the point of attachment to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof, which is in free form, bound to a monophosphate, diphosphate, or triphosphate, optionally containing an α-thiophosphate, β-thiophosphate, or γ-thiophosphate group, or nucleobase pair contained in RNA or DNA, or an RNA or DNA analog.

12. A nucleobase pair comprising a first nucleobase analogue having either formula β9a or β9b and a second nucleobase analogue having either formula α16a or α16b, 【Chemistry 4】 During the ceremony, each X is independently carbon or nitrogen; R １ are each independently hydrogen, an alkyl group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of complementary reactivity to the reactive center, or a coupled linker having a cargo attached thereto; R ２ are each optional and, when present, independently represent hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center suitable for coupling to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is attached; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently oxygen, sulfur, or selenium; E ２ are each independently sulfur or selenium; The wavy line indicates the point of attachment to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof, which is in free form, bound to a monophosphate, diphosphate, or triphosphate, optionally containing an α-thiophosphate, β-thiophosphate, or γ-thiophosphate group, or nucleobase pair contained in RNA or DNA, or an RNA or DNA analog.

13. A double-stranded oligonucleotide duplex, A double-stranded oligonucleotide duplex, wherein a first oligonucleotide strand comprises the compound of claim 1 and a second, complementary oligonucleotide strand comprises complementary base-paired nucleobases at complementary base-pairing sites thereof.

14. A double-stranded oligonucleotide duplex, A double-stranded oligonucleotide duplex, wherein a first oligonucleotide strand comprises the compound of claim 2 and a second, complementary oligonucleotide strand comprises complementary base-paired nucleobases at complementary base-pairing sites thereof.

15. 14. The double-stranded oligonucleotide duplex of claim 13, wherein the first strand comprises dTPT3 and the second complementary oligonucleotide strand comprises dNaM, dDMO, or dMM02 at the complementary base-pairing site.

16. 1. A method for performing site-specific functionalization of double-stranded oligonucleotide duplexes, comprising:

10. A method comprising: incorporating an unnatural nucleobase selected from the compounds of claim 1, wherein the unnatural nucleobase comprises a reactive linker comprising a reactive center, into a first oligonucleotide strand; then synthesizing a second strand complementary to the first strand under conditions such that the first and second strands form a double-stranded oligonucleotide duplex, wherein the second strand comprises a nucleobase complementary to the unnatural nucleobase at a site-specific complementary position thereof; and then contacting the double-stranded oligonucleotide duplex incorporating the unnatural nucleobase comprising a reactive linker moiety with a cargo reagent comprising a reactive group complementary to the cargo under conditions suitable for reaction of the reactive linker with the complementary reactive group to obtain a linked linker, thereby obtaining a double-stranded oligonucleotide duplex functionalized with a cargo attached by a linked linker.

17. 1. A method for performing site-specific functionalization of double-stranded oligonucleotide duplexes, comprising:

10. A method comprising: incorporating an unnatural nucleobase selected from the compounds of claim 2, which comprises a reactive linker comprising a reactive center, into a first oligonucleotide strand; then synthesizing a second strand complementary to the first strand under conditions such that the first and second strands form a double-stranded oligonucleotide duplex, wherein the second strand comprises a nucleobase complementary to the unnatural nucleobase at a site-specific complementary position thereof; and then contacting the double-stranded oligonucleotide duplex incorporating the unnatural nucleobase comprising a reactive linker moiety with a cargo reagent comprising a reactive group complementary to the cargo under conditions suitable for reaction of the reactive linker with the complementary reactive group to obtain a linked linker, thereby obtaining a double-stranded oligonucleotide duplex functionalized with a cargo attached by a linked linker.

18. Formula: N １ -Z ｘ -N ２ A structure comprising: In the formula, N １ is a nucleotide or its analog, or a terminal phosphate group, and N ２ is a nucleotide or analog thereof, or a terminal hydroxyl group; Z is a compound of claim 1 or 2; and x is an integer from 1 to 20. 【Request Item 19】 【Chemistry 5】 and derivatives or analogs thereof. 【Request Item 20】 【Chemistry 6】 and derivatives or analogs thereof.

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