US20200131555A1 - Incorporation of unnatural nucleotides and methods thereof - Google Patents

Incorporation of unnatural nucleotides and methods thereof Download PDF

Info

Publication number
US20200131555A1
US20200131555A1 US16/629,255 US201816629255A US2020131555A1 US 20200131555 A1 US20200131555 A1 US 20200131555A1 US 201816629255 A US201816629255 A US 201816629255A US 2020131555 A1 US2020131555 A1 US 2020131555A1
Authority
US
United States
Prior art keywords
unnatural
sfgfp
polymerase
trna
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US16/629,255
Other languages
English (en)
Inventor
Jerod Ptacin
Carolina Caffaro
Hans AERNI
Yorke ZHANG
Emil C. FISCHER
Aaron W. FELDMAN
Vivian T. DIEN
Floyd E. Romesberg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scripps Research Institute
Synthorx Inc
Original Assignee
Scripps Research Institute
Synthorx Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scripps Research Institute, Synthorx Inc filed Critical Scripps Research Institute
Priority to US16/629,255 priority Critical patent/US20200131555A1/en
Publication of US20200131555A1 publication Critical patent/US20200131555A1/en
Assigned to THE SCRIPPS RESEARCH INSTITUTE reassignment THE SCRIPPS RESEARCH INSTITUTE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FISCHER, Emil C., ZHANG, Yorke, DIEN, Vivian T., FELDMAN, Aaron W., ROMESBERG, FLOYD E.
Assigned to Synthorx, Inc. reassignment Synthorx, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CAFFARO, CAROLINA, AERNI, Hans, PTACIN, Jerod
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C12Y601/01026—Pyrrolysine-tRNAPyl ligase (6.1.1.26)

Definitions

  • oligonucleotides and their applications have revolutionized biotechnology.
  • the oligonucleotides including both DNA and RNA each includes only the four natural nucleotides of adenosine (A), guanosine (G), cytosine (C), thymine (T) for DNA, and the four natural nucleotides of adenosine (A), guanosine (G), cytosine (C), and uridine (U) for RNA, and which significantly restricts the potential functions and applications of the oligonucleotides.
  • oligonucleotides DNA or RNA
  • polymerases for example by PCR or isothermal amplification systems (e.g., transcription with T7 RNA polymerase)
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • these applications are restricted 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).
  • Disclosed herein is an additional method of generating nucleic acids that contains an expanded genetic alphabet.
  • a protein containing an unnatural amino acid comprising: preparing a mutant tRNA wherein the mutant tRNA comprises a mutant anticodon sequence selected from Table 1 or 2; preparing a mutant mRNA wherein the mutant mRNA comprises a mutant codon sequence selected from Table 1 or 2; and synthesizing the protein containing an unnatural amino acid utilizing the mutant tRNA and the mutant mRNA.
  • the protein is synthesized in a cell-free translation system.
  • the protein is synthesized in a cell (semi-synthetic organism or SSO).
  • the semi-synthetic organism comprises a microorganism.
  • the semi-synthetic organism comprises a bacterium. In some instances, the semi-synthetic organism comprises an Escherichia coli . In some instances, the mutant anticodon of the mutant tRNA pairs with a mutant codon selected from Tables 1-3. In some instances, the unnatural amino acid comprises at least one unnatural nucleotide. In some instances, the unnatural nucleotide comprises an unnatural nucleobase.
  • the unnatural base of the unnatural nucleotide is selected from the group consisting of 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, hypoxanthin-9-yl (I), 5-methyl-cytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifiuoromethyl uracils and cytosines; 5-halour
  • the unnatural nucleotide is selected from the group consisting of (only nucleobase portion shown, ribose and phosphate backbone omitted for clarity)
  • the unnatural nucleotide further comprises an unnatural sugar moiety.
  • the unnatural sugar moiety of the unnatural nucleotide is selected from the group consisting of a modification at the 2′ position: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, 2′-F, 2′-OCH 3 , 2′-O(CH 2 ) 2 OCH 3 wherein the alkyl, alkyl, alky
  • the mutant anticodon or the mutant codon further comprises an unnatural backbone. In some instances, the mutant anticodon and the mutant codon further comprises an unnatural backbone. In some instances, the unnatural nucleotides are recognized by a DNA polymerase, an RNA polymerase, or a reverse transcriptase. In some instances, an unnatural nucleotide is incorporated by the RNA polymerase into the mRNA during transcription to generate a mutant mRNA containing a mutant codon. In some instances, an unnatural nucleotide is incorporated by the RNA polymerase into the tRNA during transcription to generate a mutant tRNA containing a mutant anticodon.
  • an unnatural nucleotide is incorporated by the RNA polymerase into the mRNA during transcription to generate a mutant mRNA. In some instances, an unnatural nucleotide is incorporated by the RNA polymerase into the tRNA during transcription to generate a mutant tRNA. In some instances, the mutant tRNA is charged with an unnatural amino acid residue. In some instances, a protein containing an unnatural amino acid is generated during translation utilizing the mutant tRNA and the mutant mRNA.
  • FIG. 1A illustrates the chemical structure of the dNaM-dTPT3 UBP and a natural dA-dT base pair.
  • FIG. 1B illustrates the gene cassette used to express sfGFP(AXC) 151 and tRNA(GYT) Ser .
  • P T7 and T T7 denote the T7 RNAP promoter and terminator, respectively.
  • the sequence following the sfGFP T7 terminator is absent.
  • FIG. 1C illustrates a graph of fluorescence of cells expressing sfGFP and tRNA Ser with the indicated position 151-codon and anticodon, respectively.
  • Minus sign denotes the absence of serT in the expression cassette.
  • AGT natural Ser codon; TAG, amber stop codon; CTA amber suppressor anticodon.
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • FIG. 1D illustrates a graph of growth of cells expressing sfGFP and tRNA Ser with the indicated position 151-codon and anticodon, respectively.
  • Minus sign denotes the absence of serT in the expression cassette.
  • AGT natural Ser codon; TAG, amber stop codon; CTA amber suppressor anticodon.
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • FIG. 1E illustrates a Western blot of lysates (normalized by OD 600 ) from cells collected at the last time point shown in FIG. 1C and FIG. 1D , probed with an ⁇ -GFP antibody (N-terminal epitope).
  • FIG. 2A illustrates a graph of fluorescence of cells expressing sfGFP with the indicated position 151-codon, in the presence (+) or absence ( ⁇ ) of a tRNA Py1 with a cognate anticodon, Py1RS, or 20 mM PrK (N 6 -[(2-propynyloxy)carbonyl]-L-lysine) in the media. Fluorescence was determined at the last time point shown in FIG. 2B . Asterisk denotes the absence of tRNA Py1 in cells expressing sfGFP(TAC) 151 . TAC, natural Tyr codon; TAG, amber stop codon; n.d., not determined. Data shown as mean with individual data points, each propagated from an individual colony.
  • FIG. 2B illustrates a timecourse analysis of a subset of the data shown in FIG. 2A .
  • Plus and minus signs denote the presence or absence, respectively, of 20 mM PrK in the media.
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • FIG. 2C illustrates Western blots of sfGFP purified from cells expressing sfGFP and tRNA Py1 with the indicated position-151 codon and anticodon, respectively, with or without click conjugation of TAMRA and/or addition of 20 mM PrK to the media.
  • tRNA Py1 is absent ( ⁇ ) in cells expressing sfGFP(TAC) 151 .
  • sfGFP was purified from cultures collected at the last time point shown in FIG. 2B . Western blots were probed with an ⁇ -GFP antibody and imaged to detect both sfGFP and the conjugated TAMRA.
  • FIG. 3A illustrates a graph of fluorescence of cells expressing sfGFP(TAC) 151 or sfGFP and tRNA pAzF with the indicated position-151 codon and a cognate anticodon, respectively, in the presence (+) or absence ( ⁇ ) of 5 mM pAzF in the media.
  • TAC natural Tyr codon
  • TAG amber stop codon.
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • the fluorescence observed with sfGFP(AXC) 151 in the absence of pAzF is attributed to charging of tRNA pAzF (GYT) with a natural amino acid (likely Tyr).
  • FIG. 3B illustrates a Western blot of sfGFP purified from cells expressing sfGFP and tRNA pAzF with the indicated position-151 codon and anticodon, respectively, with or without click conjugation of TAMRA and/or addition of 5 mM pAzF to the media.
  • the minus sign denotes the absence of tRNA pAzF in cells expressing sfGFP(TAC) 151 .
  • sfGFP was purified from cultures collected at the last time point shown in FIG. 3A . Western blots were probed with an ⁇ -GFP antibody and imaged to detect both sfGFP and the conjugated TAMRA.
  • FIG. 4 illustrates fluorescence of cells expressing sfGFP with various codons at position 151.
  • FIG. 5A illustrates decoding of the AXC codon with natural near-cognate anticodons, with a graph of fluorescence of cells expressing sfGFP(AXC) 151 with or without tRNA Ser with the indicated anticodon.
  • Cells were induced as described in FIG. 1C and FIG. 1D and fluorescence measurements correspond to the last time point shown in FIG. 1C .
  • Values for the GYT anticodon and in the absence of tRNA Ser ( ⁇ tRNA) correspond to the same values in FIG. 1 c,d .
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • FIG. 5B illustrates decoding of the AXC codon with natural near-cognate anticodons, with a graph of growth of cells expressing sfGFP(AXC) 151 with or without tRNA Ser with the indicated anticodon.
  • Cells were induced as described in FIG. 1C and FIG. 1D and fluorescence measurements correspond to the last time point shown in FIG. 1C .
  • Values for the GYT anticodon and in the absence of tRNA Ser ( ⁇ tRNA) correspond to the same values in FIG. 1C and FIG. 1D .
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • FIG. 6A illustrates Western blots and growth of cells decoding AXC and GXC codons with tRNA Py1 .
  • Western blot of lysates normalized by OD 600 ) from cells expressing sfGFP with the indicated position 151-codon, in the presence (+) or absence ( ⁇ ) of a tRNA Py1 with a cognate anticodon, Py1RS, or 20 mM PrK in the media. Blots were probed with an ⁇ -GFP antibody (N-terminal epitope). Cells were induced and collected at an equivalent time point as described in FIG. 2B .
  • FIG. 6B illustrates growth of cultures analyzed in FIG. 6A .
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • FIG. 7A illustrates decoding AXC and GXC codons with tRNA Py1 and cell growth as a function of added unnatural ribotriphosphates. Fluorescence of purified sfGFP (lower panel) from cells expressing sfGFP and tRNA Py1 with the position 151-codon/anticodon indicated, in the presence (+) or absence ( ⁇ ) of each unnatural ribotriphosphate in the media, and with or without 20 mM PrK. Cells were induced as described in FIG. 2B and fluorescence measurements were taken at the end of induction ( ⁇ 3.5 h), prior to collecting the cells and purifying the sfGFP protein for click conjugation of TAMRA and western blotting.
  • FIG. 7B illustrates a gel of decoding AXC and GXC codons with tRNA Py1 as a function of added unnatural ribotriphosphates.
  • FIG. 7C illustrates graphs of fluorescence and growth of cells expressing sfGFP(TAC) 151 in the presence (+) or absence ( ⁇ ) of both unnatural deoxyribotriphosphates and each unnatural ribotriphosphate.
  • Data shown as mean ⁇ s.d., n 3 cultures, each propagated from an individual colony. At the concentrations used (see Methods), dNaMTP and dTPT3TP do not inhibit cell growth, whereas both unnatural ribotriphosphates, particularly TPT3TP, show some inhibition of growth.
  • FIG. 7D illustrates a graph of cell growth corresponding to the cultures with added PrK (20 mM) whose fluorescence is shown in FIG. 2B .
  • Cells expressing sfGFP with natural codons were grown without any unnatural triphosphates, whereas cells expressing sfGFP with unnatural codons were grown with both unnatural deoxy- and ribotriphosphates.
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • FIG. 8A illustrates a gel of decoding AXC and GXC codons with tRNA Py1 as a function of PrK concentration in the media.
  • sfGFP was induced and purified from cells collected as described in FIG. 2B .
  • Western blots were probed with an ⁇ -GFP antibody and imaged to detect both sfGFP and the conjugated TAMRA.
  • FIG. 8B illustrates a graph of decoding AXC and GXC codons with tRNA Py1 as a function of PrK concentration in the media.
  • Fluorescence of cells (measured at the last time point shown in c) expressing sfGFP and tRNA Py1 with the indicated position-151 codon and anticodon, respectively, as a function of PrK concentration in the media.
  • Fluorescence values for 0 and 20 mM PrK are the same as the ( ⁇ ) and (+) PrK values, respectively, shown in FIG. 2B .
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony.
  • FIG. 8C illustrates a timecourse analysis of fluorescence. For clarity, only one representative culture is shown for each codon/anticodon pair and PrK concentration. Without being bound by theory, we attribute the low level of sfGFP produced in the absence of PrK to decoding by endogenous tRNAs and loss of UBP retention in sfGFP (Table 5). However, the relative amount of sfGFP that contains PrK ( FIG. 8A ) and absolute amount of sfGFP expressed ( FIG. 8B and FIG.
  • FIG. 8D illustrates a timecourse analysis of cell growth at various concentrations of PrK for the experiment shown in FIG. 8C .
  • FIG. 9 illustrates cell growth of the cultures whose fluorescence is shown in FIG. 3A .
  • Data shown as mean ⁇ s.d., n 4 cultures, each propagated from an individual colony
  • ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 ⁇ L” means “about 5 ⁇ L” and also “5 ⁇ L.” Generally, the term “about” includes an amount that would be expected to be within experimental error.
  • the information of life is encoded by a four letter genetic alphabet, which is made possible by the selective formation of two base pairs: (d)G-(d)C and (d)A-dT/U.
  • a third, unnatural base pair (UBP) formed between two synthetic nucleotides expands this system, thereby increasing the potential for information storage, and has profound academic and practical implications.
  • UBP unnatural base pair
  • UBPs that are replicated, transcribed, and translated into protein in vitro provide insights into the forces underlying the storage and retrieval of natural information, and also enable wide ranging applications in chemical and synthetic biology.
  • SSO semi-synthetic organism
  • an SSO has revolutionary practical applications, including for human health.
  • an SSO revolutionizes the growing field of protein therapeutics.
  • protein therapeutics are severely limited in their molecular properties due to the finite chemical diversity available with the twenty natural amino acids.
  • the protein is synthesized in a cell-free translation system. In some instances, the protein is synthesized in a cell or semi-synthetic organism (SSO). In some instances, the semi-synthetic organism comprises a microorganism. In some instances, the semi-synthetic organism comprises a bacterium. In some instances, the semi-synthetic organism comprises an Escherichia coli . In some instances, the mutant tRNA contains a mutant anticodon sequence. In some instances, the mutant anticodon sequence is an anticodon sequence illustrated in Table 1. In some instances, the mutant anticodon sequence is an anticodon sequence illustrated in Table 2. In some instances, the mutant anticodon sequence is an anticodon sequence illustrated in Table 3.
  • the mutant anticodon of the mutant tRNA pairs with a mutant codon.
  • the mutant codon is a mutant codon illustrated in Table 1.
  • the mutant codon is a mutant codon illustrated in Table 2.
  • the mutant codon is a mutant codon illustrated in Table 3.
  • the Y and X illustrated in Table 1, Table 2, and Table 3 represent unnatural bases of the unnatural nucleotide.
  • the unnatural base is selected from the group consisting of 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, hypoxanthin-9-yl (I), 5-methyl-cytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 5-
  • the unnatural nucleotide is selected from the group consisting of (only nucleobase portion shown, ribose and phosphate backbone omitted for clarity)
  • the unnatural nucleotide is selected from the group consisting of (only nucleobase portion shown, ribose and phosphate backbone omitted for clarity)
  • the unnatural nucleotide further comprises an unnatural sugar moiety.
  • the unnatural sugar moiety is selected from the group consisting of a modification at the 2′ position: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, 2′-F, 2′-OCH 3 , 2′-O(CH 2 ) 2 OCH 3 wherein the alkyl, alkenyl and alkynyl
  • the mutant anticodon or the mutant codon further comprises an unnatural backbone. In some instances, the mutant anticodon further comprises an unnatural backbone. In some instances, the mutant codon further comprises an unnatural backbone. In some instances, the unnatural backbone is selected from the group consisting of a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, phosphonates, 3′-alkylene phosphonate, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • the unnatural nucleotides are recognized by a polymerase.
  • the polymerase is a DNA polymerase, an RNA polymerase, or a reverse transcriptase.
  • the polymerase comprises ⁇ 29, B103, GA-1, PZA, ⁇ 15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, ThermoSequenase®, 9° NmTM TherminatorTM DNA polymerase, Tne, Tma, TfI, Tth, TIi, Stoffel fragment, VentTM and Deep VentTM DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, Pfu, Taq, T7 DNA polymerase, T7 RNA polymerase, PGB-D, UlTma DNA polymerase, E.
  • coli DNA polymerase I E. coli DNA polymerase III, archaeal DP1I/DP2 DNA polymerase II, 9° N DNA Polymerase, Taq DNA polymerase, Phusion® DNA polymerase, Pfu DNA polymerase, SP6 RNA polymerase, RB69 DNA polymerase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, and SuperScript® III reverse transcriptase.
  • AMV Avian Myeloblastosis Virus
  • MMLV Moloney Murine Leukemia Virus
  • the polymerase is DNA polymerase 1-Klenow fragment, Vent polymerase, Phusion® DNA polymerase, KOD DNA polymerase, Taq polymerase, T7 DNA polymerase, T7 RNA polymerase, TherminatorTM DNA polymerase, POLB polymerase, SP6 RNA polymerase, E. coli DNA polymerase I, E. coli DNA polymerase III, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, or SuperScript® III reverse transcriptase.
  • AMV Avian Myeloblastosis Virus
  • MMLV Moloney Murine Leukemia Virus
  • an unnatural nucleotide is incorporated by the polymerase into the mRNA during transcription to generate a mutant mRNA containing a mutant codon. In some instances, an unnatural nucleotide is incorporated by the polymerase into the mRNA during transcription to generate a mutant mRNA.
  • an unnatural nucleotide is incorporated by the polymerase into the tRNA during transcription to generate a mutant tRNA containing a mutant anticodon. In some instances, an unnatural nucleotide is incorporated by the polymerase into the tRNA during transcription to generate a mutant tRNA.
  • the mutant tRNA represents an unnatural amino acid residue.
  • an unnatural amino acid residue is a non-natural amino acid such as those described in Liu C. C., Schultz, P. G. Annu. Rev. Biochem. 2010, 79, 413.
  • a protein containing an unnatural amino acid is generated during translation utilizing the mutant tRNA and the mutant mRNA. In some instances, the protein containing an unnatural amino acid is generated under a cell free translation system. In some instances, the protein is synthesized in a cell or semi-synthetic organism (SSO). In some instances, the semi-synthetic organism comprises a microorganism. In some instances, the semi-synthetic organism comprises a bacterium. In some instances, the semi-synthetic organism comprises an Escherichia coli.
  • a nucleic acid (e.g., also referred to herein as target nucleic acid, target nucleotide sequence, nucleic acid sequence of interest or nucleic acid region of interest) can be from any source or composition, such as DNA, cDNA, gDNA (genomic DNA), RNA, siRNA (short inhibitory RNA), RNAi, tRNA or mRNA, for example, and can be in any form (e.g., linear, circular, supercoiled, single-stranded, double-stranded, and the like). Nucleic acids can comprise nucleotides, nucleosides, or polynucleotides. Nucleic acids can comprise natural and unnatural nucleic acids.
  • a nucleic acid can also comprise unnatural nucleic acids, such as DNA or RNA analogs (e.g., containing base analogs, sugar analogs and/or a non-native backbone and the like). It is understood that the term “nucleic acid” does not refer to or infer a specific length of the polynucleotide chain, thus polynucleotides and oligonucleotides are also included in the definition.
  • Exemplary natural nucleotides include, without limitation, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP.
  • Exemplary natural deoxyribonucleotides include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP.
  • Exemplary natural ribonucleotides include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.
  • the uracil base is uridine.
  • a nucleic acid sometimes is a vector, plasmid, phage, autonomously replicating sequence (ARS), centromere, artificial chromosome, yeast artificial chromosome (e.g., YAC) or other nucleic acid able to replicate or be replicated.
  • An unnatural nucleic acid can be a nucleic acid analogue.
  • a nucleotide analog, or unnatural nucleotide comprises a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties.
  • a modification can comprise a chemical modification. Modifications may be, for example, of the 3′OH or 5′OH group, of the backbone, of the sugar component, or of the nucleotide base. Modifications may include addition of non-naturally occurring linker molecules and/or of interstrand or intrastrand cross links.
  • the modified nucleic acid comprises modification of one or more of the 3′OH or 5′OH group, the backbone, the sugar component, or the nucleotide base, and/or addition of non-naturally occurring linker molecules.
  • a modified backbone comprises a backbone other than a phosphodiester backbone.
  • a modified sugar comprises a sugar other than deoxyribose (in modified DNA) or other than ribose (modified RNA).
  • a modified base comprises a base other than adenine, guanine, cytosine or thymine (in modified DNA) or a base other than adenine, guanine, cytosine or uracil (in modified RNA).
  • the nucleic acid may comprise at least one modified base. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases. In some embodiments, a modification is to a modified form of adenine, guanine cytosine or thymine (in modified DNA) or a modified form of adenine, guanine cytosine or uracil (modified RNA).
  • a modified base of a unnatural nucleic acid includes but is not limited to uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
  • Certain unnatural nucleic acids such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, 0-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (—C ⁇ C—CI1 ⁇ 4) uracil, 5-propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-
  • nucleic acids comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acid can include one or several heterocyclic bases other than the principal five base components of naturally-occurring nucleic acids.
  • the heterocyclic base may include uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo [2.3-d] pyrimidin-5-yl, 2-amino-4-oxopyrolo [2,3-d] pyrimidin-5-yl, 2-amino-4-oxopyrrolo [2.3-d] pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the nucleic acid via the 9-position, the pyrimidines via the 1-position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1-position.
  • Nucleotide analogs can also be modified at the phosphate moiety.
  • Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3′-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • these phosphate or modified phosphate linkage between two nucleotides can be through a 3′-5′ linkage or a 2′-5′ linkage, and the linkage can contain inverted polarity such as 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′.
  • Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, U.S. Pat. Nos.
  • Unnatural nucleic acids can include 2′,3′-dideoxy-2′,3′-didehydro-nucleosides (PCT/US2002/006460), 5′-substituted DNA and RNA derivatives (PCT/US2011/033961; Saha et al, J.
  • Unnatural nucleic acids can include modifications at the 5′-position and the 2′-position of the sugar ring (PCT/US94/02993), such as 5′-CH 2 substituted 2′-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al. Bioconjugate Chem. 1999, 10, 921-924).
  • Unnatural nucleic acids can include amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3′ linked nucleoside in the dimer (5′ to 3′) comprises a 2′-OCH 3 and a 5′-(S)—CH 3 (Mesmaeker et al, Synlett, 1997, 1287-1290).
  • Unnatural nucleic acids can include 2′-substituted 5′-CH 2 (or O) modified nucleosides (PCT/US92/01020).
  • Unnatural nucleic acids can include 5′methylenephosphonate DNA and RNA monomers, and dimers (Bohringer et al, Tet.
  • Unnatural nucleic acids can include 5′-phosphonate monomers having a 2′-substitution (US 2006/0074035) and other modified 5′-phosphonate monomers (WO 97/35869).
  • Unnatural nucleic acids can include 5′-modified methylenephosphonate monomers (EP614907 and EP629633).
  • Unnatural nucleic acids can include analogs of 5′ or 6′-phosphonate ribonucleosides comprising a hydroxyl group at the 5′ and or 6′ position (Chen et al, Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al, Bioorg. Med. Chem., 2000, 8, 2501-2509, Gallier et al, Eur. J. Org. Chem., 2007, 925-933 and Hampton et al, J. Med. Chem., 1976, 19(8), 1029-1033).
  • Unnatural nucleic acids can include 5′-phosphonate deoxyribonucleoside monomers and dimers having a 5′-phosphate group (Nawrot et al, Oligonucleotides, 2006, 16(1), 68-82).
  • Unnatural nucleic acids can include nucleosides having a 6′-phosphonate group wherein the 5′ or/and 6′-position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH 3 ) 3 ) (and analogs thereof); a methyleneamino group (CH 2 NH 2 ) (and analogs thereof) or a cyano group (CN) (and analogs thereof) (Fairhurst et al, Synlett, 2001, 4, 467-472; Kappler et al, J.
  • Unnatural nucleic acids can also include modifications of the sugar moiety.
  • Nucleic acids of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property.
  • nucleic acids comprise a chemically modified ribofuranose ring moiety.
  • Examples of chemically modified ribofuranose rings include, without limitation, addition of substitutent groups (including 5′ and/or 2′ substituent groups; bridging of two ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R 1 )(R 2 ) (R ⁇ H, C 1 -C 12 alkyl or a protecting group); and combinations thereof.
  • Examples of chemically modified sugars can be found in WO 2008/101157, US 2005/0130923, and WO 2007/134181.
  • a modified nucleic acid may comprise modified sugars or sugar analogs.
  • the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar “analog” cyclopentyl group.
  • the sugar can be in pyranosyl or in a furanosyl form.
  • the sugar moiety may be the furanoside of ribose, deoxyribose, arabinose or 2′-O-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in [alpha] or [beta] anomeric configuration.
  • Sugar modifications include, but are not limited to, 2′-alkoxy-RNA analogs, 2′-amino-RNA analogs, 2′-fluoro-DNA, and 2′-alkoxy- or amino-RNA/DNA chimeras.
  • a sugar modification may include, 2′-O-methyl-uridine and 2′-O-methyl-cytidine.
  • Sugar modifications include 2′-O-alkyl-substituted deoxyribonucleosides and 2′-O-ethyleneglycol like ribonucleosides.
  • the preparation of these sugars or sugar analogs and the respective “nucleosides” wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known.
  • Sugar modifications may also be made and combined with other modifications.
  • Modifications to the sugar moiety include natural modifications of the ribose and deoxy ribose as well as unnatural modifications.
  • Sugar modifications include but are not limited to the following modifications at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 , alkyl or C 2 to C 10 alkenyl and alkynyl.
  • 2′ sugar modifications also include but are not limited to —O[(CH 2 ) n O] m CH 3 , —O(CH 2 ) n OCH 3 , —O(CH 2 ) n NH 2 , —O(CH 2 ) n CH 3 , —O(CH 2 ) n —ONH 2 , and —O(CH 2 ) n ON[(CH 2 )n CH 3 )J 2 , where n and m are from 1 to about 10.
  • modifications at the 2′ position include but are not limited to: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • Similar modifications may also be made at other positions on the sugar, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide.
  • Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH 2 and S.
  • Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • nucleic acids having modified sugar moieties include, without limitation, nucleic acids comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH 3 , and 2′-O(CH 2 ) 2 OCH 3 substituent groups.
  • the substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C C 10 alkyl, OCF 3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 —O—N(R m )(R n ), and O—CH 2 —C( ⁇ O)—N(R m )(R n ), where each R m and R n is, independently, H or substituted or unsubstituted C 1 -C 10 alkyl.
  • nucleic acids of the present invention include one or more bicyclic nucleic acids.
  • the bicyclic nucleic acid comprises a bridge between the 4′ and the 2′ ribosyl ring atoms.
  • nucleic acids provided herein include one or more bicyclic nucleic acids wherein the bridge comprises a 4′ to 2′ bicyclic nucleic acid.
  • 4′ to 2′ bicyclic nucleic acids include, but are not limited to, one of the formulae: 4′-(CH 2 )—O-2′ (LNA); 4′-(CH 2 )—S-2′; 4′-(CH 2 ) 2 —O-2′ (ENA); 4′-CH(CH 3 )—O-2′ and 4′-CH(CH 2 OCH 3 )—O-2′, and analogs thereof (see, U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH 3 )(CH 3 )—O-2′ and analogs thereof, (see WO2009/006478, WO2008/150729, US2004/0171570, U.S. Pat. No.
  • PCT/US2008/064591 PCT US2008/066154, and PCT US2008/068922, PCT/DK98/00393; and U.S. Pat. Nos. 4,849,513; 5,015,733; 5,118,800; and 5,118,802.
  • nucleic acids can comprise linked nucleic acids.
  • Nucleic acids can be linked together using any inter nucleic acid linkage.
  • the two main classes of inter nucleic acid linking groups are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing inter nucleic acid linking groups include, but are not limited to, methylenemethylimino (—CH 2 —N(CH 3 )—O—CH 2 —), thiodiester (—O—C(O)—S—), thionocarbamate (—O— C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); and N,N*-dimethylhydrazine (—CH 2 —N(CH 3 )—N(CH 3 )—).
  • inter nucleic acids linkages having a chiral atom can be prepared a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates.
  • Unnatural nucleic acids can contain a single modification.
  • Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.
  • Backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and may be used in any combination. Other non-phosphate linkages may also be used.
  • backbone modifications e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages
  • backbone modifications can confer immunomodulatory activity on the modified nucleic acid and/or enhance their stability in vivo.
  • a phosphorous derivative can be attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like.
  • Exemplary polynucleotides containing modified phosphate linkages or non-phosphate linkages can be found in Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids Res.
  • Backbone modification may comprise replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group.
  • modifications include: anionic internucleoside linkage; N3′ to P5′ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos, Micklefield, J.
  • anionic internucleoside linkage N3′ to P5′ phosphoramidate modification
  • boranophosphate DNA prooligonucleotides
  • neutral internucleoside linkages such as methylphosphonates
  • amide linked DNA methylene(methyli
  • a modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, e.g. a combination of phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages.
  • Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA).
  • PNA aminoethylglycine
  • U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. (See also Nielsen et al., Science, 1991, 254, 1497-1500).
  • Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
  • a particularly useful function of a polymerase is to catalyze the polymerization of a nucleic acid strand using an existing nucleic acid as a template. Other functions that are useful are described elsewhere herein. Examples of useful polymerases include DNA polymerases and RNA polymerases.
  • the ability to improve specificity, processivity, or other features of polymerases unnatural nucleic acids would be highly desirable in a variety of contexts where, e.g., unnatural nucleic acid incorporation is desired, including amplification, sequencing, labeling, detection, cloning, and many others.
  • the present invention provides polymerases with modified properties for unnatural nucleic acids, methods of making such polymerases, methods of using such polymerases, and many other features that will become apparent upon a complete review of the following.
  • polymerases that incorporate unnatural nucleic acids into a growing template copy, e.g., during DNA amplification.
  • polymerases can be modified such that the active site of the polymerase is modified to reduce steric entry inhibition of the unnatural nucleic acid into the active site.
  • polymerases can be modified to provide complementarity with one or more unnatural features of the unnatural nucleic acids. Accordingly, the invention includes compositions that include a heterologous or recombinant polymerase and methods of use thereof.
  • Polymerases can be modified using methods pertaining to protein engineering. For example, molecular modeling can be carried out based on crystal structures to identify the locations of the polymerases where mutations can be made to modify a target activity. A residue identified as a target for replacement can be replaced with a residue selected using energy minimization modeling, homology modeling, and/or conservative amino acid substitutions, such as described in Bordo, et al. J Mol Biol 217: 721-729 (1991) and Hayes, et al. Proc Natl Acad Sci, USA 99: 15926-15931 (2002).
  • polymerases can be used in a method or composition set forth herein including, for example, protein-based enzymes isolated from biological systems and functional variants thereof. Reference to a particular polymerase, such as those exemplified below, will be understood to include functional variants thereof unless indicated otherwise.
  • a polymerase is a wild type polymerase. In some embodiments, a polymerase is a modified, or mutant, polymerase.
  • a modified polymerase has a modified nucleotide binding site.
  • a modified polymerase has a specificity for an unnatural nucleic acid that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild type polymerase toward the unnatural nucleic acid.
  • a modified or wild type polymerase has a specificity for an unnatural nucleic acid comprising 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% the specificity of the wild type polymerase toward a natural nucleic acid and/or the unnatural nucleic acid without the modified sugar.
  • a modified or wild type polymerase has a specificity for an unnatural nucleic acid comprising a modified base that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild type polymerase toward a natural nucleic acid and/or the unnatural nucleic acid without the modified base.
  • a modified or wild type polymerase has a specificity for an unnatural nucleic acid comprising a triphosphate that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild type polymerase toward a nucleic acid comprising a triphosphate and/or the unnatural nucleic acid without the triphosphate.
  • a modified or wild type polymerase can have a specificity for an unnatural nucleic acid comprising a triphosphate that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild type polymerase toward the unnatural nucleic acid with a diphosphate or monophosphate, or no phosphate, or a combination thereof.
  • a modified or wild type polymerase has a relaxed specificity for an unnatural nucleic acid. In some embodiments, a modified or wild type polymerase has a specificity for an unnatural nucleic acid and a specificity to a natural nucleic acid that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild type polymerase toward the natural nucleic acid.
  • a modified or wild type polymerase has a specificity for an unnatural nucleic acid comprising a modified sugar and a specificity to a natural nucleic acid that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild type polymerase toward the natural nucleic acid.
  • a modified or wild type polymerase has a specificity for an unnatural nucleic acid comprising a modified base and a specificity to a natural nucleic acid that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild type polymerase toward the natural nucleic acid.
  • Absence of exonuclease activity can be a wild type characteristic or a characteristic imparted by a variant or engineered polymerase.
  • an exo minus Klenow fragment is a mutated version of Klenow fragment that lacks 3′ to 5′ proofreading exonuclease activity.
  • the method of the invention may be used to expand the substrate range of any DNA polymerase which lacks an intrinsic 3 to 5′ exonuclease proofreading activity or where a 3 to 5′ exonuclease proofreading activity has been disabled, e.g. through mutation.
  • DNA polymerases include polA, polB (see e.g. Parrel & Loeb, Nature Struc Biol 2001) polC, polD, polY, polX and reverse transcriptases (RT) but preferably are processive, high-fidelity polymerases (PCT/GB2004/004643).
  • a modified or wild type polymerase substantially lacks 3′ to 5′ proofreading exonuclease activity.
  • a modified or wild type polymerase substantially lacks 3′ to 5′ proofreading exonuclease activity for an unnatural nucleic acid. In some embodiments, a modified or wild type polymerase has a 3′ to 5′ proofreading exonuclease activity. In some embodiments, a modified or wild type polymerase has a 3′ to 5′ proofreading exonuclease activity for a natural nucleic acid and substantially lacks 3′ to 5′ proofreading exonuclease activity for an unnatural nucleic acid.
  • a modified polymerase has a 3′ to 5′ proofreading exonuclease activity that is at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the proofreading exonuclease activity of the wild type polymerase.
  • a modified polymerase has a 3′ to 5′ proofreading exonuclease activity for an unnatural nucleic acid that is at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the proofreading exonuclease activity of the wild type polymerase to a natural nucleic acid.
  • a modified polymerase has a 3′ to 5′ proofreading exonuclease activity for an unnatural nucleic acid and a 3′ to 5′ proofreading exonuclease activity for a natural nucleic acid that is at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the proofreading exonuclease activity of the wild type polymerase to a natural nucleic acid.
  • a modified polymerase has a 3′ to 5′ proofreading exonuclease activity for a natural nucleic acid that is at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the proofreading exonuclease activity of the wild type polymerase to the natural nucleic acid.
  • the invention provides methods of making a modified polymerase that include structurally modeling a parental polymerase, e.g., a DNA polymerase, identifying one or more complex stability or nucleotide interaction feature affecting complex stability or nucleotide access or binding in the active site or a complementarity feature for a nucleotide analog at the active site, and mutating the parental polymerase to include or remove these features.
  • a parental polymerase e.g., a DNA polymerase
  • identifying one or more complex stability or nucleotide interaction feature affecting complex stability or nucleotide access or binding in the active site or a complementarity feature for a nucleotide analog at the active site mutating the parental polymerase to include or remove these features.
  • the polymerase can be mutated to improve steric access of the unnatural nucleotide to the active site or to improve charge-charge or hydrophobic interactions between the unnatural nucleotide and the polymerase.
  • Polymerases can be characterized according to their rate of dissociation from nucleic acids.
  • a polymerase has a relatively low dissociation rate for one or more natural and unnatural nucleic acids.
  • a polymerase has a relatively high dissociation rate for one or more natural and unnatural nucleic acids.
  • the dissociation rate is an activity of a polymerase that can be adjusted to tune reaction rates in methods set forth herein.
  • Polymerases can be characterized according to their fidelity when used with a particular natural and/or unnatural nucleic acid or collections of natural and/or unnatural nucleic acid. Fidelity generally refers to the accuracy with which a polymerase incorporates correct nucleic acids into a growing nucleic acid chain when making a copy of a nucleic acid template. DNA polymerase fidelity can be measured as the ratio of correct to incorrect natural and unnatural nucleic acid incorporations when the natural and unnatural nucleic acid are present, e.g., at equal concentrations, to compete for strand synthesis at the same site in the polymerase-strand-template nucleic acid binary complex.
  • DNA polymerase fidelity can be calculated as the ratio of (k cat /K m ) for the natural and unnatural nucleic acid and (kc at /K m ) for the incorrect natural and unnatural nucleic acid; where k cat and K m are Michaelis-Menten parameters in steady state enzyme kinetics (Fersht, A. R. (1985) Enzyme Structure and Mechanism, 2nd ed., p 350, W. H. Freeman & Co., New York., incorporated herein by reference).
  • a polymerase has a fidelity value of at least about 100, 1000, 10,000, 100,000, or 1 ⁇ 10 6 , with or without a proofreading activity.
  • Polymerases from native sources or variants thereof can be screened using an assay that detects incorporation of an unnatural nucleic acid having a particular structure.
  • polymerases can be screened for the ability to incorporate an unnatural nucleic acid or UBP; e.g., d5SICSTP, dNaMTP, or d5SICSTP-dNaMTP UBP.
  • a polymerase e.g., a heterologous polymerase, can be used that displays a modified property for the unnatural nucleic acid as compared to the wild-type polymerase.
  • the modified property can be, e.g., K m , k cat , V max , polymerase processivity in the presence of an unnatural nucleic acid (or of a naturally occurring nucleotide), average template read-length by the polymerase in the presence of an unnatural nucleic acid, specificity of the polymerase for an unnatural nucleic acid, rate of binding of an unnatural nucleic acid, rate of product (pyrophosphate, triphosphate, etc.) release, branching rate, or any combination thereof.
  • the modified property is a reduced K m for an unnatural nucleic acid and/or an increased k cat /K m or V max /K m for an unnatural nucleic acid.
  • the polymerase optionally has an increased rate of binding of an unnatural nucleic acid, an increased rate of product release, and/or a decreased branching rate, as compared to a wild-type polymerase.
  • a polymerase can incorporate natural nucleic acids, e.g., A, C, G, and T, into a growing nucleic acid copy.
  • a polymerase optionally displays a specific activity for a natural nucleic acid that is at least about 5% as high (e.g., 5%, 10%, 25%, 50%, 75%, 100% or higher), as a corresponding wild-type polymerase and a processivity with natural nucleic acids in the presence of a template that is at least 5% as high (e.g., 5%, 10%, 25%, 50%, 75%, 100% or higher) as the wild-type polymerase in the presence of the natural nucleic acid.
  • the polymerase displays a k cat /K m or V max /K m for a naturally occurring nucleotide that is at least about 5% as high (e.g., about 5%, 10%, 25%, 50%, 75% or 100% or higher) as the wild-type polymerase.
  • Polymerases used herein that can have the ability to incorporate an unnatural nucleic acid of a particular structure can also be produced using a directed evolution approach.
  • a nucleic acid synthesis assay can be used to screen for polymerase variants having specificity for any of a variety of unnatural nucleic acids.
  • polymerase variants can be screened for the ability to incorporate an unnatural nucleic acid or UBP; e.g., dTPT3, dNaM analog, or dTPT3-dNaM UBP into nucleic acids.
  • such an assay is an in vitro assay, e.g., using a recombinant polymerase variant.
  • Such directed evolution techniques can be used to screen variants of any suitable polymerase for activity toward any of the unnatural nucleic acids set forth herein.
  • Modified polymerases of the compositions described can optionally be a modified and/or recombinant ⁇ 29-type DNA polymerase.
  • the polymerase can be a modified and/or recombinant ⁇ 29, B103, GA-1, PZA, ⁇ 15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.
  • Nucleic acid polymerases generally useful in the invention include DNA polymerases, RNA polymerases, reverse transcriptases, and mutant or altered forms thereof. DNA polymerases and their properties are described in detail in, among other places, DNA Replication 2 nd edition, Kornberg and Baker, W. H. Freeman, New York, N. Y. (1991).
  • Known conventional DNA polymerases useful in the invention include, but are not limited to, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108: 1, Stratagene), Pyrococcus woesei (Pwo) DNA polymerase (Hinnisdaels et al., 1996, Biotechniques, 20:186-8, Boehringer Mannheim), Thermus thermophilus (Tth) DNA polymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32), Thermococcus litoralis (TIi) DNA polymerase (also referred to as VentTM DNA polymerase, Cariello et al, 1991, Polynucleotides Res, 19: 4193, New England Biolabs), 9° NmTM DNA polymerase (New England Biolabs), Stoffe
  • thermococcus sp Thermus aquaticus (Taq) DNA polymerase (Chien et al, 1976, J. Bacteoriol, 127: 1550), DNA polymerase, Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (from thermococcus sp.
  • Thermophilic DNA polymerases include, but are not limited to, ThermoSequenase®, 9° NmTM, TherminatorTM, Taq, Tne, Tma, Pfu, TfI, Tth, TIi, Stoffel fragment, VentTM and Deep VentTM DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, and mutants, variants and derivatives thereof.
  • a polymerase that is a 3 exonuclease-deficient mutant is also contemplated.
  • Reverse transcriptases useful in the invention include, but are not limited to, reverse transcriptases from HIV, HTLV-I, HTLV-1I, FeLV, FIV, SIV, AMV, MMTV, MoMuLV and other retroviruses (see Levin, Cell 88:5-8 (1997); Verma, Biochim Biophys Acta. 473:1-38 (1977); Wu et al, CRC Crit Rev Biochem. 3:289-347(1975)).
  • Further examples of polymerases include, but are not limited to 9° N DNA Polymerase, Taq DNA polymerase, Phusion® DNA polymerase, Pfu DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase, and VentR® DNA polymerase Gardner et al.
  • Polymerases isolated from non-thermophilic organisms can be heat inactivatable. Examples are DNA polymerases from phage. It will be understood that polymerases from any of a variety of sources can be modified to increase or decrease their tolerance to high temperature conditions.
  • a polymerase can be thermophilic.
  • a thermophilic polymerase can be heat inactivatable. Thermophilic polymerases are typically useful for high temperature conditions or in thermocycling conditions such as those employed for polymerase chain reaction (PCR) techniques.
  • the polymerase comprises 129, B103, GA-1, PZA, ⁇ 15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, ThermoSequenase®, 9° NmTM, TherminatorTM DNA polymerase, Tne, Tma, TfI, Tth, TIi, Stoffel fragment, VentTM and Deep VentTM DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, Pfu, Taq, T7 DNA polymerase, T7 RNA polymerase, PGB-D, UlTma DNA polymerase, E.
  • coli DNA polymerase I E. coli DNA polymerase III, archaeal DP1I/DP2 DNA polymerase II, 9° N DNA Polymerase, Taq DNA polymerase, Phusion® DNA polymerase, Pfu DNA polymerase, SP6 RNA polymerase, RB69 DNA polymerase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, and SuperScript® III reverse transcriptase.
  • AMV Avian Myeloblastosis Virus
  • MMLV Moloney Murine Leukemia Virus
  • the polymerase is DNA polymerase 1-Klenow fragment, Vent polymerase, Phusion® DNA polymerase, KOD DNA polymerase, Taq polymerase, T7 DNA polymerase, T7 RNA polymerase, TherminatorTM DNA polymerase, POLB polymerase, SP6 RNA polymerase, E. coli DNA polymerase I, E. coli DNA polymerase III, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, or SuperScript® III reverse transcriptase.
  • AMV Avian Myeloblastosis Virus
  • MMLV Moloney Murine Leukemia Virus
  • such polymerases can be used for DNA amplification and/or sequencing applications, including real-time applications, e.g., in the context of amplification or sequencing that include incorporation of unnatural nucleic acid residues into DNA by the polymerase.
  • the unnatural nucleic acid that is incorporated can be the same as a natural residue, e.g., where a label or other moiety of the unnatural nucleic acid is removed by action of the polymerase during incorporation, or the unnatural nucleic acid can have one or more feature that distinguishes it from a natural nucleic acid.
  • Green fluorescent protein and variants such as sfGFP have served as model systems for the study of ncAA incorporation using the amber suppression system, including at position Y151, which has been shown to tolerate a variety of natural and ncAAs ( FIG. 4 ).
  • Ser at position 151 of sfGFP we first focused on the incorporation of Ser at position 151 of sfGFP, as E. coli serine aminoacyl-tRNA synthetase (SerRS) does not rely on anticodon recognition for tRNA aminoacylation, thus eliminating the potential complications of inefficient charging.
  • SSO strain YZ3 was transformed with a plasmid encoding sfGFP and an E.
  • Transformants were grown in media supplemented with dNaMTP and dTPT3TP, then supplemented further with NaMTP and TPT3TP, as well as isopropyl- ⁇ -D-thiogalactoside (IPTG) to induce expression of T7 RNA polymerase (T7 RNAP) and tRNA Ser (GYT). After a brief period of tRNA induction, anhydrotetracycline (aTc) was added to induce expression of sfGFP(AXC) 151 .
  • TAC sfGFP codon 151
  • X Na
  • Lysates of these cells were subjected to western blotting with an anti-GFP antibody, which revealed a significant reduction in sfGFP expression and the presence of sfGFP truncated at the position of the unnatural codon ( FIG. 1E ).
  • cells transformed with the plasmid encoding both sfGFP(AXC) 151 and tRNA Ser (GYT) exhibited fluorescence that was nearly equal to that of control cells expressing sfGFP(AGT) 151 ( FIG. 1C ), cell growth did not plateau upon induction of sfGFP(AXC) 151 ( FIG.
  • FIGS. 5A and 5B western blots of lysates from these cells revealed only full-length sfGFP protein.
  • tRNA Ser GNT
  • N G, C, A, or T
  • tRNA Py1 can be selectively charged by the Methanosarcina barkeri pyrrolysine aminoacyl tRNA synthetase (Py1RS) with the ncAA N 6 -[(2-propynyloxy)carbonyl]-L-lysine (PrK).
  • Py1RS Methanosarcina barkeri pyrrolysine aminoacyl tRNA synthetase
  • PrK Methanosarcina barkeri pyrrolysine aminoacyl tRNA synthetase
  • GYC Methanosarcina barkeri pyrrolysine aminoacyl tRNA synthetase
  • the SSO carrying a separate plasmid encoding an IPTG-inducible Py1RS, was transformed with the required plasmids and grown with or without added PrK.
  • the cognate unnatural tRNA Py1 , or PrK we observed only low cellular fluorescence ( FIG. 2A ), truncation of sfGFP ( FIGS. 6A and 6B ), and a plateau in cell growth ( FIG. 6B ).
  • sfGFP was affinity purified from cell lysates using a C-terminal Strep-tag II and subjected to copper-catalyzed click chemistry to attach a carboxytetramethylrhodamine (TAMRA) dye (TAMRA-PEG 4 -N 3 ), which was found to shift the electrophoretic mobility of sfGFP during SDS-PAGE, thus allowing us to assess the fidelity of PrK incorporation by western blotting ( FIG. 2C ).
  • TAMRA carboxytetramethylrhodamine
  • DBCO dibenzocyclooctyl
  • the UBP is unlikely to be limited to these, and when combined with a recently reported Cas9 editing system that reinforces UBP retention, it will likely make available more codons than can ever be used.
  • the reported SSO may be just the first of a new form of semi-synthetic life that is able to access a broad range of forms and functions not available to natural organisms.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US16/629,255 2017-07-11 2018-07-10 Incorporation of unnatural nucleotides and methods thereof Abandoned US20200131555A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/629,255 US20200131555A1 (en) 2017-07-11 2018-07-10 Incorporation of unnatural nucleotides and methods thereof

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201762531325P 2017-07-11 2017-07-11
PCT/US2018/041509 WO2019014267A1 (fr) 2017-07-11 2018-07-10 Incorporation de nucléotides non naturels et procédés associés
US16/629,255 US20200131555A1 (en) 2017-07-11 2018-07-10 Incorporation of unnatural nucleotides and methods thereof

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/041509 A-371-Of-International WO2019014267A1 (fr) 2017-07-11 2018-07-10 Incorporation de nucléotides non naturels et procédés associés

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/196,151 Continuation US11834689B2 (en) 2017-07-11 2021-03-09 Incorporation of unnatural nucleotides and methods thereof

Publications (1)

Publication Number Publication Date
US20200131555A1 true US20200131555A1 (en) 2020-04-30

Family

ID=65002471

Family Applications (4)

Application Number Title Priority Date Filing Date
US16/629,255 Abandoned US20200131555A1 (en) 2017-07-11 2018-07-10 Incorporation of unnatural nucleotides and methods thereof
US16/530,742 Abandoned US20190376054A1 (en) 2017-07-11 2019-08-02 Incorporation of unnatural nucleotides and methods thereof
US17/196,151 Active 2039-01-08 US11834689B2 (en) 2017-07-11 2021-03-09 Incorporation of unnatural nucleotides and methods thereof
US18/481,086 Pending US20240043823A1 (en) 2017-07-11 2023-10-04 Incorporation of unnatural nucleotides and methods thereof

Family Applications After (3)

Application Number Title Priority Date Filing Date
US16/530,742 Abandoned US20190376054A1 (en) 2017-07-11 2019-08-02 Incorporation of unnatural nucleotides and methods thereof
US17/196,151 Active 2039-01-08 US11834689B2 (en) 2017-07-11 2021-03-09 Incorporation of unnatural nucleotides and methods thereof
US18/481,086 Pending US20240043823A1 (en) 2017-07-11 2023-10-04 Incorporation of unnatural nucleotides and methods thereof

Country Status (15)

Country Link
US (4) US20200131555A1 (fr)
EP (1) EP3652316A4 (fr)
JP (2) JP7325341B2 (fr)
KR (2) KR20240038157A (fr)
CN (1) CN111051512A (fr)
AR (1) AR112756A1 (fr)
AU (1) AU2018300069A1 (fr)
CA (1) CA3069321A1 (fr)
EA (1) EA202090090A1 (fr)
IL (1) IL271903A (fr)
MA (1) MA49578A (fr)
NZ (1) NZ761479A (fr)
SG (1) SG11202000167SA (fr)
TW (1) TWI821192B (fr)
WO (1) WO2019014267A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11466279B2 (en) 2014-04-09 2022-10-11 The Scripps Research Institute Import of unnatural or modified nucleoside triphosphates into cells via nucleic acid triphosphate transporters
US11634451B2 (en) 2013-08-08 2023-04-25 The Scripps Research Institute Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides
US11761007B2 (en) 2015-12-18 2023-09-19 The Scripps Research Institute Production of unnatural nucleotides using a CRISPR/Cas9 system
US11834479B2 (en) 2016-06-24 2023-12-05 The Scripps Research Institute Nucleoside triphosphate transporter and uses thereof
US11834689B2 (en) 2017-07-11 2023-12-05 The Scripps Research Institute Incorporation of unnatural nucleotides and methods thereof
US11879145B2 (en) 2019-06-14 2024-01-23 The Scripps Research Institute Reagents and methods for replication, transcription, and translation in semi-synthetic organisms

Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019014262A1 (fr) * 2017-07-11 2019-01-17 The Scripps Research Institute Incorporation de nucléotides non naturels et procédés d'utilisation in vivo de ceux-ci
US20200181220A1 (en) 2017-08-03 2020-06-11 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
CN114949240A (zh) 2019-02-06 2022-08-30 新索思股份有限公司 Il-2缀合物及其使用方法
TW202120128A (zh) 2019-08-15 2021-06-01 美商欣爍克斯公司 使用ｉｌ－２接合物之免疫腫瘤學組合療法
TW202122401A (zh) 2019-08-23 2021-06-16 美商欣爍克斯公司 新穎之ｉｌ－１５接合物及其用途
JP2022547078A (ja) 2019-09-10 2022-11-10 シンソークス， インコーポレイテッド Ｉｌ－２コンジュゲートおよび自己免疫疾患を治療するための使用方法
TW202128994A (zh) * 2019-09-30 2021-08-01 美商史基普研究協會 真核半合成生物
TW202128996A (zh) * 2019-10-10 2021-08-01 美商史基普研究協會 用於活體內合成非天然多肽的組合物及方法
JP2022554272A (ja) 2019-11-04 2022-12-28 シンソークス， インコーポレイテッド インターロイキン１０コンジュゲートおよびその使用
CA3159944A1 (fr) 2019-12-02 2021-06-10 David HUSS Edition therapeutique
KR20230004682A (ko) 2020-04-22 2023-01-06 머크 샤프 앤드 돔 엘엘씨 인터류킨-2 수용체 베타 감마c 이량체에 대해 편향되고 비펩티드성 수용성 중합체에 접합된 인간 인터류킨-2 접합체
IL299074A (en) 2020-06-25 2023-02-01 Synthorx Inc Immuno-oncology therapeutic combination with IL-2 and anti-AGFR antibody conjugates
WO2022076859A1 (fr) 2020-10-09 2022-04-14 Synthorx, Inc. Thérapies immuno-oncologiques avec des conjugués d'il-2
BR112023006364A2 (pt) 2020-10-09 2023-05-09 Synthorx Inc Terapia de combinação de imuno-oncologia com conjugados de il-2 e pembrolizumabe
WO2022098160A1 (fr) * 2020-11-06 2022-05-12 한국과학기술원 Protéine éditée par un gène dans laquelle un acide aminé non naturel est incorporé spécifiquement à un site, et procédé d'édition de gène l'utilisant
CN114807154A (zh) * 2020-12-10 2022-07-29 深圳市瑞吉生物科技有限公司 一种修饰的核酸及其应用
EP4291243A1 (fr) 2021-02-12 2023-12-20 Synthorx, Inc. Polythérapie contre le cancer du poumon avec des conjugués d'il-2 et un anticorps anti-pd-1 ou un fragment de liaison à l'antigène de ce dernier
TW202245843A (zh) 2021-02-12 2022-12-01 美商欣爍克斯公司 Ｉｌ－２接合物及西米普利單抗（ｃｅｍｉｐｌｉｍａｂ）之皮膚癌組合療法
WO2022200525A1 (fr) 2021-03-26 2022-09-29 Innate Pharma Protéines multi-spécifiques comprenant un site de liaison à nkp46, un site de liaison à un antigène tumoral fusionné à une cytokine pour la liaison à des cellules nk
EP4346904A1 (fr) 2021-06-03 2024-04-10 Synthorx, Inc. Polythérapie contre le cancer de la tête et du cou comprenant un conjugué d'il-2 et du cétuximab
WO2022258691A1 (fr) 2021-06-09 2022-12-15 Innate Pharma Protéines multispécifiques se liant à nkg2d, récepteur de cytokine, antigène tumoral et cd16a
IL308531A (en) 2021-06-09 2024-01-01 Innate Pharma Multiple specific antibodies that bind to CD20, NKP46, CD16 and are equipped with IL-2
EP4352098A1 (fr) 2021-06-09 2024-04-17 Innate Pharma Protéines multispécifiques se liant à nkp46, récepteur de cytokine, antigène tumoral et cd16a
WO2022258678A1 (fr) 2021-06-09 2022-12-15 Innate Pharma Protéines multispécifiques se liant à nkp30, un récepteur de cytokine, un antigène tumoral et cd16a
CN117980481A (zh) 2021-07-16 2024-05-03 阿普塔生物公司 用于基因表达调节的多核苷酸组合物和方法
CN114196714B (zh) * 2021-11-04 2023-09-29 华南理工大学 利用末端脱氧核糖核苷酸转移酶无模板合成含非天然碱基寡核苷酸链的方法及其应用
WO2023122573A1 (fr) 2021-12-20 2023-06-29 Synthorx, Inc. Polythérapie contre le cancer de la tête et du cou comprenant un conjugué d'il-2 et du pembrolizumab
WO2023122750A1 (fr) 2021-12-23 2023-06-29 Synthorx, Inc. Polythérapie contre le cancer avec des conjugués d'il-2 et du cétuximab

Family Cites Families (294)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
JPS5927900A (ja) 1982-08-09 1984-02-14 Wakunaga Seiyaku Kk 固定化オリゴヌクレオチド
FR2540122B1 (fr) 1983-01-27 1985-11-29 Centre Nat Rech Scient Nouveaux composes comportant une sequence d'oligonucleotide liee a un agent d'intercalation, leur procede de synthese et leur application
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US5015733A (en) 1983-12-20 1991-05-14 California Institute Of Technology Nucleosides possessing blocked aliphatic amino groups
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US4849513A (en) 1983-12-20 1989-07-18 California Institute Of Technology Deoxyribonucleoside phosphoramidites in which an aliphatic amino group is attached to the sugar ring and their use for the preparation of oligonucleotides containing aliphatic amino groups
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
DE3572982D1 (en) 1984-03-06 1989-10-19 Takeda Chemical Industries Ltd Chemically modified lymphokine and production thereof
US4569790A (en) 1984-03-28 1986-02-11 Cetus Corporation Process for recovering microbially produced interleukin-2 and purified recombinant interleukin-2 compositions
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
FR2567892B1 (fr) 1984-07-19 1989-02-17 Centre Nat Rech Scient Nouveaux oligonucleotides, leur procede de preparation et leurs applications comme mediateurs dans le developpement des effets des interferons
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5430136A (en) 1984-10-16 1995-07-04 Chiron Corporation Oligonucleotides having selectably cleavable and/or abasic sites
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
FR2575751B1 (fr) 1985-01-08 1987-04-03 Pasteur Institut Nouveaux nucleosides de derives de l'adenosine, leur preparation et leurs applications biologiques
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5656493A (en) 1985-03-28 1997-08-12 The Perkin-Elmer Corporation System for automated performance of the polymerase chain reaction
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US5206344A (en) 1985-06-26 1993-04-27 Cetus Oncology Corporation Interleukin-2 muteins and polymer conjugation thereof
US4766106A (en) 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US5576195A (en) 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US4910300A (en) 1985-12-11 1990-03-20 Chiron Corporation Method for making nucleic acid probes
US5093232A (en) 1985-12-11 1992-03-03 Chiron Corporation Nucleic acid probes
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
JPS638396A (ja) 1986-06-30 1988-01-14 Wakunaga Pharmaceut Co Ltd ポリ標識化オリゴヌクレオチド誘導体
US4931544A (en) 1986-09-04 1990-06-05 Cetus Corporation Succinylated interleukin-2 for pharmaceutical compositions
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
AU598946B2 (en) 1987-06-24 1990-07-05 Howard Florey Institute Of Experimental Physiology And Medicine Nucleoside derivatives
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US4924624A (en) 1987-10-22 1990-05-15 Temple University-Of The Commonwealth System Of Higher Education 2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof
US5525465A (en) 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
DE3738460A1 (de) 1987-11-12 1989-05-24 Max Planck Gesellschaft Modifizierte oligonukleotide
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
JPH03503894A (ja) 1988-03-25 1991-08-29 ユニバーシィティ　オブ　バージニア　アランミ　パテンツ　ファウンデイション オリゴヌクレオチド　ｎ‐アルキルホスホラミデート
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
CA2008096A1 (fr) 1989-01-19 1990-07-19 Samuel Rose Amplification de l'acide nucleique a l'aide d'une amorce unique
US4902502A (en) 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5089261A (en) 1989-01-23 1992-02-18 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5599923A (en) 1989-03-06 1997-02-04 Board Of Regents, University Of Tx Texaphyrin metal complexes having improved functionalization
US5457183A (en) 1989-03-06 1995-10-10 Board Of Regents, The University Of Texas System Hydroxylated texaphyrins
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5591722A (en) 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
EP0942000B1 (fr) 1989-10-24 2004-06-23 Isis Pharmaceuticals, Inc. Oligonucléotides modifiés en position 2'
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5177198A (en) 1989-11-30 1993-01-05 University Of N.C. At Chapel Hill Process for preparing oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5130302A (en) 1989-12-20 1992-07-14 Boron Bilogicals, Inc. Boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using same
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US5587470A (en) 1990-01-11 1996-12-24 Isis Pharmaceuticals, Inc. 3-deazapurines
US5578718A (en) 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
US5670633A (en) 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
US5646265A (en) 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5681941A (en) 1990-01-11 1997-10-28 Isis Pharmaceuticals, Inc. Substituted purines and oligonucleotide cross-linking
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
AU7579991A (en) 1990-02-20 1991-09-18 Gilead Sciences, Inc. Pseudonucleosides and pseudonucleotides and their polymers
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
WO1991014781A1 (fr) 1990-03-19 1991-10-03 Henkel Research Corporation METHODE POUR AUGMENTER L'ACTIVITE D'OMEGA-HYDROXYLASE CHEZ LE $i(CANDIDA TROPICALIS)
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
GB9009980D0 (en) 1990-05-03 1990-06-27 Amersham Int Plc Phosphoramidite derivatives,their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
JPH04126084A (ja) 1990-05-11 1992-04-27 Hoechst Japan Ltd 蛋白質の製造法
EP0455905B1 (fr) 1990-05-11 1998-06-17 Microprobe Corporation Bâtonnets d'immersion pour l'essai d'hybridition des acides nucléiques et procédés pour l'immobilisation covalente d'oligonucléotides
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5688941A (en) 1990-07-27 1997-11-18 Isis Pharmaceuticals, Inc. Methods of making conjugated 4' desmethyl nucleoside analog compounds
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
BR9106702A (pt) 1990-07-27 1993-06-08 Isis Pharmaceuticals Inc Analogo de oligonucleotideos e processos para modular a producao de uma proteina por um organismo e para tratar um organismo
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
IL99066A (en) 1990-08-03 1996-01-31 Sterling Winthrop Inc Nuclease-resistant compounds containing oligonucleotide sequences having either or both of the ends, and preparations containing them
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
JPH06505704A (ja) 1990-09-20 1994-06-30 ギリアド　サイエンシズ，インコーポレイテッド 改変ヌクレオシド間結合
NZ239893A (en) 1990-09-25 1993-11-25 Hoechst Japan A method for introducing a foreign dna into a cell
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
CA2095212A1 (fr) 1990-11-08 1992-05-09 Sudhir Agrawal Incorporation de multiples groupes de ligands sur des oligonucleotides synthetiques
US5268273A (en) 1990-12-14 1993-12-07 Phillips Petroleum Company Pichia pastoris acid phosphatase gene, gene regions, signal sequence and expression vectors comprising same
US5672697A (en) 1991-02-08 1997-09-30 Gilead Sciences, Inc. Nucleoside 5'-methylene phosphonates
US5595732A (en) 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5389529A (en) 1991-06-12 1995-02-14 Regeneron Pharmaceuticals, Inc. Modified lamβ signal sequence and processes for producing recombinant neurotrophins
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5612199A (en) 1991-10-11 1997-03-18 Behringwerke Ag Method for producing a polynucleotide for use in single primer amplification
DE59208572D1 (de) 1991-10-17 1997-07-10 Ciba Geigy Ag Bicyclische Nukleoside, Oligonukleotide, Verfahren zu deren Herstellung und Zwischenprodukte
US5594121A (en) 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
DE637965T1 (de) 1991-11-26 1995-12-14 Gilead Sciences Inc Gesteigerte bildung von triple- und doppelhelices aus oligomeren mit modifizierten pyrimidinen.
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
TW393513B (en) 1991-11-26 2000-06-11 Isis Pharmaceuticals Inc Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5359044A (en) 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
DE9115660U1 (fr) 1991-12-18 1992-07-30 Hoechst Ag, 6230 Frankfurt, De
US5595726A (en) 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
FR2687679B1 (fr) 1992-02-05 1994-10-28 Centre Nat Rech Scient Oligothionucleotides.
US5229109A (en) 1992-04-14 1993-07-20 Board Of Regents, The University Of Texas System Low toxicity interleukin-2 analogues for use in immunotherapy
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
EP0577558A2 (fr) 1992-07-01 1994-01-05 Ciba-Geigy Ag Nucléosides carbocycliques contenant des noyaux bicycliques, oligonucléotides en dérivant, procédé pour leur préparation, leur application et des intermédiaires
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
TW263437B (fr) 1992-09-24 1995-11-21 Takeda Pharm Industry Co Ltd
US6288302B1 (en) 1992-11-04 2001-09-11 National Science Council Of R.O.C. Application of α-amylase gene promoter and signal sequence in the production of recombinant proteins in transgenic plants and transgenic plant seeds
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
GB9304618D0 (en) 1993-03-06 1993-04-21 Ciba Geigy Ag Chemical compounds
ATE155467T1 (de) 1993-03-30 1997-08-15 Sanofi Sa Acyclische nucleosid analoge und sie enthaltende oligonucleotidsequenzen
AU6412794A (en) 1993-03-31 1994-10-24 Sterling Winthrop Inc. Oligonucleotides with amide linkages replacing phosphodiester linkages
HU9501994D0 (en) 1993-03-31 1995-09-28 Sterling Winthrop Inc Novel 5'-substituted nucleosides and oligomers produced therefrom
DE4311944A1 (de) 1993-04-10 1994-10-13 Degussa Umhüllte Natriumpercarbonatpartikel, Verfahren zu deren Herstellung und sie enthaltende Wasch-, Reinigungs- und Bleichmittelzusammensetzungen
US6294323B1 (en) 1993-04-14 2001-09-25 Behringwerke Ag Self initiating single primer amplification of nucleic acids
GB9311682D0 (en) 1993-06-05 1993-07-21 Ciba Geigy Ag Chemical compounds
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
US5446137B1 (en) 1993-12-09 1998-10-06 Behringwerke Ag Oligonucleotides containing 4'-substituted nucleotides
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5470719A (en) 1994-03-18 1995-11-28 Meng; Shi-Yuan Modified OmpA signal sequence for enhanced secretion of polypeptides
US5596091A (en) 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US5627053A (en) 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5525711A (en) 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5580731A (en) 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5597909A (en) 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
WO1996010089A1 (fr) 1994-09-29 1996-04-04 Ajinomoto Co., Inc. Modification d'un peptide et d'une proteine
JPH0889278A (ja) 1994-09-29 1996-04-09 Ajinomoto Co Inc 遺伝子導入用修飾蛋白質及びその製法
US5712114A (en) 1995-06-06 1998-01-27 Basf Aktiengesellschaft Compositions for expression of proteins in host cells using a preprocollagen signal sequence
US6143557A (en) 1995-06-07 2000-11-07 Life Technologies, Inc. Recombination cloning using engineered recombination sites
AU724922B2 (en) 1995-06-07 2000-10-05 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6261797B1 (en) 1996-01-29 2001-07-17 Stratagene Primer-mediated polynucleotide synthesis and manipulation techniques
GB9606158D0 (en) 1996-03-23 1996-05-29 Ciba Geigy Ag Chemical compounds
JPH09324000A (ja) 1996-06-05 1997-12-16 Suntory Ltd インターロイキン−２受容体を特異的に認識する因子とリボヌクレアーゼとの接合体
JP3756313B2 (ja) 1997-03-07 2006-03-15 武 今西 新規ビシクロヌクレオシド及びオリゴヌクレオチド類縁体
US6770748B2 (en) 1997-03-07 2004-08-03 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogue
CN1273476C (zh) 1997-09-12 2006-09-06 埃克西康有限公司 寡核苷酸类似物
US6794499B2 (en) 1997-09-12 2004-09-21 Exiqon A/S Oligonucleotide analogues
US6054271A (en) 1997-10-21 2000-04-25 The Regents Of The University Of California Methods of using synthetic molecules and target sequences
EP1032837B1 (fr) 1997-10-21 2006-05-03 The Regents Of The University Of California Sequences cibles pour molecules synthetiques et procedes d'utilisation de ces dernieres
US5932474A (en) 1997-10-21 1999-08-03 The Regents Of The University Of California Target sequences for synthetic molecules
US6008378A (en) 1997-10-21 1999-12-28 The Regents Of The University Of California Synthetic molecules that specifically react with target sequences
CA2307016A1 (fr) 1997-10-24 1999-05-06 Life Technologies, Inc. Clonage recombinatoire au moyen d'acides nucleiques possedant des sites de recombinaison
DE19813317A1 (de) 1998-03-26 1999-09-30 Roche Diagnostics Gmbh Verbessertes Verfahren zur Primer Extension Präamplifikations-PCR
US6955807B1 (en) 1998-05-15 2005-10-18 Bayer Pharmaceuticals Corporation IL-2 selective agonists and antagonists
US6391544B1 (en) 1998-05-15 2002-05-21 Abbott Laboratories Method for using unequal primer concentrations for generating nucleic acid amplification products
US6562798B1 (en) 1998-06-05 2003-05-13 Dynavax Technologies Corp. Immunostimulatory oligonucleotides with modified bases and methods of use thereof
WO2000011028A2 (fr) 1998-08-21 2000-03-02 Yeda Research And Development Co. Ltd. Peptides anti-inflammatoires derives d'il-2 et analogues correspondants
US6175001B1 (en) 1998-10-16 2001-01-16 The Scripps Research Institute Functionalized pyrimidine nucleosides and nucleotides and DNA's incorporating same
AU774643B2 (en) 1999-03-02 2004-07-01 Invitrogen Corporation Compositions and methods for use in recombinational cloning of nucleic acids
NZ514348A (en) 1999-05-04 2004-05-28 Exiqon As L-ribo-LNA analogues
US6525191B1 (en) 1999-05-11 2003-02-25 Kanda S. Ramasamy Conformationally constrained L-nucleosides
AU5088600A (en) 1999-06-03 2000-12-28 Bioinnovation Limited Gene therapy products
EP1114827B1 (fr) 1999-07-15 2010-11-17 Japan Science and Technology Agency Nouvelle paire de bases d'acide nucleique
EP1226255B1 (fr) 1999-10-29 2006-03-29 Stratagene California Compositions et procedes utilisant des adn polymerases
ATE391789T1 (de) 1999-12-10 2008-04-15 Invitrogen Corp Verwendung einer vielzahl von rekombinationsstellen mit einzigartiger spezifität beim rekombinatorischen klonen
WO2003070918A2 (fr) 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Inhibition mediee par interference arn d'une expression genique faisant appel a des acides nucleiques interferants courts chimiquement modifies (sina)
US20020001804A1 (en) 2000-02-25 2002-01-03 Wayne Mitchell Genomic analysis of tRNA gene sets
WO2002036626A1 (fr) 2000-11-02 2002-05-10 Maxygen Aps Polypeptides multimeres comportant une seule chaine
US20040122223A1 (en) 2001-02-07 2004-06-24 Davis Jeremy Martin Non-natural nucleotides and dinucleotides
JP2004527504A (ja) 2001-03-01 2004-09-09 フアーマセツト・リミテツド ２’，３’−ジデオキシ−２’，３’−ジデヒドロヌクレオシドの合成法
EP2796546B1 (fr) * 2001-04-19 2017-08-09 The Scripps Research Institute Incorporation d'acides aminés artificiels
EP2292271A3 (fr) 2001-10-10 2011-09-14 BioGeneriX AG Remodelage et glycoconjugation des anticorps
KR100948532B1 (ko) 2001-11-07 2010-03-23 넥타르 테라퓨틱스 분지형 중합체 및 그의 공액체
AUPR975201A0 (en) 2001-12-24 2002-01-24 Unisearch Limited Enzymatic redox labelling of nucleic acids
US20060074035A1 (en) 2002-04-17 2006-04-06 Zhi Hong Dinucleotide inhibitors of de novo RNA polymerases for treatment or prevention of viral infections
US7745417B2 (en) 2002-07-17 2010-06-29 Riken Nucleosides or nucleotides having novel unnatural bases and use thereof
US7977049B2 (en) 2002-08-09 2011-07-12 President And Fellows Of Harvard College Methods and compositions for extending the life span and increasing the stress resistance of cells and organisms
EP1562971B1 (fr) 2002-11-05 2014-02-12 Isis Pharmaceuticals, Inc. Composes oligomeres renfermant un substitut de sucre polycyclique et compositions intervenant dans la modulation genique
AU2003290598A1 (en) 2002-11-05 2004-06-03 Isis Pharmaceuticals, Inc. Modified oligonucleotides for use in rna interference
CA2507189C (fr) 2002-11-27 2018-06-12 Sequenom, Inc. Procedes et systemes de detection et d'analyse de variations de sequences bases sur la fragmentation
RS20050501A (en) 2002-12-26 2007-08-03 Mountain View Pharmaceuticals Inc., Polymer conjugates of cytokines,chemokines,growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity
CA2519092C (fr) 2003-03-14 2014-08-05 Neose Technologies, Inc. Polymeres hydrosolubles ramifies et conjugues de ceux-ci
PL1615945T3 (pl) 2003-04-09 2012-03-30 Ratiopharm Gmbh Sposoby glikopegylacji i białka/peptydy wytwarzane tymi sposobami
AU2004233083B2 (en) 2003-04-17 2010-09-16 The Scripps Research Institute Expanding the eukaryotic genetic code
WO2004106356A1 (fr) 2003-05-27 2004-12-09 Syddansk Universitet Derives de nucleotides fonctionnalises
WO2005007121A2 (fr) 2003-07-18 2005-01-27 Massachusetts Institute Of Technology Polypeptides de l'interleukine-2(il-2) mutante
DK1661905T3 (da) 2003-08-28 2012-07-23 Takeshi Imanishi Hidtil ukendte syntetiske nukleinsyrer af N-O-krydsbindingstype
JP4956802B2 (ja) 2003-09-10 2012-06-20 独立行政法人理化学研究所 非天然型塩基を有するヌクレオシド又はヌクレオチド及びその利用
CN1852740B (zh) 2003-09-17 2011-05-11 耐科塔医药公司 多支链聚合物的药物前体
JP5379347B2 (ja) 2003-09-18 2013-12-25 アイシス ファーマシューティカルズ， インコーポレーテッド ４’−チオヌクレオシドおよびオリゴマー化合物
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
DK2145956T3 (da) 2003-11-03 2015-09-21 Medical Res Council Polymerase
AU2005211385B2 (en) 2004-02-02 2008-12-11 Ambrx, Inc. Modified human growth hormone polypeptides and their uses
WO2005092928A1 (fr) 2004-03-11 2005-10-06 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon d'hydroxyaklyle et d'une proteine, prepares par l'amination reductrice
US20060084136A1 (en) 2004-07-14 2006-04-20 Invitrogen Corporation Production of fusion proteins by cell-free protein synthesis
US7579451B2 (en) 2004-07-21 2009-08-25 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising a modified or non-natural nucleobase
WO2006049297A1 (fr) 2004-11-08 2006-05-11 Riken Nouveau derive de nucleoside ou de nucleotide et son utilisation
CA2595959A1 (fr) 2005-01-27 2006-08-03 Novartis Vaccines And Diagnostics, Inc. Procedes pour traiter un carcinome cellulaire renal
TWI376234B (en) 2005-02-01 2012-11-11 Msd Oss Bv Conjugates of a polypeptide and an oligosaccharide
JP5649018B2 (ja) 2005-08-04 2015-01-07 タグシクス・バイオ株式会社 新規人工塩基対及びその利用
US8030478B2 (en) 2005-12-09 2011-10-04 Riken Method for nucleic acid replication and novel artificial base pairs
PL2314594T3 (pl) 2006-01-27 2014-12-31 Isis Pharmaceuticals Inc Zmodyfikowane w pozycji 6 analogi bicykliczne kwasów nukleinowych
JP2009524419A (ja) 2006-01-27 2009-07-02 サンタリス ファーマ アー／エス Ｌｎａ修飾したホスホロチオール化オリゴヌクレオチド
US20090069179A1 (en) 2006-02-14 2009-03-12 Basf Se Pyridin-4-ylmethylamides
CA2648009A1 (fr) 2006-02-22 2007-09-07 Riken Procede de synthese d'arnt suppresseur, de construction d'adn et de production de proteine a laquelle un acide amine non naturel est integre au moyen de la construction d'adn
US20140314864A1 (en) 2006-03-31 2014-10-23 Massachusetts Institute Of Technology System for Targeted Delivery of Therapeutic Agents
EP2018437A2 (fr) 2006-05-02 2009-01-28 Allozyne, Inc. Molécules substituées par des acides aminés
DK2066684T3 (da) 2006-05-11 2012-10-22 Isis Pharmaceuticals Inc 5´-Modificerede bicycliske nukleinsyreanaloge
JP5451390B2 (ja) 2006-09-08 2014-03-26 アンブルックス，インコーポレイテッド 脊椎動物細胞内におけるサプレッサーｔｒｎａの転写
CA2858907A1 (fr) 2006-09-08 2008-03-13 Bayer Schering Pharma Aktiengesellschaft Composes et procedes associes a des agents etiquetes 18f
WO2008065372A2 (fr) 2006-11-28 2008-06-05 Nautilus Biotech, S.A. Polypeptides d'érythropoïétine modifiés et utilisation de ceux-ci dans des traitements
WO2008067825A1 (fr) 2006-12-06 2008-06-12 Erik Bjerregaard Pedersen Formation de triplex de type hoogsteen de sondes marquées au pyrène pour une détection d'acide nucléique dans un dosage par fluorescence
US20100190837A1 (en) 2007-02-15 2010-07-29 Isis Pharmaceuticals, Inc. 5'-Substituted-2-F' Modified Nucleosides and Oligomeric Compounds Prepared Therefrom
JP5615558B2 (ja) 2007-02-28 2014-10-29 セリナ・セラピユーテイツクス・インコーポレーテツド 活性ポリオキサゾリンおよびそれを含む組成物
KR101551985B1 (ko) 2007-03-09 2015-09-09 리가가쿠 겐큐쇼 모노뉴클레오시드 또는 모노뉴클레오티드로부터 유도되는 구조를 갖는 화합물, 핵산, 표지 물질, 핵산 검출 방법 및 키트
ES2388590T3 (es) 2007-05-30 2012-10-16 Isis Pharmaceuticals, Inc. Análogos de ácidos nucleicos bicíclicos con puente aminometileno N-sustituido.
ES2386492T3 (es) 2007-06-08 2012-08-21 Isis Pharmaceuticals, Inc. Análogos de ácidos nucleicos bicíclicos carbocíclicos
ES2376507T5 (es) 2007-07-05 2015-08-31 Isis Pharmaceuticals, Inc. Análogos de ácidos nucleicos bicíclicos 6-disustituidos
CA2707840A1 (fr) 2007-08-20 2009-02-26 Allozyne, Inc. Molecules substituees par des acides amines
BRPI0817212A2 (pt) * 2007-09-20 2014-10-14 Riken Pirrolisil-trna sintetase mutante, e método para a produção de proteína tendo aminoácido não-natural integrado nela por sua utilização
US8546556B2 (en) 2007-11-21 2013-10-01 Isis Pharmaceuticals, Inc Carbocyclic alpha-L-bicyclic nucleic acid analogs
WO2009099073A1 (fr) 2008-02-07 2009-08-13 The University Of Tokyo Arnt modifié contenant un nucléotide non naturel et son utilisation
WO2009123216A1 (fr) 2008-03-31 2009-10-08 独立行政法人理化学研究所 Nouvel adn susceptible d'être amplifié par pcr avec une sélectivité élevée et une efficacité élevée
US20130333068A1 (en) 2008-04-29 2013-12-12 Marie Coffin Genes and uses for plant enhancement
US20100112660A1 (en) 2008-05-30 2010-05-06 Barofold, Inc. Method for Derivatization of Proteins Using Hydrostatic Pressure
US20110150820A1 (en) 2008-08-28 2011-06-23 Insight Biopharmaceuticals Ltd. Methods for covalently attaching a polymer to a methionine residue in proteins and peptides
EP2356129B1 (fr) 2008-09-24 2013-04-03 Isis Pharmaceuticals, Inc. Nucléosides alpha-l-bicycliques substitués
CN102307905B (zh) 2008-12-10 2015-11-25 斯克利普斯研究院 利用化学反应性非天然氨基酸产生载体-肽偶联物
CA2749539C (fr) 2009-01-21 2022-07-19 Amgen Inc. Compositions et methodes comprenant des mutants d'interleukine-2 destinees au traitement de maladies inflammatoires et auto-immunes
CN102741266A (zh) 2009-10-06 2012-10-17 塔古西库斯生物株式会社 形成特异碱基对的人工碱基对
EP2495258B1 (fr) 2009-10-29 2016-12-07 Korea Advanced Institute Of Science And Technology Dérivé de poly(éthylèneglycol) avec catéchol et conjugués protéiques ou peptidiques, et leur procédé de préparation
US9127033B2 (en) 2010-04-28 2015-09-08 Isis Pharmaceuticals, Inc. 5′ modified nucleosides and oligomeric compounds prepared therefrom
US20130183761A1 (en) * 2010-09-24 2013-07-18 North Carolina State University Methods for Incorporating Unnatural Amino Acids in Eukaryotic Cells
PT2637694T (pt) 2010-11-12 2021-05-05 Nektar Therapeutics Conjugados de uma fração de il-2 e um polímero
DK3489255T3 (da) 2011-02-10 2021-08-23 Roche Glycart Ag Muterede interleukin-2-polypeptider
US8343752B2 (en) 2011-05-03 2013-01-01 Verdezyne, Inc. Biological methods for preparing adipic acid
LT3401400T (lt) 2012-05-25 2019-06-10 The Regents Of The University Of California Būdai ir kompozicijos, skirtos rnr molekulės nukreipiamai tikslinės dnr modifikacijai ir rnr molekulės nukreipiamam transkripcijos moduliavimui
EP4074728A1 (fr) 2012-08-31 2022-10-19 Sutro Biopharma, Inc. Peptides modifiés comprenant un groupe azido
DK2906592T3 (en) 2012-10-12 2018-12-10 Sutro Biopharma Inc Proteolytic inactivation of selected proteins in bacterial extracts for enhanced expression
SG11201504887TA (en) 2012-12-21 2015-07-30 Bioalliance Cv Hydrophilic self-immolative linkers and conjugates thereof
EP2972327B1 (fr) 2013-03-13 2018-11-21 Trustees of Boston University Commande personnalisable de dégradation protéique dans des systèmes bactériens synthétiques et endogènes
US9234213B2 (en) 2013-03-15 2016-01-12 System Biosciences, Llc Compositions and methods directed to CRISPR/Cas genomic engineering systems
AU2014253785B2 (en) 2013-04-19 2018-07-19 Sutro Biopharma, Inc. Expression of biologically active proteins in a bacterial cell-free synthesis system using cell extracts with elevated levels of exogenous chaperones
EA202191796A2 (ru) 2013-08-08 2022-03-31 Дзе Скриппс Рисёч Инститьют Способ сайт-специфического ферментативного мечения нуклеиновых кислот in vitro введением не встречающихся в природе нуклеотидов
WO2015038426A1 (fr) 2013-09-13 2015-03-19 Asana Biosciences, Llc Lieurs auto-immolables contenant des dérivés d'acide mandélique, conjugués médicament-ligand pour thérapies ciblées, et leurs utilisations
WO2015054658A1 (fr) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Acides aminés modifiés comprenant des groupes fonctionnels de tétrazine, procédés de préparation et procédés d'utilisation associés
WO2015054590A2 (fr) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Arnt synthétases chargées d'amino-acides non naturels pour la production de pyridyl-tétrazine
PL3055321T3 (pl) 2013-10-11 2019-02-28 Sutro Biopharma, Inc. SYNTETAZY tRNA DOŁĄCZAJĄCE AMINOKWAS NIENATURALNY DLA PARA-METYLOAZYDO-L-FENYLOALANINY
WO2015086795A1 (fr) 2013-12-13 2015-06-18 Cellectis Plateforme nucléase cas9 pour ingénierie génomique de micro-algues
US10513706B2 (en) * 2014-04-09 2019-12-24 The Scripps Research Institute Import of unnatural or modified nucleoside triphosphates into cells via nucleic acid triphosphate transporters
CA2946398A1 (fr) 2014-04-24 2015-10-29 The Board Of Trustees Of The Leland Stanford Junior University Superagonistes, agonistes et antagonistes partiels de l'interleukine-2
SG11201700629TA (en) 2014-08-11 2017-02-27 Delinia Inc Modified il-2 variants that selectively activate regulatory t cells for the treatment of autoimmune diseases
AU2015343307B2 (en) 2014-11-06 2021-05-20 Iff Us Holding, Llc Peptide-mediated delivery of RNA-guided endonuclease into cells
EP3985115A1 (fr) 2014-12-12 2022-04-20 The Broad Institute, Inc. Arn guides protégés (pgrnas)
WO2016100889A1 (fr) 2014-12-19 2016-06-23 Sutro Biopharma, Inc. Optimisation de codons en vue d'améliorer les titres et la fidélité
US20170369871A1 (en) 2015-01-12 2017-12-28 Synthorx, Inc. Incorporation of unnatural nucleotides and methods thereof
WO2017106767A1 (fr) * 2015-12-18 2017-06-22 The Scripps Research Institute Production de nucléotides non naturels par l'utilisation d'un système crispr/cas9
US20180369399A1 (en) 2015-12-21 2018-12-27 Duke University Polymer conjugates having reduced antigenicity and methods of using the same
HUE060123T2 (hu) 2016-06-24 2023-01-28 Scripps Research Inst Új nukleozid-trifoszfát-transzporter és alkalmazásai
WO2019014262A1 (fr) 2017-07-11 2019-01-17 The Scripps Research Institute Incorporation de nucléotides non naturels et procédés d'utilisation in vivo de ceux-ci
AU2018300069A1 (en) 2017-07-11 2020-02-27 Synthorx, Inc. Incorporation of unnatural nucleotides and methods thereof
US20200181220A1 (en) 2017-08-03 2020-06-11 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
BR112020013090A2 (pt) 2017-12-29 2020-12-01 The Scripps Research Institute composições de pares não naturais e métodos de uso
AU2020291535A1 (en) 2019-06-14 2022-01-20 The Scripps Research Institute Reagents and methods for replication, transcription, and translation in semi-synthetic organisms
TW202128994A (zh) 2019-09-30 2021-08-01 美商史基普研究協會 真核半合成生物
TW202128996A (zh) 2019-10-10 2021-08-01 美商史基普研究協會 用於活體內合成非天然多肽的組合物及方法
IL302243A (en) 2020-10-23 2023-06-01 Scripps Research Inst Reverse transcription of polynucleotides that include unnatural nucleotides

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11634451B2 (en) 2013-08-08 2023-04-25 The Scripps Research Institute Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides
US11466279B2 (en) 2014-04-09 2022-10-11 The Scripps Research Institute Import of unnatural or modified nucleoside triphosphates into cells via nucleic acid triphosphate transporters
US11761007B2 (en) 2015-12-18 2023-09-19 The Scripps Research Institute Production of unnatural nucleotides using a CRISPR/Cas9 system
US11834479B2 (en) 2016-06-24 2023-12-05 The Scripps Research Institute Nucleoside triphosphate transporter and uses thereof
US11834689B2 (en) 2017-07-11 2023-12-05 The Scripps Research Institute Incorporation of unnatural nucleotides and methods thereof
US11879145B2 (en) 2019-06-14 2024-01-23 The Scripps Research Institute Reagents and methods for replication, transcription, and translation in semi-synthetic organisms

Also Published As

Publication number Publication date
KR102649135B1 (ko) 2024-03-18
WO2019014267A1 (fr) 2019-01-17
MA49578A (fr) 2021-04-07
US20210222147A1 (en) 2021-07-22
KR20200029531A (ko) 2020-03-18
AR112756A1 (es) 2019-12-11
JP2020526197A (ja) 2020-08-31
EP3652316A1 (fr) 2020-05-20
CN111051512A (zh) 2020-04-21
EA202090090A1 (ru) 2020-06-09
TWI821192B (zh) 2023-11-11
US20190376054A1 (en) 2019-12-12
TW201920681A (zh) 2019-06-01
US11834689B2 (en) 2023-12-05
JP2023175678A (ja) 2023-12-12
JP7325341B2 (ja) 2023-08-14
KR20240038157A (ko) 2024-03-22
NZ761479A (en) 2024-03-22
SG11202000167SA (en) 2020-02-27
IL271903A (en) 2020-02-27
AU2018300069A1 (en) 2020-02-27
CA3069321A1 (fr) 2019-01-17
US20240043823A1 (en) 2024-02-08
EP3652316A4 (fr) 2021-04-07

Similar Documents

Publication Publication Date Title
US11834689B2 (en) Incorporation of unnatural nucleotides and methods thereof
WO2019014262A1 (fr) Incorporation de nucléotides non naturels et procédés d'utilisation in vivo de ceux-ci
US20200024597A1 (en) Incorporation of unnatural nucleotides and methods thereof
EP3475295B1 (fr) Nouveau transporteur de nucléoside triphosphate et ses utilisations
US11879145B2 (en) Reagents and methods for replication, transcription, and translation in semi-synthetic organisms
US20230392140A1 (en) Reverse transcription of polynucleotides comprising unnatural nucleotides
AU2020363962A1 (en) Compositions and methods for in vivo synthesis of unnatural polypeptides
EA042937B1 (ru) Включение неприродных нуклеотидов и способы с ними

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

AS Assignment

Owner name: SYNTHORX, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:PTACIN, JEROD;CAFFARO, CAROLINA;AERNI, HANS;SIGNING DATES FROM 20180801 TO 20180806;REEL/FRAME:056035/0613

Owner name: THE SCRIPPS RESEARCH INSTITUTE, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ZHANG, YORKE;FISCHER, EMIL C.;FELDMAN, AARON W.;AND OTHERS;SIGNING DATES FROM 20180716 TO 20180806;REEL/FRAME:056035/0812

STCB Information on status: application discontinuation

Free format text: EXPRESSLY ABANDONED -- DURING EXAMINATION