WO2022104001A1 - Expanded protein libraries and uses thereof - Google Patents
Expanded protein libraries and uses thereof Download PDFInfo
- Publication number
- WO2022104001A1 WO2022104001A1 PCT/US2021/059049 US2021059049W WO2022104001A1 WO 2022104001 A1 WO2022104001 A1 WO 2022104001A1 US 2021059049 W US2021059049 W US 2021059049W WO 2022104001 A1 WO2022104001 A1 WO 2022104001A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- natural amino
- composition
- codon
- amino acids
- complementary
- Prior art date
Links
- 102000004169 proteins and genes Human genes 0.000 title description 5
- 108090000623 proteins and genes Proteins 0.000 title description 5
- 150000001413 amino acids Chemical class 0.000 claims abstract description 255
- 108020004705 Codon Proteins 0.000 claims abstract description 187
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 94
- 108010067902 Peptide Library Proteins 0.000 claims abstract description 34
- 229940024606 amino acid Drugs 0.000 claims description 252
- 235000001014 amino acid Nutrition 0.000 claims description 250
- 108020004566 Transfer RNA Proteins 0.000 claims description 171
- 230000000295 complement effect Effects 0.000 claims description 92
- 239000000203 mixture Substances 0.000 claims description 71
- 230000002068 genetic effect Effects 0.000 claims description 67
- 108020005098 Anticodon Proteins 0.000 claims description 57
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 47
- 108020004999 messenger RNA Proteins 0.000 claims description 37
- 238000013519 translation Methods 0.000 claims description 35
- 108010069514 Cyclic Peptides Proteins 0.000 claims description 14
- 102000001189 Cyclic Peptides Human genes 0.000 claims description 14
- 241000984622 Leucodon Species 0.000 claims description 12
- 150000002678 macrocyclic compounds Chemical class 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- JCZLABDVDPYLRZ-AWEZNQCLSA-N biphenylalanine Chemical compound C1=CC(C[C@H](N)C(O)=O)=CC=C1C1=CC=CC=C1 JCZLABDVDPYLRZ-AWEZNQCLSA-N 0.000 claims description 9
- NILQLFBWTXNUOE-UHFFFAOYSA-N 1-aminocyclopentanecarboxylic acid Chemical compound OC(=O)C1(N)CCCC1 NILQLFBWTXNUOE-UHFFFAOYSA-N 0.000 claims description 8
- DAQPDSNNUNDKNX-UHFFFAOYSA-N 2-(hexylamino)acetic acid Chemical compound CCCCCCNCC(O)=O DAQPDSNNUNDKNX-UHFFFAOYSA-N 0.000 claims description 8
- DGYHPLMPMRKMPD-UHFFFAOYSA-N L-propargyl glycine Natural products OC(=O)C(N)CC#C DGYHPLMPMRKMPD-UHFFFAOYSA-N 0.000 claims description 8
- DGYHPLMPMRKMPD-BYPYZUCNSA-N L-propargylglycine Chemical compound OC(=O)[C@@H](N)CC#C DGYHPLMPMRKMPD-BYPYZUCNSA-N 0.000 claims description 8
- KTHDTJVBEPMMGL-VKHMYHEASA-N N-acetyl-L-alanine Chemical compound OC(=O)[C@H](C)NC(C)=O KTHDTJVBEPMMGL-VKHMYHEASA-N 0.000 claims description 8
- 238000000338 in vitro Methods 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 7
- RDFMDVXONNIGBC-UHFFFAOYSA-N (+-)-2-amino-heptanoic acid Natural products CCCCCC(N)C(O)=O RDFMDVXONNIGBC-UHFFFAOYSA-N 0.000 claims description 6
- SCGJGNWMYSYORS-YFKPBYRVSA-N (2s)-2-azaniumylhex-5-ynoate Chemical compound OC(=O)[C@@H](N)CCC#C SCGJGNWMYSYORS-YFKPBYRVSA-N 0.000 claims description 6
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 claims description 6
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 claims description 6
- RDFMDVXONNIGBC-LURJTMIESA-N L-2-aminoheptanoic acid Chemical compound CCCCC[C@H](N)C(O)=O RDFMDVXONNIGBC-LURJTMIESA-N 0.000 claims description 6
- KTHDTJVBEPMMGL-UHFFFAOYSA-N N-acetyl-L-alanine Natural products OC(=O)C(C)NC(C)=O KTHDTJVBEPMMGL-UHFFFAOYSA-N 0.000 claims description 6
- 229960005190 phenylalanine Drugs 0.000 claims description 6
- 108091081024 Start codon Proteins 0.000 claims description 5
- 229950010131 puromycin Drugs 0.000 claims description 5
- FUHGSOAURCZWCC-SECBINFHSA-N (2r)-2-[(2-chloroacetyl)amino]-3-phenylpropanoic acid Chemical compound ClCC(=O)N[C@@H](C(=O)O)CC1=CC=CC=C1 FUHGSOAURCZWCC-SECBINFHSA-N 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 claims description 4
- 238000010348 incorporation Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 230000000063 preceeding effect Effects 0.000 claims 1
- 239000003446 ligand Substances 0.000 abstract description 3
- 230000003389 potentiating effect Effects 0.000 abstract description 3
- 230000008685 targeting Effects 0.000 abstract description 2
- 230000003993 interaction Effects 0.000 abstract 1
- 239000000178 monomer Substances 0.000 abstract 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 29
- 101710196623 Stimulator of interferon genes protein Proteins 0.000 description 21
- 230000027455 binding Effects 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 101000643024 Homo sapiens Stimulator of interferon genes protein Proteins 0.000 description 9
- 238000006471 dimerization reaction Methods 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 102000050022 human STING1 Human genes 0.000 description 7
- 239000003999 initiator Substances 0.000 description 7
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 6
- HTAQFYLADZNZHZ-VKHMYHEASA-N (2s)-2-[(2-chloroacetyl)amino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)CCl HTAQFYLADZNZHZ-VKHMYHEASA-N 0.000 description 5
- 230000010933 acylation Effects 0.000 description 5
- 238000005917 acylation reaction Methods 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 108090000994 Catalytic RNA Proteins 0.000 description 4
- 102000053642 Catalytic RNA Human genes 0.000 description 4
- 101100043703 Mus musculus Sting1 gene Proteins 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 238000000159 protein binding assay Methods 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 108091092562 ribozyme Proteins 0.000 description 4
- 238000007363 ring formation reaction Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 108090000364 Ligases Proteins 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- -1 N-a-Methyl-L-glycine Chemical compound 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 230000006229 amino acid addition Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 229960001153 serine Drugs 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 102100035533 Stimulator of interferon genes protein Human genes 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 238000007481 next generation sequencing Methods 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 230000004850 protein–protein interaction Effects 0.000 description 2
- ZCCUUQDIBDJBTK-UHFFFAOYSA-N psoralen Chemical compound C1=C2OC(=O)C=CC2=CC2=C1OC=C2 ZCCUUQDIBDJBTK-UHFFFAOYSA-N 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 150000007970 thio esters Chemical class 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- XRILCFTWUCUKJR-INFSMZHSSA-N 2'-3'-cGAMP Chemical compound C([C@H]([C@H]1O)O2)OP(O)(=O)O[C@H]3[C@@H](O)[C@H](N4C5=NC=NC(N)=C5N=C4)O[C@@H]3COP(O)(=O)O[C@H]1[C@@H]2N1C=NC2=C1NC(N)=NC2=O XRILCFTWUCUKJR-INFSMZHSSA-N 0.000 description 1
- VXGRJERITKFWPL-UHFFFAOYSA-N 4',5'-Dihydropsoralen Natural products C1=C2OC(=O)C=CC2=CC2=C1OCC2 VXGRJERITKFWPL-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 102100039496 Choline transporter-like protein 4 Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101000889282 Homo sapiens Choline transporter-like protein 4 Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- RFCBNSCSPXMEBK-INFSMZHSSA-N c-GMP-AMP Chemical class C([C@H]1O2)OP(O)(=O)O[C@H]3[C@@H](O)[C@H](N4C5=NC=NC(N)=C5N=C4)O[C@@H]3COP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 RFCBNSCSPXMEBK-INFSMZHSSA-N 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000054766 genetic haplotypes Human genes 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000003402 intramolecular cyclocondensation reaction Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- LGAILEFNHXWAJP-BMEPFDOTSA-N macrocycle Chemical group N([C@H]1[C@@H](C)CC)C(=O)C(N=2)=CSC=2CNC(=O)C(=C(O2)C)N=C2[C@H]([C@@H](C)CC)NC(=O)C2=CSC1=N2 LGAILEFNHXWAJP-BMEPFDOTSA-N 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 125000002950 monocyclic group Chemical group 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000006231 tRNA aminoacylation Effects 0.000 description 1
- 230000005851 tumor immunogenicity Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
-
- 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
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
Definitions
- Said ASCII copy, created on November 10, 2021, is named 13378-WO-PCT_SL.txt and is 56,782 bytes in size.
- Macrocycles mimicking natural compounds have emerged as a class of biomolecules that can effectively target protein-protein interaction interface with high affinity and specificity.
- RaPID powerful in vitro display methods
- a vast library >10 12
- novel amino acids bearing useful functionalities can further expand the range of chemical diversity of the selection library beyond what 20 natural amino acids can offer.
- the presence of nonnatural amino acids may confer proteolytic stability of macrocycles and may lead to increase in oral bioavailability.
- the present disclosure is based, in part, on the surprising discovery that degenerate codons for multiple amino acids (e.g., Met, Ser, Thr, Leu, Arg and/or Vai) can simultaneously be split to accommodate 6 or more non-natural amino acids in the same library, thus providing significant increase in library diversity compared to prior methods.
- degenerate codons for multiple amino acids e.g., Met, Ser, Thr, Leu, Arg and/or Vai
- composition comprising: a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; and c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non- natural amino acid or a different natural amino acid.
- the composition further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
- the composition further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids.
- one or more tRNAs charged with natural amino acids are not naturally occurring.
- one or more tRNAs charged with non-natural amino acids are not naturally occurring.
- one or more tRNAs charged with non-natural amino acids is tRNA AsnE2 .
- the anti-codon on one or more tRNA AsnE2 is CAA, GGA, CGA, CUA, CAG, CCG, CAU, GGU, CCU or CAC.
- one or more tRNAs charged with a natural amino acid is a tRNA that is naturally charged with that natural amino acid.
- the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and/or Vai or any combination thereof.
- the composition comprises tRNAPhe.
- the composition comprises at least 30 different tRNAs for incorporation of 19 different natural amino acids.
- the composition does not comprise tRNA Met .
- the composition comprises a tRNA with an anticodon complementary to initiation codon that is charged with a non-natural amino acid.
- the tRNA with an anticodon complementary to initiation codon is charged with N-a-Chloroacetyl-L-phenylalanine (ClAc-L-Phe), N-a- Chloroacetyl-L-alanine (ClAc-L-Ala), N-acetyl-L-alanine (Ac-L-Ala) or N-a- Chloroacetyl-D-phenylalanine (ClAc-D-Phe).
- one or more non-natural amino acids encoded by a codon that encodes for Met, Ser, Thr, Leu, Arg, Vai and/or termination of translation under universal genetic code is (S)-l,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic), N-a-Methyl-L-phenylalanine (mF), 4-Phenyl-L-phenylalanine (Bip), (S)-2- Aminoheptanoic acid (Ahp), P-Cyclohexyl-L-alanine (Cha), D-Alanine (dA), Cycloleucine (Cle), N-a-Methyl-L-alanine (mA), N-a-Methyl-L-norleucine (mNle), N-n- Hexyl-glycine (HxG), N-a-Methyl-L-glycine (mG), L-a
- the Ser codons under universal genetic code encode 3 different natural or non-natural amino acids including Ser.
- a non-natural amino acid encoded by a Ser codon is N- a-Methyl-L-phenylalanine (mF), (S)-l,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic) or 4-Phenyl-L-phenylalanine (Bip).
- the Arg codons under universal genetic code encode 2 or 3 different natural or non-natural amino acids including Arg.
- a non-natural amino acid encoded by an Arg codon is N-a-Methyl-L-alanine (mA), N-a-Methyl-L-glycine (mG), N-n-Hexyl-glycine (HxG) or Cycloleucine (Cle).
- the Thr codons under universal genetic code encode Thr and a different natural or non-natural amino acid.
- a non-natural amino acid encoded by a Thr codon is N- a-Methyl-L-alanine (mA) or N-a-Methyl-L-norleucine (mNle).
- the Leu codons under universal genetic code encode Leu and a different natural or non-natural amino acid.
- a non-natural amino acid encoded by a Leu codon is L- propargylglycine (Pra).
- Vai codons under universal genetic code encode Vai and a different natural or non-natural amino acid.
- a non-natural amino acid encoded by a Vai codon is N- a-Methyl-L-glycine (mG).
- the composition further comprises purified translation components.
- the composition is cell-free.
- the tRNA component in the translation system is chemically synthesized.
- the tRNA complementary to a Met codon under universal genetic code is charged with a non-natural amino acid.
- a tRNA complementary to Ser codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Ser.
- the tRNA complementary to the Ser codon has an anticodon complementary to UCU and/or UCC.
- the tRNA complementary to the Ser codon has an anticodon complementary to UCG and/or UCA.
- the tRNA complementary to the Ser codon has an anticodon complementary to AGU and/or AGC.
- a tRNA complementary to Arg codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Arg.
- the tRNA complementary to the Arg codon has an anticodon complementary to CGA and/or CGG.
- the tRNA complementary to the Arg codon has an anticodon complementary to AGA and/or AGG.
- the tRNA complementary to the Arg codon has an anticodon complementary to CGU and/or CGC.
- a tRNA complementary to Thr codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Thr.
- the tRNA complementary to the Thr codon has an anticodon complementary to ACU and/or ACC.
- the tRNA complementary to the Thr codon has an anticodon complementary to ACG and/or ACA.
- a tRNA complementary to Leu codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Leu.
- the tRNA complementary to the Leu codon has an anticodon complementary to UUA and/or UUG.
- the tRNA complementary to the Leu codon has an anticodon complementary to CUU and/or CUC.
- the tRNA complementary to the Leu codon has an anticodon complementary to CUG and/or CUA.
- a tRNA complementary to Vai codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Vai.
- the tRNA complementary to the Vai codon has an anticodon complementary to GUA and/or GUG.
- the tRNA complementary to the Vai codon has an anticodon complementary to GUU and/or GUC.
- the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
- the invention provides a composition comprising a plurality of the following tRNAs:
- the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
- the composition comprises a plurality of the following tRNAs: [0057]
- the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
- the composition comprises a plurality of the following tRNAs:
- the invention provides a peptide library comprising 10 12 or more peptides containing both natural and non-natural amino acids, wherein each peptide comprises an amino acid selected from the group consisting of N-a-Chloroacetyl-L- phenylalanine (ClAc-L-Phe), N-a-Chloroacetyl-L-alanine (ClAc-L-Ala), N-acetyl-L- alanine (Ac-L-Ala) and N-a-Chloroacetyl-D-phenylalanine (ClAc-D-Phe), wherein one or more of the non-natural amino acids is (S)-l,2,3,4-Tetrahydroisoquinoline-3- carboxylic acid (Tic), N-a-Methyl-L-phenylalanine (mF), 4-Phenyl-L-phenylalanine (Bip), (S)-2-Amino
- the invention provides a peptide library comprising 10 12 or more peptides containing both natural and non-natural amino acids generated by an in vitro translation system reconstituted with purified translation components comprising: a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; and c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non-natural amino acid or a different natural amino acid.
- the peptide library further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
- the peptide library further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids.
- the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and Vai or a subset thereof.
- each peptide is complexed with a template nucleic acid that codes for the peptide when it is synthesized.
- the template nucleic acid is mRNA.
- the peptide-mRNA complex comprises puromycin or an analogue thereof.
- each peptide forms a macrocycle.
- each peptide is 6-18 amino acids in size.
- each peptide is 6-16 amino acids in size. [0071] In some embodiments, each peptide is 6-14 amino acids in size.
- the library comprises less than 10 16 , less than 10 15 or less than 10 14 peptides.
- the invention provides a method of screening the peptide library to identify a peptide that binds to a biological target comprising: (a) reverse transcribing the mRNA of the peptide-mRNA complex in the peptide library described above to generate the peptide-DNA conjugate; (b) incubating the peptide-DNA conjugate with the biological target; (c) isolating the peptide-DNA conjugate bound to the target; and (d) amplifying the DNA by PCR to determine the amino sequence of the peptide.
- the inventions provides a kit for translation of peptides containing both natural and non-natural amino acids comprising: (a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; (b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; (c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or nonnatural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non-natural amino acid or a different natural amino acid; and (d) a translation system reconstituted with purified translation components.
- the kit further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
- the kit further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids.
- the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and Vai or a subset thereof.
- the invention provides a peptide having an amino acid sequence shown in Fig. 10B.
- the peptide is a cyclic peptide.
- the invention provides a cyclic peptide selected from the peptides in Figs. 11 A and 1 IB.
- the peptide is isolated and/or purified.
- the peptide is a linear peptide.
- Fig. 1A illustrates a degenerate codon split for insertion of multiple amino acids according to an embodiment of the disclosure (NNK-a library): the degenerate codons of Arg, Ser, and Thr split into multiple amino acids. Codons with re-assigned amino acids are shaded in gray. Codons not listed in the chart represent universal genetic code codons. Multiple degenerate codon split for non-natural amino acids are: (1) Ser: mF, Bip; (2) Arg: mG, mA; and (3) Thr: mNle. The AUG codon has been reassigned to code for ClAcF when used to initiate translation and to He when used during translation. Codons not listed in the chart represent universal genetic code codons.
- Fig. IB illustrates a degenerate codon split for insertion of multiple amino acids according to an embodiment of the disclosure (T NK-P library). Codons with reassigned amino acids are shaded in gray. Codons not listed in the chart represent universal genetic code codons. Multiple degenerate codon split for non-natural amino acids are: (1) Ser: mF, Tic; (2) Arg: HxG, Cle; (3) Thr: mA; (4) Leu: Pra; and (5) Vai: mG. The AUG codon has been reassigned to code for ClAcF when used to initiate translation and to mS when used during translation. Codons not listed in the chart represent universal genetic code codons.
- Fig. 1C illustrates codon usage of a NNU library. Codons with reassigned amino acids are shaded in gray. The AUG codon has been reassigned to code for ClAcF when used to initiate translation and to Cys when used during translation. Codons not listed in the chart represent universal genetic code codons.
- Fig. 2 shows an example of a schematic workflow for peptide library generation.
- Fig. 3 shows the increase in diversity of NNK-a and NNK-P libraries vs. the library of Passioura et al. (2016) as a function of peptide size.
- Fig. 4A illustrates exemplary cyclic peptide library members according to one embodiment of the disclosure.
- Each N-mer cyclic peptide contains N-2 randomized amino acids.
- Fig. 4B shows the intramolecular cyclization reaction of ClAcF (N-a- Chloroacetyl-L-phenylalanine) with the sulfhydryl group of Cys in the same peptide, during which the -Cl group is lost and ClAcF becomes AcF (N-a- Acetyl -L- phenylalanine).
- Fig. 5 shows an exemplary peptide cyclization process and attachment of the mRNA coding sequence for each peptide that may be used to generate a library of (e.g., with >10 12 ) macrocyclic peptides, each of which is covalently linked to its coding mRNA via a puromycin-containing linker.
- Fig. 6 shows structures of a subset of non-natural amino acids useful for peptide library generation. These amino acids are ClAcF, Bip, mF, mNle, mA and mG.
- Fig. 7A shows MALDI detection of a model linear peptide (ClAcF)RGRGRGRG(mG)G(mA)G (SEQ ID NO: 1), where R, mG, and mA were simultaneously assigned to three split Arg codon positions.
- Fig. 7A discloses SEQ ID NOS: 44 and 1, respectively, in order of appearance.
- Fig. 7B shows MALDI detection of a model linear peptide (ClAcF)GG(mA)RGSRGR(Bip)G (SEQ ID NO: 2), where S, mA, and Bip were simultaneously assigned to three split Ser codon positions.
- Fig 7B discloses SEQ ID NOS: 46 and 2, respectively, in order of appearance.
- Figs. 7C and 7D illustrate: representative acylation spectra (Fig. 7C) and acylation data summary (Fig. 7D) of LC/MS analysis of engineered ribozyme (flexizyme) mediated aminoacylation of tRNAs.
- Fig. 8A is a schematic representation of a selection cycle, exemplified with human STING protein.
- Fig. 8B shows profiles of cyclic peptide selection vs. an exemplary target (STING protein) for the NNK-a and NNU libraries of peptides of different sizes.
- Fig. 9 shows that selections with macrocyclic peptide libraries yielded potent hits vs. human STING target.
- Fig. 9 discloses SEQ ID NOS: 48-113, respectively, in order of appearance.
- Fig. 10A shows Schematic representation of the NNK- and NNU-derived macrocyclic peptide libraries and the cyclization process.
- Fig. 10A discloses SEQ ID NOS 132, 134, and 133, respectively, in order of appearance.
- Fig. 10B shows a summary of representative STING-binding peptides and their characterization results.
- Fig. 10B discloses SEQ ID NOS: 114, 50, 115-117, 49, 52, 118-131, 45, and 47, respectively in order of appearance.
- Fig. 11A shows the structures of cyclic peptides NNK-1 to NNK-13. Nonnatural amino acids are shown in light gray.
- Fig. 11B shows the structures of cyclic peptides NNU-1 to NNU-10. Nonnatural amino acids are shown in light gray.
- Fig. 12A shows a schematic of the competition binding assay to evaluate screening hits (e.g., for hSTING protein).
- Fig. 12B shows a schematic of the dimerization assay to evaluate screening hits (e.g., for hSTING protein).
- a or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
- the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
- naturally-occurring refers to the fact that an object can be found in nature.
- a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally- occurring.
- a “polypeptide” refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain.
- One or more amino acid residues in the protein can contain a modification such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation.
- a “protein” can comprise one or more polypeptides.
- nucleic acid molecule is intended to include DNA molecules and RNA molecules.
- a nucleic acid molecule can be single- stranded or double- stranded, and can be cDNA.
- ug and uM are used interchangeably with “pg” and “pM,” respectively.
- variable As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range.
- a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values > 0 and ⁇ 2 if the variable is inherently continuous.
- Peptide Libraries Comprising Non-Natural Amino Acids and Generation Thereof [0117]
- This disclosure describes the construction and application of new peptide libraries bearing more than 20, e.g., 25, amino acids (19 natural and 6 non-natural) via splitting of degenerate universal codons for multiple natural amino acids simultaneously.
- the codons that are split naturally code for Met, Ser, Thr, Leu, Arg and/or Vai or any subset thereof. This is accomplished by codon reassignments.
- Figs. 1A and IB Examples of codon tables that can be used to create a peptide library with more than 20 amino acids are shown in Figs. 1A and IB.
- Fig. 1A Arg, Ser, and Thr degenerate codons and the Met codon were split to allow for introduction of 6 non-natural amino acids, i.e., mF, Bip, mG, mNle, mA and ClAcF (as initiator), while retaining all natural amino acids except Met.
- the AUG codon has been reassigned to code for isoleucine (He), as shown in Fig. 1A.
- He isoleucine
- codons for Met, Ser, Thr, Leu, Arg and Vai are split to allow for introduction of 11 non-natural amino acids, i.e., mF, Pra, Tic, Bip, mNle, HxG, mA, ClAcF (as an initiator), mS (assigned to codon AUG after initiation of translation), Cle and mG.
- 11 non-natural amino acids i.e., mF, Pra, Tic, Bip, mNle, HxG, mA, ClAcF (as an initiator), mS (assigned to codon AUG after initiation of translation), Cle and mG.
- Fig. 2 shows an example of a library construction workflow.
- Bold text indicates a molecular entity that is added or formed during the library construction.
- the degenerate oligonucleotide library template and the reagents (e.g., amino acids, tRNAs, synthetases) underlying in vitro translation may be specific to the chosen library (e.g., NNK-a, NNK-P) and its corresponding codon table, as explained further below.
- Peptides in such libraries may be a range of sizes, generally between 6 to 18 amino acids. In some embodiments, the peptides can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids in size.
- Diversity of NNK libraries described herein relative to previous libraries is shown in Fig. 3. As shown in Fig. 3, this disclosure provides libraries with orders of magnitude greater diversity than those described in Passioura et al. (2016). The greater diversity of the NNK libraries described herein compared to the Passioura et al. (2018) library allows for exploration of much greater chemical space and increases the probability of fining a peptide that will bind strongly and selectively to the desired target.
- Peptides in the library may form macrocycles, as shown in Fig. 4A.
- Such peptides are N-mers, where N is the number of amino acids in each peptide, N-2 positions in the library are randomized, shown as “X” in Fig. 4A. The remaining two amino acids are typically used for cyclization. Examples of such amino acid pairs include Cys and ClAcF, Cys and ClAcA, and Cys and ClAc-D-F, which can be intramolecularly cyclized as shown in Fig. 4B to form cyclic peptides. In the example shown in Fig. 4B, amino acid ClAcF in the linear peptide is converted to amino acid AcF upon reaction with a sulfhydryl group of Cys, thereby forming a circular peptide.
- non-natural amino acids may be introduced into libraries described above. This may be accomplished by reprograming the universal genetic code and using suitable tRNAs to allow for introduction of non-natural amino acids as well as to change the codons for certain natural amino acids.
- Non-limiting examples of non-natural amino acids that can be introduced into such peptide libraries include (S)-l,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic), N-a-Methyl-L-phenylalanine (mF), 4-Phenyl-L-phenylalanine (Bip), (S)-2- Aminoheptanoic acid (Ahp), P-Cyclohexyl-L-alanine (Cha), D-Alanine (dA), Cycloleucine (Cle), N-a-Methyl-L-alanine (mA), N-a-Methyl-L-norleucine (mNle), N-n- Hexyl-glycine (HxG), N-a-Methyl-L-glycine (mG), L-a,P-Diaminopropionic acid (Dap), N-a-Methyl-L-serine
- non-natural amino acids are useful for initiation of translation, e.g., to replace a codon naturally coding for Met.
- These non-natural amino acids may include, but are not limited to, N-a-Chloroacetyl-L-phenylalanine (ClAc-L-F or simply ClAcF), N-a-Chloroacetyl-L-alanine (ClAcA), and N-a-Chloroacetyl-D-phenylalanine (ClAc-D- F).
- ClAc-L-F N-a-Chloroacetyl-L-phenylalanine
- ClAcA N-a-Chloroacetyl-D-phenylalanine
- N-a- Chloroacetyl-L-alanine converts to N-a-Acetyl-L-alanine (AcA)
- N-a- Chloroacetyl-D-phenylalanine converts to N-a-Acetyl-D-phenylalanine (Ac- D-F).
- non-natural amino acid are useful for internal positions of the peptide.
- These non-natural amino acids may include, but are not limited to, (S)-l, 2,3,4- Tetrahydroisoquinoline-3-carboxylic acid, N-a-Methyl-L-phenylalanine, 4-Phenyl-L- phenylalanine, Biphenylalanine, (S)-2-Aminoheptanoic acid, P-Cyclohexyl-L-alanine, Hexahydro-L-phenylalanine, D-Alanine, Cycloleucine, N-a-Methyl-L-alanine, N-a- Methyl-L-norleucine, N-n-Hexyl-glycine, N-a-Methyl-L-glycine, Sarcosine, L-a, P- Diaminopropionic acid, N-a-Methyl-L-serine, 4-Azi
- a library may contain anywhere from about 10 12 to about 10 16 peptides.
- Exemplary library sizes are 10 12 , 10 13 , 10 14 , 10 15 and 10 16 peptides, as well as any ranges in-between.
- tRNA is aminoacylated with an amino acid by the engineered ribozyme, followed by ethanol precipitation. After resuspension and RNAse T1 digestion, the reaction mixture is then separated and analyzed on LC/MS.
- tRNA aminoacylation efficiency e.g., as shown in Figs. 7C and 7D.
- tRNAs may be used for library generation. Examples of tRNAs that may be used in an NNK library construction are provided in Table 1.
- tRNA AsnE2 is used for loading of some or all nonnatural amino acids.
- FIG. 2 A general workflow for library construction is illustrated in Fig. 2.
- coli tRNAs was replaced with individual, chemically synthesized tRNAs for in vitro translation: a) tRNA with primary nucleotide sequences identical to aaRS substrates for charging natural amino acids (Gly, Ser, Arg), and b) tRNA with unique, engineered anti-codon loops for flexizyme-mediated acylation of reassigned amino acids and directing them to the desired codons.
- tRNA sequences that may be used to construct libraries described herein are provided in Table 2.
- Table 2 List of exemplary tRNA sequences
- tRNAs are either AsnE2 tRNAs (for re-programmed amino acids) or as surrogate substrates for amino acid synthetases (for natural amino acids Gly, Ser, Arg).
- AsnE2 tRNAs for re-programmed amino acids
- surrogate substrates for amino acid synthetases for natural amino acids Gly, Ser, Arg.
- each reprogrammed amino acid was enzymatically acylated onto the acceptor AsnE2 tRNAs by engineered ribozyme as acylation catalysts (see W02007/066627, incorporated by reference herein), and their acylation efficiency was further confirmed to be more than 10% for all amino acids, tracked by an LC/MS analysis analyzing the 3'- trinucleotide fragments (un-acylated or acylated) that were liberated by RNAse T1 digestion, as shown in Figs. 7C-7D.
- tRNAs used to construct the NNK-a library are shown in Table 3.
- This library extended the total number of amino acids encoded to 25, thus allowing for a greater diversity of peptides in the library. Moreover, the amino acids forming the peptides in this library cover a spectrum of polarity instead of exclusively biasing toward hydrophobic amino acids.
- This library allows for introduction of 6 non-natural amino acids, e.g., mF, mNle, mG, Bip, ClAcF and mA.
- this library allows generation of libraries of peptides made up of 25 different amino acids: 19 natural (all except Met) and 6 non- natural.
- the composition of this library is dramatically different from the library reported by Passioura et al. (2016), who eliminated all hydrophilic natural amino acids, for the combination of 12 natural and 11 non-natural amino acids.
- NNK-P library NNK-P library
- codons for Met, Ser, Thr, Leu, Arg and Vai were split to allow for introduction of 11 non-natural amino acids, i.e., mF, Pra, Tic, Bip, mNle, HxG, mA, ClAcF (as an initiator), mS, Cle and mG.
- tRNAs used to construct the NNK-P library are shown in Table 4.
- a NNU library was generally constructed using the principles of Yamagishi et al., Chemistry and Biology (2011): 1562-1570 to be used as a comparator to the NNK-a and NNK-P libraries, but with certain improvements described below.
- the NNU library unlike in the NNK libraries, only nucleotide U, but not G, is allowed in the third position.
- the NNU library thus encodes 6 non-natural amino acids and 11 natural amino acids.
- the same 6 amino acids are also present in the NNK-a and NNK-P libraries, and the NNU library described herein preserves the important aromatic residues (Bip, Trp) and long aliphatic side-chains (Leu, mNle) that are absent in the library of Yamagishi et al.
- the two degenerate Ser codons, UCU and AGU, can be further split to accommodate two different amino acids using reconstituted synthetic tRNAs, the same method described for splitting codons of the NNK libraries.
- the codon table of the NNU library described herein is shown in Fig. 1C.
- STING is an emerging target of interest for cancer immunotherapy.
- NNK and NNU libraries were applied to produce thioester-linked mono-cyclic peptides with varying ring sizes (7-14 amino acids) and numbers of exocyclic residues (0-2 randomized amino acids) by in vitro translation, followed by c-terminal anti-FLAG purification of full-length mRNA-tagged cyclic peptides.
- the diversity of each purified library was approximately 5 x 10 12 , as estimated by quantitative PCR.
- the STING- binding enriched populations were split and tested for binding against additional proteins including human STING haplotype (AQ) and mouse STING in parallel.
- NGS nextgeneration DNA sequencing
- Fig. 9 sequences of individual selection were triaged via abundance filtering and clustered into related families.
- No crosstalk was observed between NNK and NNU libraries as no overlapping full-length sequences or sharing short motifs were observed, despite that the NNU codon theoretically belongs to the subset of NNK and both libraries encode the same set of non-natural amino acids.
- the NNK-a library produced fewer sequence families as it was more sequence convergent and exhibited stronger apparent population binding to STING.
- FIG. 10A shows the cyclic structures of the NNK macrocyclic peptide libraries, showing the covalent cross-link between the initiator residue (ClAcF) and the c-terminal Cys.
- Fig. 10B shows the sequence and function of the chemically synthesized peptides NNK-1 to NNK-13. The asterisks denote the thioester linkage between the initiator and the cysteine, and the non-natural amino acids are shown in bold.
- the 9-mer hits comprise the largest sequence family in the NNK library, featuring the 8-mer cycle with one exocyclic amino acid overhang (Sequences NNK-1 to NNK-7 in Fig. 10B).
- sequences NNK-1 to NNK-7 in Fig. 10B there is at least one charged amino acid closer to the c-terminal Cysteine amongst the rather hydrophobic residues.
- some of these charged amino acids are unique to NNK library and absent in the NNU library such as (1) His, which is replaced by mNle in the NNU library, and (2) Lys, which is precluded by codon selection.
- NNU library produced diverse families of the 10-14-mer macrocycles, and many of which exhibited strong preference of non-natural aromatic amino acids such as Bip and mF at position two. Similar to that of the NNK library, 1 to 3 residues of Asn, Asp, and Arg existing intermittently with aromatic amino acids were also prevalent across all the families (NNU-1 to NNK10 in Fig. 10B, extended sequence families in Fig. 9).
- Figs. HA and 11B Structures of macrocyclic peptides NNK-1 to NNK-13 and NNU-1 to NNU-10 are shown in Figs. HA and 11B, respectively. These peptides may be linearized to form linear peptides. Furthermore, the amino acids forming the circular linkage (e.g., ClAcF and Cys) can be removed from the linearized peptides to form linear peptides without these amino acids. Of course, such linear peptides can also synthesized de novo using standard techniques. Such peptides can have any amino acid sequence within the amino acid sequences of Fig.
- linear peptides can be 6-12 amino acids long, having the amino acid sequences from position 2-7, 2-10, 2-11, 2-12, and 2-13 of peptides NNK-1 to NNK-13 and NNU-1 to NNU-10, shown in Fig. 10B.
- the information in the sequences of selected macrocycles with the highest affinity for STING contributes to a validation of the degenerate-codon splitting approach when used in large peptide libraries.
- peptides NNK-1 and NNK-2 both contain two amino-acid residues that were encoded by codons split from the degenerate codon for serine: serine, in position 2 (encoded by AGU), and mF, in position 9 (encoded by UCU).
- the presence of these two amino-acid residues in the STING-binding peptides was first inferred from the sequence of the DNA encoding the peptides, and then confirmed when peptides with the inferred sequence were synthesized chemically and shown to have a high affinity for STING.
- a time resolved FRET-based competition binding assay was used to assess test article binding to the cytoplasmic domain (amino acids 155-341) of WT (232R) STING, G230A-R293Q STING, and mouse STING. His-tagged STING cytoplasmic domain at a concentration of 20 nM was incubated with 2.5 nM Tb-labeled anti-His antibody, test compound, and fluorescein-labeled cGAMP analog probe (BioLog cat. no. Cl 95) at a concentration of 200 nM (WT STING) or 40 nM (G230A-R293Q STING and mouse STING) in PBS containing 0.005% Tween-20 and 0.1% BSA.
- Fluorescence at 495 nm and 520 nm was measured using an EnVision microplate reader to quantify FRET between Tb-labeled anti-His antibody and fluorescein-labeled probe. Background was defined as the signal obtained in the absence of STING protein, and background subtracted FRET ratios were normalized to the maximum signal obtained in the absence of test compound. These values were converted to a percent inhibition. Percent inhibition was determined for test compounds at 11 concentrations. The IC50, defined as the concentration of competing test compound needed to reduce specific binding of the probe by 50%, was calculated using the 4-parameter logistic equation to fit the data. A schematic of the competition binding assay is shown in Fig. 12A.
- a time resolved FRET-based assay was used to assess test article-induced dimerization of the cytoplasmic domain (amino acids 155-341) of WT (232R) STING, G230A-R293Q STING, and mouse STING. His-tagged STING and enzymatically biotinylated STING at a concentration of 10 nM each were incubated with 2.5 nM Tb- labeled anti-His antibody, 5 nM d2-labeled Streptavidin (Cisbio cat. no. 610SADLF), and test compound for one hour in PBS containing 0.005% Tween-20 and 0.1% BSA.
- Fluorescence at 660 nm and 610 nm was measured using an EnVision microplate reader to quantify FRET between Tb-labeled anti-His antibody and d2-labeled Streptavidin. Background was defined as the signal obtained in the absence of STING protein, and background subtracted FRET ratios were normalized to the maximum signal obtained with complete dimerization in the presence of 150 nM 2’ 3 ’-cGAMP (InvivoGen cat. no. tlrl-nacga23-02). These values were converted to percent dimerization. Percent dimerization was determined for test compounds at 11 concentrations. The ECso, defined as the concentration of test compound needed to induce 50% dimerization, was calculated using the 4-parameter logistic equation to fit the data. A schematic of the dimerization assay is shown in Fig. 12B.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
Provided herein are peptide libraries with increased expanded monomer diversity. Such libraries may be constructed by splitting degenerate codons of natural amino acids to make room for non-natural amino acids. Such libraries may be used to construct and identify high-affinity, potent macrocyclic peptide ligands that may be useful for targeting protein-protein or similar interaction interfaces with high affinity and specificity.
Description
EXPANDED PROTEIN LIBRARIES AND USES THEREOF
REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Pat. App. No. 63/113,495, filed Nov. 13, 2020, which is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0001.1] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.
Said ASCII copy, created on November 10, 2021, is named 13378-WO-PCT_SL.txt and is 56,782 bytes in size.
BACKGROUND
[0002] Macrocycles mimicking natural compounds have emerged as a class of biomolecules that can effectively target protein-protein interaction interface with high affinity and specificity. Through powerful in vitro display methods (e.g., RaPID), a vast library (>1012) of different genetically encoded peptides can be profiled and identified as potent ligand binding to the chosen target. Introduction of novel amino acids bearing useful functionalities can further expand the range of chemical diversity of the selection library beyond what 20 natural amino acids can offer. For example, the presence of nonnatural amino acids may confer proteolytic stability of macrocycles and may lead to increase in oral bioavailability.
[0003] In order to genetically reprogram and direct one new amino acid to a given codon, one prerequisite is to remove the competing pair of natural amino acid and its recognition synthetase (aaRS) underlying the same codon. This replacement procedure results in the mutually exclusive presence of either native natural amino acid or the reprogrammed amino acid, but not both, in a library, thus imposing an ultimate upper limit on the total numbers of amino acids allowed.
[0004] Passioura et al., J Am Chem Soc, 140: 11551-11555 (2018), have disclosed the splitting the arginine (Arg) degenerate codons via reconstituting the arginyl-tRNAs to successful encode two (in addition to Arg) or three (replacing Arg) amino acids in the library. However, there is a need to generate libraries that allow for even greater diversity and introduction of additional non-natural amino acids, while also retaining a greater diversity provided by the natural amino acids.
SUMMARY
[0005] The present disclosure is based, in part, on the surprising discovery that degenerate codons for multiple amino acids (e.g., Met, Ser, Thr, Leu, Arg and/or Vai) can simultaneously be split to accommodate 6 or more non-natural amino acids in the same library, thus providing significant increase in library diversity compared to prior methods. [0006] In one aspect, the disclosure provides composition comprising: a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; and c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non- natural amino acid or a different natural amino acid.
[0007] In some embodiments, the composition further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
[0008] In some embodiments, the composition further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids.
[0009] In some embodiments, one or more tRNAs charged with natural amino acids are not naturally occurring.
[0010] In some embodiments, one or more tRNAs charged with non-natural amino acids are not naturally occurring.
[0011] In some embodiments, one or more tRNAs charged with non-natural amino acids is tRNAAsnE2.
[0012] In some embodiments, the anti-codon on one or more tRNAAsnE2 is CAA, GGA, CGA, CUA, CAG, CCG, CAU, GGU, CCU or CAC.
[0013] In some embodiments, one or more tRNAs charged with a natural amino acid is a tRNA that is naturally charged with that natural amino acid.
[0014] In some embodiments, the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and/or Vai or any combination thereof.
[0015] In some embodiments, the composition comprises tRNAPhe.
[0016] In some embodiments, the composition comprises at least 30 different tRNAs for incorporation of 19 different natural amino acids.
[0017] In some embodiments, the composition does not comprise tRNAMet.
[0018] In some embodiments, the composition comprises a tRNA with an anticodon complementary to initiation codon that is charged with a non-natural amino acid.
[0019] In some embodiments, the tRNA with an anticodon complementary to initiation codon is charged with N-a-Chloroacetyl-L-phenylalanine (ClAc-L-Phe), N-a- Chloroacetyl-L-alanine (ClAc-L-Ala), N-acetyl-L-alanine (Ac-L-Ala) or N-a- Chloroacetyl-D-phenylalanine (ClAc-D-Phe).
[0020] In some embodiments, one or more non-natural amino acids encoded by a codon that encodes for Met, Ser, Thr, Leu, Arg, Vai and/or termination of translation under universal genetic code is (S)-l,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic), N-a-Methyl-L-phenylalanine (mF), 4-Phenyl-L-phenylalanine (Bip), (S)-2- Aminoheptanoic acid (Ahp), P-Cyclohexyl-L-alanine (Cha), D-Alanine (dA), Cycloleucine (Cle), N-a-Methyl-L-alanine (mA), N-a-Methyl-L-norleucine (mNle), N-n- Hexyl-glycine (HxG), N-a-Methyl-L-glycine (mG), L-a,P-Diaminopropionic acid (Dap), N-a-Methyl-L-serine (mS), 4-Azido-L-homoalanine (Aha), L-propargylglycine (Pra) or L-Homopropargylglycine (Hpg).
[0021] In some embodiments, the Ser codons under universal genetic code encode 3 different natural or non-natural amino acids including Ser.
[0022] In some embodiments, a non-natural amino acid encoded by a Ser codon is N- a-Methyl-L-phenylalanine (mF), (S)-l,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic) or 4-Phenyl-L-phenylalanine (Bip).
[0023] In some embodiments, the Arg codons under universal genetic code encode 2 or 3 different natural or non-natural amino acids including Arg.
[0024] In some embodiments, a non-natural amino acid encoded by an Arg codon is N-a-Methyl-L-alanine (mA), N-a-Methyl-L-glycine (mG), N-n-Hexyl-glycine (HxG) or Cycloleucine (Cle).
[0025] In some embodiments, the Thr codons under universal genetic code encode Thr and a different natural or non-natural amino acid.
[0026] In some embodiments, a non-natural amino acid encoded by a Thr codon is N- a-Methyl-L-alanine (mA) or N-a-Methyl-L-norleucine (mNle).
[0027] In some embodiments, the Leu codons under universal genetic code encode Leu and a different natural or non-natural amino acid.
[0028] In some embodiments, a non-natural amino acid encoded by a Leu codon is L- propargylglycine (Pra).
[0029] In some embodiments, the Vai codons under universal genetic code encode Vai and a different natural or non-natural amino acid.
[0030] In some embodiments, a non-natural amino acid encoded by a Vai codon is N- a-Methyl-L-glycine (mG).
[0031] In some embodiments, the composition further comprises purified translation components.
[0032] In some embodiments, the composition is cell-free.
[0033] In some embodiments, the tRNA component in the translation system is chemically synthesized.
[0034] In some embodiments, the tRNA complementary to a Met codon under universal genetic code is charged with a non-natural amino acid.
[0035] In some embodiments, a tRNA complementary to Ser codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Ser.
[0036] In some embodiments, the tRNA complementary to the Ser codon has an anticodon complementary to UCU and/or UCC.
[0037] In some embodiments, the tRNA complementary to the Ser codon has an anticodon complementary to UCG and/or UCA.
[0038] In some embodiments, the tRNA complementary to the Ser codon has an anticodon complementary to AGU and/or AGC.
[0039] In some embodiments, a tRNA complementary to Arg codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Arg.
[0040] In some embodiments, the tRNA complementary to the Arg codon has an anticodon complementary to CGA and/or CGG.
[0041] In some embodiments, the tRNA complementary to the Arg codon has an anticodon complementary to AGA and/or AGG.
[0042] In some embodiments, the tRNA complementary to the Arg codon has an anticodon complementary to CGU and/or CGC.
[0043] In some embodiments, a tRNA complementary to Thr codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Thr.
[0044] In some embodiments, the tRNA complementary to the Thr codon has an anticodon complementary to ACU and/or ACC.
[0045] In some embodiments, the tRNA complementary to the Thr codon has an anticodon complementary to ACG and/or ACA.
[0046] In some embodiments, a tRNA complementary to Leu codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Leu.
[0047] In some embodiments, the tRNA complementary to the Leu codon has an anticodon complementary to UUA and/or UUG.
[0048] In some embodiments, the tRNA complementary to the Leu codon has an anticodon complementary to CUU and/or CUC.
[0049] In some embodiments, the tRNA complementary to the Leu codon has an anticodon complementary to CUG and/or CUA.
[0050] In some embodiments, a tRNA complementary to Vai codon under universal genetic code is charged with a non-natural amino acid or a natural amino acid that is not Vai.
[0051] In some embodiments, the tRNA complementary to the Vai codon has an anticodon complementary to GUA and/or GUG.
[0052] In some embodiments, the tRNA complementary to the Vai codon has an anticodon complementary to GUU and/or GUC.
[0053] In some embodiments, the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
Second Position
wherein the NNN codons not listed in the chart represent universal genetic code codons. [0054] In some embodiments, the invention provides a composition comprising a plurality of the following tRNAs:
[0055] In some embodiments, the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
Seeawd Position
wherein the NNN codons not listed in the chart represent universal genetic code codons.
[0056] In some embodiments, the composition comprises a plurality of the following tRNAs:
[0057]
[0058] In some embodiments, the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
Position
wherein the NNN codons not listed in the chart represent universal genetic code codons.
[0059] In some embodiments, the composition comprises a plurality of the following tRNAs:
[0060] In another aspect, the invention provides a peptide library comprising 1012 or more peptides containing both natural and non-natural amino acids, wherein each peptide comprises an amino acid selected from the group consisting of N-a-Chloroacetyl-L- phenylalanine (ClAc-L-Phe), N-a-Chloroacetyl-L-alanine (ClAc-L-Ala), N-acetyl-L- alanine (Ac-L-Ala) and N-a-Chloroacetyl-D-phenylalanine (ClAc-D-Phe), wherein one or more of the non-natural amino acids is (S)-l,2,3,4-Tetrahydroisoquinoline-3- carboxylic acid (Tic), N-a-Methyl-L-phenylalanine (mF), 4-Phenyl-L-phenylalanine (Bip), (S)-2-Aminoheptanoic acid (Ahp), P-Cyclohexyl-L-alanine (Cha), D-Alanine (dA), Cycloleucine (Cle), N-a-Methyl-L-alanine (mA), N-a-Methyl-L-norleucine (mNle), N-n- Hexyl-glycine (HxG), N-a-Methyl-L-glycine (mG), L-a,P-Diaminopropionic acid (Dap),
N-a-Methyl-L-serine (mS), 4-Azido-L-homoalanine (Aha), L-propargylglycine (Pra) or L-Homopropargylglycine (Hpg); and wherein each peptide is 6-20 amino acids in size. [0061] In another aspect, the invention provides a peptide library comprising 1012 or more peptides containing both natural and non-natural amino acids generated by an in vitro translation system reconstituted with purified translation components comprising: a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; and c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non-natural amino acid or a different natural amino acid.
[0062] In some embodiments, the peptide library further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
[0063] In some embodiments, the peptide library further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids.
[0064] In some embodiments, the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and Vai or a subset thereof.
[0065] In some embodiments, each peptide is complexed with a template nucleic acid that codes for the peptide when it is synthesized.
[0066] In some embodiments, the template nucleic acid is mRNA.
[0067] In some embodiments, the peptide-mRNA complex comprises puromycin or an analogue thereof.
[0068] In some embodiments, each peptide forms a macrocycle.
[0069] In some embodiments, each peptide is 6-18 amino acids in size.
[0070] In some embodiments, each peptide is 6-16 amino acids in size.
[0071] In some embodiments, each peptide is 6-14 amino acids in size.
[0072] In some embodiments, the library comprises less than 1016, less than 1015 or less than 1014 peptides.
[0073] In another aspect, the invention provides a method of screening the peptide library to identify a peptide that binds to a biological target comprising: (a) reverse transcribing the mRNA of the peptide-mRNA complex in the peptide library described above to generate the peptide-DNA conjugate; (b) incubating the peptide-DNA conjugate with the biological target; (c) isolating the peptide-DNA conjugate bound to the target; and (d) amplifying the DNA by PCR to determine the amino sequence of the peptide. [0074] In yet another aspect, the inventions provides a kit for translation of peptides containing both natural and non-natural amino acids comprising: (a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; (b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; (c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or nonnatural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non-natural amino acid or a different natural amino acid; and (d) a translation system reconstituted with purified translation components.
[0075] In some embodiments, the kit further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
[0076] In some embodiments, the kit further comprises a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids.
[0077] In some embodiments, the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and Vai or a subset thereof.
[0078] In another aspect, the invention provides a peptide having an amino acid sequence shown in Fig. 10B.
[0079] In some embodiments, the peptide is a cyclic peptide.
[0080] In one aspect, the invention provides a cyclic peptide selected from the peptides in Figs. 11 A and 1 IB.
[0081] In some embodiments, the peptide is isolated and/or purified.
[0082] In some embodiments, the peptide is a linear peptide.
[0083] Other features and advantages of the instant disclosure will be apparent from the following description and examples, which should not be construed as limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0084] Fig. 1A illustrates a degenerate codon split for insertion of multiple amino acids according to an embodiment of the disclosure (NNK-a library): the degenerate codons of Arg, Ser, and Thr split into multiple amino acids. Codons with re-assigned amino acids are shaded in gray. Codons not listed in the chart represent universal genetic code codons. Multiple degenerate codon split for non-natural amino acids are: (1) Ser: mF, Bip; (2) Arg: mG, mA; and (3) Thr: mNle. The AUG codon has been reassigned to code for ClAcF when used to initiate translation and to He when used during translation. Codons not listed in the chart represent universal genetic code codons.
[0085] Fig. IB illustrates a degenerate codon split for insertion of multiple amino acids according to an embodiment of the disclosure (T NK-P library). Codons with reassigned amino acids are shaded in gray. Codons not listed in the chart represent universal genetic code codons. Multiple degenerate codon split for non-natural amino acids are: (1) Ser: mF, Tic; (2) Arg: HxG, Cle; (3) Thr: mA; (4) Leu: Pra; and (5) Vai: mG. The AUG codon has been reassigned to code for ClAcF when used to initiate translation and to mS when used during translation. Codons not listed in the chart represent universal genetic code codons.
[0086] Fig. 1C illustrates codon usage of a NNU library. Codons with reassigned amino acids are shaded in gray. The AUG codon has been reassigned to code for ClAcF when used to initiate translation and to Cys when used during translation. Codons not listed in the chart represent universal genetic code codons.
[0087] Fig. 2 shows an example of a schematic workflow for peptide library generation.
[0088] Fig. 3 shows the increase in diversity of NNK-a and NNK-P libraries vs. the library of Passioura et al. (2018) as a function of peptide size.
[0089] Fig. 4A illustrates exemplary cyclic peptide library members according to one embodiment of the disclosure. Each N-mer cyclic peptide contains N-2 randomized amino acids. Fig. 4B shows the intramolecular cyclization reaction of ClAcF (N-a- Chloroacetyl-L-phenylalanine) with the sulfhydryl group of Cys in the same peptide, during which the -Cl group is lost and ClAcF becomes AcF (N-a- Acetyl -L- phenylalanine).
[0090] Fig. 5 shows an exemplary peptide cyclization process and attachment of the mRNA coding sequence for each peptide that may be used to generate a library of (e.g., with >1012) macrocyclic peptides, each of which is covalently linked to its coding mRNA via a puromycin-containing linker.
[0091] Fig. 6 shows structures of a subset of non-natural amino acids useful for peptide library generation. These amino acids are ClAcF, Bip, mF, mNle, mA and mG.
[0092] Fig. 7A shows MALDI detection of a model linear peptide (ClAcF)RGRGRG(mG)G(mA)G (SEQ ID NO: 1), where R, mG, and mA were simultaneously assigned to three split Arg codon positions. Fig. 7A discloses SEQ ID NOS: 44 and 1, respectively, in order of appearance.
[0093] Fig. 7B shows MALDI detection of a model linear peptide (ClAcF)GG(mA)RGSRGR(Bip)G (SEQ ID NO: 2), where S, mA, and Bip were simultaneously assigned to three split Ser codon positions. Fig 7B discloses SEQ ID NOS: 46 and 2, respectively, in order of appearance.
[0094] Figs. 7C and 7D illustrate: representative acylation spectra (Fig. 7C) and acylation data summary (Fig. 7D) of LC/MS analysis of engineered ribozyme (flexizyme) mediated aminoacylation of tRNAs.
[0095] Fig. 8A is a schematic representation of a selection cycle, exemplified with human STING protein.
[0096] Fig. 8B shows profiles of cyclic peptide selection vs. an exemplary target (STING protein) for the NNK-a and NNU libraries of peptides of different sizes.
[0097] Fig. 9 shows that selections with macrocyclic peptide libraries yielded potent hits vs. human STING target. Fig. 9 discloses SEQ ID NOS: 48-113, respectively, in order of appearance.
[0098] Fig. 10A shows Schematic representation of the NNK- and NNU-derived macrocyclic peptide libraries and the cyclization process. Fig. 10A discloses SEQ ID NOS 132, 134, and 133, respectively, in order of appearance.
[0099] Fig. 10B shows a summary of representative STING-binding peptides and their characterization results. Fig. 10B discloses SEQ ID NOS: 114, 50, 115-117, 49, 52, 118-131, 45, and 47, respectively in order of appearance.
[0100] Fig. 11A shows the structures of cyclic peptides NNK-1 to NNK-13. Nonnatural amino acids are shown in light gray.
[0101] Fig. 11B shows the structures of cyclic peptides NNU-1 to NNU-10. Nonnatural amino acids are shown in light gray.
[0102] Fig. 12A shows a schematic of the competition binding assay to evaluate screening hits (e.g., for hSTING protein).
[0103] Fig. 12B shows a schematic of the dimerization assay to evaluate screening hits (e.g., for hSTING protein).
DETAILED DESCRIPTION
[0104] In order that the present description can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
Definitions and Abbreviations
[0105] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
[0106] Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A
and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone);
B (alone); and C (alone).
[0107] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided.
[0108] The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally- occurring.
[0109] A “polypeptide” refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein can contain a modification such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation. A “protein” can comprise one or more polypeptides.
[0110] The term “nucleic acid molecule,” as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single- stranded or double- stranded, and can be cDNA.
[OHl] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.
[0112] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5’ to 3’ orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the
disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
[0113] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).
[0114] As used herein, the terms “ug” and “uM” are used interchangeably with “pg” and “pM,” respectively.
[0115] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values > 0 and < 2 if the variable is inherently continuous.
[0116] All of the references cited herein are incorporated herein by reference in their entireties.
Peptide Libraries Comprising Non-Natural Amino Acids and Generation Thereof [0117] This disclosure describes the construction and application of new peptide libraries bearing more than 20, e.g., 25, amino acids (19 natural and 6 non-natural) via splitting of degenerate universal codons for multiple natural amino acids simultaneously. In some embodiments, the codons that are split naturally code for Met, Ser, Thr, Leu, Arg and/or Vai or any subset thereof. This is accomplished by codon reassignments.
[0118] Examples of codon tables that can be used to create a peptide library with more than 20 amino acids are shown in Figs. 1A and IB. In Fig. 1A, Arg, Ser, and Thr degenerate codons and the Met codon were split to allow for introduction of 6 non-natural
amino acids, i.e., mF, Bip, mG, mNle, mA and ClAcF (as initiator), while retaining all natural amino acids except Met. After initiation, the AUG codon has been reassigned to code for isoleucine (He), as shown in Fig. 1A. In Fig. IB, codons for Met, Ser, Thr, Leu, Arg and Vai are split to allow for introduction of 11 non-natural amino acids, i.e., mF, Pra, Tic, Bip, mNle, HxG, mA, ClAcF (as an initiator), mS (assigned to codon AUG after initiation of translation), Cle and mG. The non-natural amino acids and their incorporation are discussed further below.
[0119] Fig. 2 shows an example of a library construction workflow. Bold text indicates a molecular entity that is added or formed during the library construction. The degenerate oligonucleotide library template and the reagents (e.g., amino acids, tRNAs, synthetases) underlying in vitro translation may be specific to the chosen library (e.g., NNK-a, NNK-P) and its corresponding codon table, as explained further below.
[0120] Peptides in such libraries may be a range of sizes, generally between 6 to 18 amino acids. In some embodiments, the peptides can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids in size. Diversity of NNK libraries described herein relative to previous libraries is shown in Fig. 3. As shown in Fig. 3, this disclosure provides libraries with orders of magnitude greater diversity than those described in Passioura et al. (2018). The greater diversity of the NNK libraries described herein compared to the Passioura et al. (2018) library allows for exploration of much greater chemical space and increases the probability of fining a peptide that will bind strongly and selectively to the desired target. [0121] Peptides in the library may form macrocycles, as shown in Fig. 4A. Such peptides are N-mers, where N is the number of amino acids in each peptide, N-2 positions in the library are randomized, shown as “X” in Fig. 4A. The remaining two amino acids are typically used for cyclization. Examples of such amino acid pairs include Cys and ClAcF, Cys and ClAcA, and Cys and ClAc-D-F, which can be intramolecularly cyclized as shown in Fig. 4B to form cyclic peptides. In the example shown in Fig. 4B, amino acid ClAcF in the linear peptide is converted to amino acid AcF upon reaction with a sulfhydryl group of Cys, thereby forming a circular peptide.
[0122] Formation of peptide macrocycles and linkers useful for this purpose is well known and has been described in U.S. Pat. No. 6,258,558, U.S. Pat. Pub. No. 2010/0105569, and Kurz, et al., “Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein
fusions,” Nucleic Acids Research 28(18): pp. e83 (2000), each of which is incorporated by reference herein. An example of use of the puromycin linkers in the library generation is shown in Fig. 5.
Peptide Library Diversity
[0123] Many non-natural amino acids may be introduced into libraries described above. This may be accomplished by reprograming the universal genetic code and using suitable tRNAs to allow for introduction of non-natural amino acids as well as to change the codons for certain natural amino acids.
[0124] Non-limiting examples of non-natural amino acids that can be introduced into such peptide libraries include (S)-l,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic), N-a-Methyl-L-phenylalanine (mF), 4-Phenyl-L-phenylalanine (Bip), (S)-2- Aminoheptanoic acid (Ahp), P-Cyclohexyl-L-alanine (Cha), D-Alanine (dA), Cycloleucine (Cle), N-a-Methyl-L-alanine (mA), N-a-Methyl-L-norleucine (mNle), N-n- Hexyl-glycine (HxG), N-a-Methyl-L-glycine (mG), L-a,P-Diaminopropionic acid (Dap), N-a-Methyl-L-serine (mS), 4-Azido-L-homoalanine (Aha), L-propargylglycine (Pra) or L-Homopropargylglycine (Hpg). Structures of some of these non-natural amino acids are shown in Fig. 6.
[0125] Certain non-natural amino acids are useful for initiation of translation, e.g., to replace a codon naturally coding for Met. These non-natural amino acids may include, but are not limited to, N-a-Chloroacetyl-L-phenylalanine (ClAc-L-F or simply ClAcF), N-a-Chloroacetyl-L-alanine (ClAcA), and N-a-Chloroacetyl-D-phenylalanine (ClAc-D- F). Similarly to what is shown in Fig. 4B for ClAcF, when reacting with cysteine, N-a- Chloroacetyl-L-alanine (ClAcA) converts to N-a-Acetyl-L-alanine (AcA) and N-a- Chloroacetyl-D-phenylalanine (ClAc-D-F) converts to N-a-Acetyl-D-phenylalanine (Ac- D-F).
[0126] Other non-natural amino acid are useful for internal positions of the peptide. These non-natural amino acids may include, but are not limited to, (S)-l, 2,3,4- Tetrahydroisoquinoline-3-carboxylic acid, N-a-Methyl-L-phenylalanine, 4-Phenyl-L- phenylalanine, Biphenylalanine, (S)-2-Aminoheptanoic acid, P-Cyclohexyl-L-alanine, Hexahydro-L-phenylalanine, D-Alanine, Cycloleucine, N-a-Methyl-L-alanine, N-a- Methyl-L-norleucine, N-n-Hexyl-glycine, N-a-Methyl-L-glycine, Sarcosine, L-a, P-
Diaminopropionic acid, N-a-Methyl-L-serine, 4-Azido-L-homoalanine, L- propargylglycine, and L-Homopropargylglycine.
[0127] A library may contain anywhere from about 1012 to about 1016 peptides.
Exemplary library sizes are 1012, 1013, 1014, 1015 and 1016 peptides, as well as any ranges in-between.
Aminoacylation Process and tRNAs Useful in Translation
[0128] Aminoacylation of tRNAs is accomplished with engineered ribozymes, also referred to as flexizymes. Flexizymes are described, e.g., in Ohuchi et al., Curr Opin Chem Biol, 11 : 537-42 (2007) and Lee et al., Nat Commun, 10:5097 (2019). tRNA is aminoacylated with an amino acid by the engineered ribozyme, followed by ethanol precipitation. After resuspension and RNAse T1 digestion, the reaction mixture is then separated and analyzed on LC/MS. The areas of two fragments, 5 ’-CCA ([M+H]+ = 878.2) and 5’-CCA-amino acid, for each amino acid are extracted and compared to calculate the % tRNA aminoacylation efficiency, e.g., as shown in Figs. 7C and 7D. [0129] Various tRNAs may be used for library generation. Examples of tRNAs that may be used in an NNK library construction are provided in Table 1.
Table 1
[0130] In some embodiments, tRNAAsnE2 is used for loading of some or all nonnatural amino acids.
EXAMPLES
[0131] The following examples are offered by way of illustration and not by way of limitation.
Example 1: NNK Library Construction
[0132] A general workflow for library construction is illustrated in Fig. 2.
[0133] To test the feasibility of extensive codon splitting, two model peptides that encompass three degenerate Arg codons (CGU, CGG, AGG) or three degenerate Ser codons (UCU, AGU, UCG), respectively, were designed. In each peptide, in order to insert multiple amino acids via degenerate codon split, the mixture of E. coli tRNAs was replaced with individual, chemically synthesized tRNAs for in vitro translation: a) tRNA with primary nucleotide sequences identical to aaRS substrates for charging natural amino acids (Gly, Ser, Arg), and b) tRNA with unique, engineered anti-codon loops for flexizyme-mediated acylation of reassigned amino acids and directing them to the desired codons.
[0134] tRNA sequences that may be used to construct libraries described herein are provided in Table 2.
Table 2: List of exemplary tRNA sequences
[0135] These tRNAs are either AsnE2 tRNAs (for re-programmed amino acids) or as surrogate substrates for amino acid synthetases (for natural amino acids Gly, Ser, Arg). Subsequently, each reprogrammed amino acid was enzymatically acylated onto the acceptor AsnE2 tRNAs by engineered ribozyme as acylation catalysts (see W02007/066627, incorporated by reference herein), and their acylation efficiency was further confirmed to be more than 10% for all amino acids, tracked by an LC/MS analysis analyzing the 3'- trinucleotide fragments (un-acylated or acylated) that were liberated by RNAse T1 digestion, as shown in Figs. 7C-7D.
[0136] In vitro translation successfully generated two series of peptides as desired, each bearing one arginine or serine and two additional non-natural amino acids. Mapping of the three degenerate codons to different amino acids gave rise to a range of different peptides, all of which conformed to the design, demonstrating the suitability of these redundant codons for genetic reprogramming. To extend the numbers of exogenous amino acids, Arg, Ser, and Thr degenerate codons were combined to create an library that harbors 19 natural (except methionine) and 6 additional non-natural amino acids (ClAcF, Bip, mA, mG, mF, mNle). The codon assignments for this library (NNK-a library) are shown in Fig. 1A.
[0137] Exemplary tRNAs used to construct the NNK-a library are shown in Table 3.
Table 3: tRNA usage (NNK-a library)
[0138] This library extended the total number of amino acids encoded to 25, thus allowing for a greater diversity of peptides in the library. Moreover, the amino acids forming the peptides in this library cover a spectrum of polarity instead of exclusively biasing toward hydrophobic amino acids.
[0139] Having confirmed the appropriate incorporation of reassigned amino acids at split-codon sites, we used this approach to make a complex library of macrocycles. In this library, the initiator codon (AUG) was reprogrammed to encode the non-natural amino acid ClAcF, which is used to cyclize the peptide by reacting with a cysteine further downstream in the sequence, at which point ClAcF loses the chlorine, and becomes AcF, as shown in Fig. 4B. Randomized amino-acid positions beyond the initiator were encoded by the 32 NNK codon mix, where N stands for any nucleotide and K stands for U or G. These 32 randomized elongation codons included the redundant codons for Arg, Ser, and Thr, which were split simultaneously to sample the total of 25 amino acids, including six non-natural amino acids (ClAcF as an initiator, Bip, mA, mG, mF, and mNle) (Fig. 1A). 19 of the 20 natural amino acids were retained, removing only methionine.
[0140] This library allows for introduction of 6 non-natural amino acids, e.g., mF, mNle, mG, Bip, ClAcF and mA. Thus, this library allows generation of libraries of peptides made up of 25 different amino acids: 19 natural (all except Met) and 6 non- natural.
[0141] The composition of this library is dramatically different from the library reported by Passioura et al. (2018), who eliminated all hydrophilic natural amino acids, for the combination of 12 natural and 11 non-natural amino acids.
[0142] Using the methods described above, a second NNK library was constructed (NNK-P library) with codon assignments as shown in Fig. IB. In this library, codons for Met, Ser, Thr, Leu, Arg and Vai were split to allow for introduction of 11 non-natural amino acids, i.e., mF, Pra, Tic, Bip, mNle, HxG, mA, ClAcF (as an initiator), mS, Cle and mG. Out of these amino acids, mF, Pra, Tic, Bip, mNle, HxG, mA, Cle, mG, and mS are in the internal positions of the peptide and ClAcF/mS are used for cyclization of the peptide. Exemplary tRNAs used to construct the NNK-P library are shown in Table 4.
Table 4: tRNA usage (NNK-B library)
Example 2: NNU Library Construction
[0143] A NNU library was generally constructed using the principles of Yamagishi et al., Chemistry and Biology (2011): 1562-1570 to be used as a comparator to the NNK-a and NNK-P libraries, but with certain improvements described below. In the NNU library, unlike in the NNK libraries, only nucleotide U, but not G, is allowed in the third position. The NNU library thus encodes 6 non-natural amino acids and 11 natural amino acids. The same 6 amino acids are also present in the NNK-a and NNK-P libraries, and
the NNU library described herein preserves the important aromatic residues (Bip, Trp) and long aliphatic side-chains (Leu, mNle) that are absent in the library of Yamagishi et al. (2011). The two degenerate Ser codons, UCU and AGU, can be further split to accommodate two different amino acids using reconstituted synthetic tRNAs, the same method described for splitting codons of the NNK libraries. The codon table of the NNU library described herein is shown in Fig. 1C.
Example 3: Selection of Peptides with Desired Properties from Library [0144] To demonstrate the utility of these libraries, human STING protein was selected as the target for proof-of-concept selections. STING is an emerging target of interest for cancer immunotherapy. Activation of innate immune modulators such as STING, in addition to clinically proven strategy targeting the adaptive immune modulators (e.g. PD-L1, CTL4), is expected to enhance tumor immunogenicity. Both NNK and NNU libraries were applied to produce thioester-linked mono-cyclic peptides with varying ring sizes (7-14 amino acids) and numbers of exocyclic residues (0-2 randomized amino acids) by in vitro translation, followed by c-terminal anti-FLAG purification of full-length mRNA-tagged cyclic peptides. The diversity of each purified library was approximately 5 x 1012, as estimated by quantitative PCR.
[0145] Selections of these mRNA-displayed peptides were incubated with biotinylated human STING, followed by streptavidin bead capture and PCR amplification of bound sequences (Fig. 8A). The fully assembled, purified mRNA-displayed cyclic peptide library is subjected to binding biotinylated human STING, followed by capture using streptavidin beads. After washing, the bound fraction of the library is recovered and PCR amplified, ready for entering the next round of the selection cycle. After four rounds of selection (denoted Rl, R2, R3 and R4), populations binding preferentially to STING were enriched (Fig. 8B). To further investigate the binding cross-reactivity, the STING- binding enriched populations were split and tested for binding against additional proteins including human STING haplotype (AQ) and mouse STING in parallel. Using nextgeneration DNA sequencing (NGS) analysis with Illumina MiSeq, sequences of individual selection were triaged via abundance filtering and clustered into related families (Fig. 9).
[0146] Both selection arms produced distinct families amongst otherwise diverse sequences, differing in the macrocycle ring size and the prevalence of amino acid usage in the hit sequences. No crosstalk was observed between NNK and NNU libraries as no overlapping full-length sequences or sharing short motifs were observed, despite that the NNU codon theoretically belongs to the subset of NNK and both libraries encode the same set of non-natural amino acids. Compared to the NNU library, the NNK-a library produced fewer sequence families as it was more sequence convergent and exhibited stronger apparent population binding to STING.
[0147] Selection from macrocyclic peptide libraries yielded peptides with high affinity for human STING. Fig. 10A shows the cyclic structures of the NNK macrocyclic peptide libraries, showing the covalent cross-link between the initiator residue (ClAcF) and the c-terminal Cys. Fig. 10B shows the sequence and function of the chemically synthesized peptides NNK-1 to NNK-13. The asterisks denote the thioester linkage between the initiator and the cysteine, and the non-natural amino acids are shown in bold. [0148] The 9-mer hits comprise the largest sequence family in the NNK library, featuring the 8-mer cycle with one exocyclic amino acid overhang (Sequences NNK-1 to NNK-7 in Fig. 10B). In each sequence of these 9-mers there is at least one charged amino acid closer to the c-terminal Cysteine amongst the rather hydrophobic residues. Surprisingly, some of these charged amino acids are unique to NNK library and absent in the NNU library such as (1) His, which is replaced by mNle in the NNU library, and (2) Lys, which is precluded by codon selection. In addition, Phe and He are also two frequent residues appearing in the hydrophobic core of these 9-mers and are only present in NNK but not in NNU library (Figs. 10A-B). The NNU library produced diverse families of the 10-14-mer macrocycles, and many of which exhibited strong preference of non-natural aromatic amino acids such as Bip and mF at position two. Similar to that of the NNK library, 1 to 3 residues of Asn, Asp, and Arg existing intermittently with aromatic amino acids were also prevalent across all the families (NNU-1 to NNK10 in Fig. 10B, extended sequence families in Fig. 9).
[0149] Structures of macrocyclic peptides NNK-1 to NNK-13 and NNU-1 to NNU-10 are shown in Figs. HA and 11B, respectively. These peptides may be linearized to form linear peptides. Furthermore, the amino acids forming the circular linkage (e.g., ClAcF and Cys) can be removed from the linearized peptides to form linear peptides without
these amino acids. Of course, such linear peptides can also synthesized de novo using standard techniques. Such peptides can have any amino acid sequence within the amino acid sequences of Fig. 10B, except that they do not include ClAcF and Cys and, optionally, also do not include any amino acids c-terminal and inclusive of Cys (such as mF, D, H, Y, N and G shown in Fig. 10B). Thus, such linear peptides can be 6-12 amino acids long, having the amino acid sequences from position 2-7, 2-10, 2-11, 2-12, and 2-13 of peptides NNK-1 to NNK-13 and NNU-1 to NNU-10, shown in Fig. 10B. The information in the sequences of selected macrocycles with the highest affinity for STING contributes to a validation of the degenerate-codon splitting approach when used in large peptide libraries. In particular, peptides NNK-1 and NNK-2 both contain two amino-acid residues that were encoded by codons split from the degenerate codon for serine: serine, in position 2 (encoded by AGU), and mF, in position 9 (encoded by UCU). The presence of these two amino-acid residues in the STING-binding peptides was first inferred from the sequence of the DNA encoding the peptides, and then confirmed when peptides with the inferred sequence were synthesized chemically and shown to have a high affinity for STING.
[0150] The high affinity, potency, and specificity of the 9-mer cyclic peptides selected in this example demonstrate that a 25-amino-acid alphabet (19 natural and 6 nonnatural amino acids) provides sufficient chemical and structural diversity to mimic the strength of protein-protein interactions. The dissociation constant between peptide NNK- 2 and STING is 3.4 nM, which is comparable to that of the natural ligand cGMAP (3.79 nM), despite their drastically different structures. Such high affinity binding is more typical of in vitro selected macrocycles with rings in the range of 13 to 16 amino-acid residues, which confirms the benefits and further validates the approach described herein.
Competition Binding Assays
[0151] A time resolved FRET-based competition binding assay was used to assess test article binding to the cytoplasmic domain (amino acids 155-341) of WT (232R) STING, G230A-R293Q STING, and mouse STING. His-tagged STING cytoplasmic domain at a concentration of 20 nM was incubated with 2.5 nM Tb-labeled anti-His antibody, test compound, and fluorescein-labeled cGAMP analog probe (BioLog cat. no. Cl 95) at a concentration of 200 nM (WT STING) or 40 nM (G230A-R293Q STING and mouse STING) in PBS containing 0.005% Tween-20 and 0.1% BSA. Fluorescence at 495 nm and 520 nm was measured using an EnVision microplate reader to quantify FRET between Tb-labeled anti-His antibody and fluorescein-labeled probe. Background was defined as the signal obtained in the absence of STING protein, and background subtracted FRET ratios were normalized to the maximum signal obtained in the absence of test compound. These values were converted to a percent inhibition. Percent inhibition was determined for test compounds at 11 concentrations. The IC50, defined as the concentration of competing test compound needed to reduce specific binding of the probe by 50%, was calculated using the 4-parameter logistic equation to fit the data. A schematic of the competition binding assay is shown in Fig. 12A.
Dimerization Assays
[0152] A time resolved FRET-based assay was used to assess test article-induced dimerization of the cytoplasmic domain (amino acids 155-341) of WT (232R) STING, G230A-R293Q STING, and mouse STING. His-tagged STING and enzymatically biotinylated STING at a concentration of 10 nM each were incubated with 2.5 nM Tb- labeled anti-His antibody, 5 nM d2-labeled Streptavidin (Cisbio cat. no. 610SADLF), and test compound for one hour in PBS containing 0.005% Tween-20 and 0.1% BSA. Fluorescence at 660 nm and 610 nm was measured using an EnVision microplate reader to quantify FRET between Tb-labeled anti-His antibody and d2-labeled Streptavidin. Background was defined as the signal obtained in the absence of STING protein, and background subtracted FRET ratios were normalized to the maximum signal obtained with complete dimerization in the presence of 150 nM 2’ 3 ’-cGAMP (InvivoGen cat. no. tlrl-nacga23-02). These values were converted to percent dimerization. Percent
dimerization was determined for test compounds at 11 concentrations. The ECso, defined as the concentration of test compound needed to induce 50% dimerization, was calculated using the 4-parameter logistic equation to fit the data. A schematic of the dimerization assay is shown in Fig. 12B.
Claims
What is claimed is:
1. A composition comprising: a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; and c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non-natural amino acid or a different natural amino acid.
2. The composition of the preceding claim, further comprising a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
3. The composition of any one of the preceding claims, comprising a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids.
4. The composition of any one of the preceding claims, wherein one or more tRNAs charged with natural amino acids are not naturally ocurring.
5. The composition of any one of the preceding claims, wherein one or more tRNAs charged with non-natural amino acids are not naturally ocurring.
- 37 -
6. The composition of any one of the preceding claims, wherein one or more tRNAs charged with non-natural amino acids is tRNAAsnE2.
7. The composition of any one of the preceding claims, wherein the anti-codon on one or more tRNAAsnE2 is CAA, GGA, CGA, CUA, CAG, CCG, CAU, GGU, CCU or CAC.
8. The composition of any one of the preceding claims, wherein one or more tRNAs charged with a natural amino acid is a tRNA that isnaturally charged with that natural amino acid.
9. The composition of any one of the preceding claims, wherein the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and/or Vai or any combination thereof.
10. The composition of any one of the preceding claims, comprising tRNAphe.
11. The composition of any one of the preceding claims, comprising at least 30 different tRNAs for incorporation of 19 different natural amino acids.
12. The composition of any one of the preceding claims, wherein the composition does not comprise tRNAMet.
13. The composition of any one of the preceding claims, comprising a tRNA with an anticodon complementary to initiation codon that is charged with a non-natural amino acid.
14. The composition of the preceding claims, wherein the tRNA with an anticodon complementary to initiation codon is charged with N-a-Chloroacetyl-L- phenylalanine (ClAc-L-Phe), N-a-Chloroacetyl-L-alanine (ClAc-L-Ala), N- acetyl-L-alanine (Ac-L-Ala) or N-a-Chloroacetyl-D-phenylalanine (ClAc-D-Phe).
- 38 -
15. The composition of any one of the preceding claims, wherein one or more nonnatural amino acids encoded by a codon that encodes for Met, Ser, Thr, Leu, Arg, Vai and/or termination of translation under universal genetic code is (S)-l, 2,3,4- Tetrahydroisoquinoline-3-carboxylic acid (Tic), N-a-Methyl-L-phenylalanine (mF), 4-Phenyl-L-phenylalanine (Bip), (S)-2-Aminoheptanoic acid (Ahp), P- Cyclohexyl-L-alanine (Cha), D-Alanine (dA), Cycloleucine (Cle), N-a-Methyl-L- alanine (mA), N-a-Methyl-L-norleucine (mNle), N-n-Hexyl-glycine (HxG), N-a- Methyl-L-glycine (mG), L-a,P-Diaminopropionic acid (Dap), N-a-Methyl-L- serine (mS), 4-Azido-L-homoalanine (Aha), L-propargylglycine (Pra) or L- Homopropargylglycine (Hpg).
16. The composition of any one of the preceding claims, wherein the Ser codons under universal genetic code encode 3 different natural or non-natural amino acids including Ser.
17. The composition of the preceding claim, wherein a non-natural amino acid encoded by a Ser codon is N-a-Methyl-L-phenylalanine (mF), (S)-l, 2,3,4- Tetrahydroisoquinoline-3-carboxylic acid (Tic) or 4-Phenyl-L-phenylalanine (Bip).
18. The composition of any one of the preceding claims, wherein the Arg codons under universal genetic code encode 2 or 3 different natural or non-natural amino acids including Arg.
19. The composition of the preceding claim, wherein a non-natural amino acid encoded by an Arg codon is N-a-Methyl-L-alanine (mA), N-a-Methyl-L-glycine (mG), N-n-Hexyl-glycine (HxG) or Cycloleucine (Cle).
20. The composition of any one of the preceding claims, wherein the Thr codons under universal genetic code encode Thr and a different natural or non-natural amino acid.
21. The composition of the preceding claim, wherein a non-natural amino acid encoded by a Thr codon is N-a-Methyl-L-alanine (mA) or N-a-Methyl-L- norleucine (mNle).
22. The composition of any one of the preceding claims, wherein the Leu codons under universal genetic code encode Leu and a different natural or non-natural amino acid.
23. The composition of the preceding claim, wherein a non-natural amino acid encoded by a Leu codon is L-propargylglycine (Pra).
24. The composition of any one of the preceding claims, wherein the Vai codons under universal genetic code encode Vai and a different natural or non-natural amino acid.
25. The composition of the preceding claim, wherein a non-natural amino acid encoded by a Vai codon is N-a-Methyl-L-glycine (mG).
26. The composition of any one of the preceding claims, wherein the composition further comprises purified translation components.
27. The composition of any one of the preceding claims, wherein the composition is cell-free.
28. The composition of any one of the preceding claims, wherein the tRNA component in the translation system is chemically synthesized.
29. The composition of any one of the preceding claims, wherein the tRNA complementary to a Met codon under universal genetic code is charged with a non-natural amino acid.
30. The composition of any one of the preceding claims, wherein a tRNA complementary to Ser codon under universal genetic code is charged with a nonnatural amino acid or a natural amino acid that is not Ser.
31. The composition of any one of the preceding claims, wherein the tRNA complementary to the Ser codon has an anticodon complementary to UCU and/or ucc.
32. The composition of any one of the preceding claims, wherein the tRNA complementary to the Ser codon has an anticodon complementary to UCG and/or UCA.
33. The composition of any one of the preceding claims, wherein the tRNA complementary to the Ser codon has an anticodon complementary to AGU and/or AGC.
34. The composition of any one of the preceding claims, wherein a tRNA complementary to Arg codon under universal genetic code is charged with a nonnatural amino acid or a natural amino acid that is not Arg.
35. The composition of any one of the preceding claims, wherein the tRNA complementary to the Arg codon has an anticodon complementary to CGA and/or CGG.
36. The composition of any one of the preceding claims, wherein the tRNA complementary to the Arg codon has an anticodon complementary to AGA and/or AGG.
37. The composition of any one of the preceding claims, wherein the tRNA complementary to the Arg codon has an anticodon complementary to CGU and/or CGC.
38. The composition of any one of the preceding claims, wherein a tRNA complementary to Thr codon under universal genetic code is charged with a nonnatural amino acid or a natural amino acid that is not Thr.
39. The composition of any one of the preceding claims, wherein the tRNA complementary to the Thr codon has an anticodon complementary to ACU and/or ACC.
40. The composition of any one of the preceding claims, wherein the tRNA complementary to the Thr codon has an anticodon complementary to ACG and/or ACA.
41. The composition of any one of the preceding claims, wherein a tRNA complementary to Leu codon under universal genetic code is charged with a nonnatural amino acid or a natural amino acid that is not Leu.
42. The composition of any one of the preceding claims, wherein the tRNA complementary to the Leu codon has an anticodon complementary to UUA and/or UUG.
43. The composition of any one of the preceding claims, wherein the tRNA complementary to the Leu codon has an anticodon complementary to CUU and/or cue.
44. The composition of any one of the preceding claims, wherein the tRNA complementary to the Leu codon has an anticodon complementary to CUG and/or CUA.
45. The composition of any one of the preceding claims, wherein a tRNA complementary to Vai codon under universal genetic code is charged with a nonnatural amino acid or a natural amino acid that is not Vai.
- 42 -
46. The composition of any one of the preceding claims, wherein the tRNA complementary to the Vai codon has an anticodon complementary to GUA and/or GUG. 47. The composition of any one of the preceding claims, wherein the tRNA complementary to the Vai codon has an anticodon complementary to GUU and/or GUC.
48. The composition of any one of the preceding claims, wherein the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
wherein the NNN codons not listed in the chart represent universal genetic code codons.
49. The composition of any one of the preceding claims, comprising a plurality of the following tRNAs:
-44 -
50. The composition of any one of the preceding claims, wherein the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
Second Position
wherein the NNN codons not listed in the chart represent universal genetic code codons.
- 45 -
51. The composition of any one of the preceding claims, comprising a plurality of the following tRNAs:
52. The composition of any one of the preceding claims, wherein the tRNAs are simultaneously charged with the natural and non-natural amino acids encoded by the codons as follows:
wherein the NNN codons not listed in the chart represent universal genetic code codons.
- 47 -
53. The composition of any one of the preceding claims, comprising a plurality of the following tRNAs:
54. A peptide library comprising 1012 or more peptides containing both natural and non-natural amino acids, wherein each peptide comprises an amino acid selected from the group consisting of N-a-Chloroacetyl-L-phenylalanine (ClAc-L-Phe), N- a-Chloroacetyl-L-alanine (ClAc-L-Ala), N-acetyl-L-alanine (Ac-L-Ala) and N-a- Chloroacetyl-D-phenylalanine (ClAc-D-Phe),
wherein one or more of the non-natural amino acids is (S)-l,2,3,4- Tetrahydroisoquinoline-3-carboxylic acid (Tic), N-a-Methyl-L-phenylalanine (mF), 4-Phenyl-L-phenylalanine (Bip), (S)-2-Aminoheptanoic acid (Ahp), P- Cyclohexyl-L-alanine (Cha), D-Alanine (dA), Cycloleucine (Cle), N-a-Methyl-L- alanine (mA), N-a-Methyl-L-norleucine (mNle), N-n-Hexyl-glycine (HxG), N-a- Methyl-L-glycine (mG), L-a,P-Diaminopropionic acid (Dap), N-a-Methyl-L- serine (mS), 4-Azido-L-homoalanine (Aha), L-propargylglycine (Pra) or L- Homopropargylglycine (Hpg); and wherein each peptide is 6-20 amino acids in size. A peptide library comprising 1012 or more peptides containing both natural and non-natural amino acids generated by an in vitro translation system reconstituted with purified translation components comprising: a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; and c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non-natural amino acid or a different natural amino acid. The peptide library of the preceding claim, further comprising a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
- 49 -
The peptide library of any one of the preceding claims, comprising a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids. The peptide library of any one of the preceding claims, wherein the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and Vai or a subset thereof. The peptide library of any one of the preceding claims, wherein each peptide is complexed with a template nucleic acid that codes for the peptide when it is synthesizied. The peptide library of any one of the preceding claims, wherein the template nucleic acid is mRNA. The peptide library of any one of the preceding claims, wherein the peptide- mRNA complex comprises puromycin or an analogue thereof. The peptide library of any one of the preceding claims, wherein each peptide forms a macrocycle. The peptide library of any one of the preceding claims, wherein each peptide is 6- 18 amino acids in size. The peptide library of any one of the preceding claims, wherein each peptide is 6- 16 amino acids in size. The peptide library of any one of the preceding claims, wherein each peptide is 6- 14 amino acids in size. The peptide library of any one of the preceding claims, wherein the library comprises less than 1016, less than 1015 or less than 1014 peptides.
- 50 -
67. A method of screening the peptide library to identify a peptide that binds to a biological target comprising: a) reverse transcribing the mRNA of the peptide-mRNA complex in the peptide library of any one of claims 55-66 to generate the peptide-DNA conjugate; b) incubating the peptide-DNA conjugate with the biological target; c) isolating the peptide-DNA conjugate bound to the target; and d) amplifying the DNA by PCR to determine the amino sequence of the peptide.
68. A kit for translation of peptides containing both natural and non-natural amino acids comprising: a) a plurality of mRNA templates comprising amino acid codons represented by NNN, wherein N represents A, G, C or U; b) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with a natural amino acid that is encoded by a codon under universal genetic code; c) a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six or more different natural amino acids, wherein the codons that encode for the six or more different natural amino acids under universal genetic code each encode for a non-natural amino acid or a different natural amino acid; and d) a translation system reconstituted with purified translation components.
69. The kit of the preceding claim, further comprising a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and charged with natural or non-natural amino acids encoded by codons that under universal genetic code encodes for termination of translation.
70. The kit of any one of the preceding claims, comprising a plurality of tRNAs comprising an anticodon complementary to a codon on the mRNA template and
- 51 -
charged with natural or non-natural amino acids encoded by codons that under universal genetic code encode for six different natural amino acids.
71. The kit of any one of the preceding claims, wherein the six different natural amino acids comprise Met, Ser, Thr, Leu, Arg and Vai or a subset thereof.
72. A peptide having an amino acid sequence shown in Fig. 10B.
73. The peptide of any one of the preceding claims, which is a cyclic peptide.
74. A cyclic peptide selected from the peptides in Figs. 11 A and 1 IB.
75. The peptide of any one of the preceding claims, which is isolated and/or purifed.
76. The peptide of any one of the preceeding claims, which is a linear peptide.
- 52 -
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202063113495P | 2020-11-13 | 2020-11-13 | |
| US63/113,495 | 2020-11-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022104001A1 true WO2022104001A1 (en) | 2022-05-19 |
Family
ID=79092950
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2021/059049 WO2022104001A1 (en) | 2020-11-13 | 2021-11-12 | Expanded protein libraries and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2022104001A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4026906A4 (en) * | 2019-12-26 | 2023-12-27 | Chugai Seiyaku Kabushiki Kaisha | Composition for translation, and method for producing peptide |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6258558B1 (en) | 1997-01-21 | 2001-07-10 | The General Hospital Corporation | Method for selection of proteins using RNA-protein fusions |
| WO2007066627A1 (en) | 2005-12-06 | 2007-06-14 | The University Of Tokyo | Multi-purpose acylation catalayst and use thereof |
| US20100105569A1 (en) | 2008-09-30 | 2010-04-29 | Abbott Laboratories | Methods of RNA Display |
| JP2017216961A (en) * | 2016-06-09 | 2017-12-14 | 国立大学法人埼玉大学 | Non-natural amino acid-containing peptide library |
| WO2020138336A1 (en) * | 2018-12-26 | 2020-07-02 | 中外製薬株式会社 | Mutated trna for codon expansion |
-
2021
- 2021-11-12 WO PCT/US2021/059049 patent/WO2022104001A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6258558B1 (en) | 1997-01-21 | 2001-07-10 | The General Hospital Corporation | Method for selection of proteins using RNA-protein fusions |
| WO2007066627A1 (en) | 2005-12-06 | 2007-06-14 | The University Of Tokyo | Multi-purpose acylation catalayst and use thereof |
| US20100105569A1 (en) | 2008-09-30 | 2010-04-29 | Abbott Laboratories | Methods of RNA Display |
| JP2017216961A (en) * | 2016-06-09 | 2017-12-14 | 国立大学法人埼玉大学 | Non-natural amino acid-containing peptide library |
| WO2020138336A1 (en) * | 2018-12-26 | 2020-07-02 | 中外製薬株式会社 | Mutated trna for codon expansion |
Non-Patent Citations (14)
| Title |
|---|
| "The Dictionary of Cell and Molecular Biology", 1999, ACADEMIC PRESS |
| CUI ZHENLING ET AL: "Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, vol. 8, 28 September 2020 (2020-09-28), XP055886982, DOI: 10.3389/fbioe.2020.01031 * |
| HIBI KEITA ET AL: "Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs", COMMUNICATIONS BIOLOGY, vol. 3, no. 1, 3 July 2020 (2020-07-03), XP055894259, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334211/pdf/42003_2020_Article_1074.pdf> DOI: 10.1038/s42003-020-1074-2 * |
| JUO, PEI-SHOW: "Concise Dictionary of Biomedicine and Molecular Biology", 2002, CRC PRESS |
| KURZ ET AL.: "Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions", NUCLEIC ACIDS RESEARCH, vol. 28, no. 18, 2000, pages e83, XP002466472, DOI: 10.1093/nar/28.18.e83 |
| LEE ET AL., NAT COMMUN, vol. 10, 2019, pages 5097 |
| LEE JOONGOO ET AL: "Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro", NATURE COMMUNICATIONS, vol. 11, no. 1, 27 August 2020 (2020-08-27), pages 4304, XP055888050, Retrieved from the Internet <URL:https://www.nature.com/articles/s41467-020-18001-x.pdf> DOI: 10.1038/s41467-020-18001-x * |
| LIN CHI-WANG ET AL: "A Selection of Macrocyclic Peptides That Bind STING From an mRNA-Display Library With Split Degenerate Codons", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 60, no. 42, 11 October 2021 (2021-10-11), pages 22640 - 22645, XP055894178, ISSN: 1433-7851, Retrieved from the Internet <URL:https://onlinelibrary.wiley.com/doi/full-xml/10.1002/anie.202103043> DOI: 10.1002/anie.202103043 * |
| NITSCHE CHRISTOPH ET AL: "De Novo Discovery of Nonstandard Macrocyclic Peptides as Noncompetitive Inhibitors of the Zika Virus NS2B-NS3 Protease", ACS MEDICINAL CHEMISTRY LETTERS, vol. 10, no. 2, 14 February 2019 (2019-02-14), US, pages 168 - 174, XP055894270, ISSN: 1948-5875, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378662/pdf/ml8b00535.pdf> DOI: 10.1021/acsmedchemlett.8b00535 * |
| OHUCHI ET AL., CURR OPIN CHEM BIOL, vol. 11, 2007, pages 537 - 42 |
| PASSIOURA ET AL., JAM CHEM SOC, vol. 140, 2018, pages 11551 - 11555 |
| TAJIMA KENYA ET AL: "Genetic code expansion via integration of redundant amino acid assignment by finely tuning tRNA pools", CURRENT OPINION IN CHEMICAL BIOLOGY, CURRENT BIOLOGY LTD, LONDON, GB, vol. 46, 30 July 2018 (2018-07-30), pages 212 - 218, XP085507182, ISSN: 1367-5931, DOI: 10.1016/J.CBPA.2018.07.010 * |
| YAMAGISHI ET AL., CHEMISTRY AND BIOLOGY, 2011, pages 1562 - 1570 |
| YOSHIHIKO IWANE ET AL: "Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes", NATURE CHEMISTRY, vol. 8, no. 4, 1 February 2016 (2016-02-01), London, pages 317 - 325, XP055508589, ISSN: 1755-4330, DOI: 10.1038/nchem.2446 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4026906A4 (en) * | 2019-12-26 | 2023-12-27 | Chugai Seiyaku Kabushiki Kaisha | Composition for translation, and method for producing peptide |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Obexer et al. | Exploring sequence space: harnessing chemical and biological diversity towards new peptide leads | |
| US10195578B2 (en) | Peptide library production method, peptide library, and screening method | |
| Katoh et al. | Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation | |
| US10435439B2 (en) | Peptide with safer secondary structure, peptide library, and production methods for same | |
| AC’t Hoen et al. | Phage display screening without repetitious selection rounds | |
| AU2002213251B2 (en) | Protein scaffolds for antibody mimics and other binding proteins | |
| EP2868744B1 (en) | Screening method for peptide binding to target molecule in a ph-dependent manner | |
| US10711268B2 (en) | Method for producing peptide library, peptide library, and screening method | |
| Passioura et al. | Flexizyme‐Mediated Genetic Reprogramming As a Tool for Noncanonical Peptide Synthesis and Drug Discovery | |
| US20180171321A1 (en) | Platform for a non-natural amino acid incorporation into proteins | |
| US20030082768A1 (en) | Use of a ribozyme to join nucleic acids and peptides | |
| US11136574B2 (en) | Methods for in vitro ribosome synthesis and evolution | |
| WO2014119600A1 (en) | Flexible display method | |
| WO2022104001A1 (en) | Expanded protein libraries and uses thereof | |
| JP2017000154A (en) | Peptide library | |
| EP2593478B1 (en) | Rnf8-fha domain-modified protein and method of producing the same | |
| JP6478392B2 (en) | Nucleic acid linker | |
| US9783800B2 (en) | Method for producing peptides having azole-derived skeleton | |
| US9863936B2 (en) | Nucleic acid construct, nucleic acid-protein complex, and use thereof | |
| Wang et al. | Screening of the candidate inhibitory peptides of subtilisin by in vitro RNA display technique | |
| JP2017035063A (en) | Method for selecting (poly) peptide/protein tag capable of covalently bonding to any target substance and (poly) peptide/protein tag selected and obtained | |
| WO2022270396A1 (en) | Double-screening method | |
| Passioura et al. | Biological Synthesis and Affinity-based Selection of Small Macrocyclic Peptide Ligands | |
| Schwark et al. | Orthogonal pair-directed codon reassignment as a tool for evaluating the factors affecting translation in E. coli | |
| CN116670284A (en) | Method for screening candidate molecules capable of forming complexes with multiple target molecules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21831390 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21831390 Country of ref document: EP Kind code of ref document: A1 |