Composition for translation and method for producing peptide

Abstract

The present invention addresses one problem of providing a new means capable of reducing the percentage of unintended amino acids that are erroneously translated by misreading codons. In some aspects, the present disclosure provides a composition for translation, the composition comprising a tRNA having particular base combinations at positions 32, 33, 37 and 38 thereof (according to the tRNA numbering rule). According to a particular aspect of the present disclosure, it is possible to reduce the percentage of unintended amino acids that are introduced by misreading codons.

Description

[Technical Field]

[0001] The present disclosure relates to compositions for translation and methods for producing peptides. [Background technology]

[0002] Cell-free translation systems, artificially reconstituted by combining only the components involved in protein translation (Non-Patent Document 1), are used in a wide range of fields, from elucidating biological phenomena to developing new drugs. Because cell-free translation systems do not use microorganisms or cells, they can be used to synthesize highly toxic proteins. Furthermore, because components such as amino acids, tRNAs, and aminoacyl-tRNA synthetases can be added or removed as needed, it is possible to change the codon-to-amino acid mapping (reprogramming the genetic code). These features have led to cell-free translation systems being applied to synthesize proteins containing unnatural amino acids and to construct display libraries incorporating diverse building blocks. While cell-free translation systems are extremely useful, they can sometimes produce unintended peptides due to codon misreading. For example, when RF1 was inactivated and unnatural amino acids were assigned to the amber codon (UAG), tyrosine (UAU, UAC), lysine (AAG), and glutamine (GAG) were erroneously introduced at a certain rate into the amber codon (Non-Patent Document 2).

[0003] There have been reports of artificially dividing codon boxes and reprogramming the genetic code. Non-Patent Document 3 attempts to assign one unnatural amino acid and one natural amino acid to CGC / G, GUC / G, and GGC / G. In the same document, Gly CCCAs a measure to reduce misreading of the GGC codon in a translation reaction, it is described that the translation reaction is stopped before tRNAs precharged with amino acids and having an anticodon GCC complementary to GGC are depleted, but it is not described what kind of translation composition can reduce misreading of codons. Furthermore, Non-Patent Documents 4 to 7 and Patent Documents 1 and 2 also attempt to artificially divide the codon box, but do not describe a method for reducing misreading of codons.

[0004] It is known that specific bases are conserved at positions 32 and 38 in the anticodon loop of tRNA for each anticodon triplet. tRNA (Ala) with the anticodon GGC GGC According to a report using tRNA, natural tRNA (Ala) was found at positions 32 and 38. GGC It has been shown that when the bases conserved in the tRNA (Ala) are used, misreading of the GUC codon is unlikely to occur, whereas when the bases at positions 32 and 38 are changed, misreading of the GUC codon by the tRNA occurs, resulting in the erroneous translational incorporation of alanine (GCC) (Non-Patent Document 8). GGC The study only examines the misreading of the GUC codon by genomic DNA, and does not mention any misreading of codons that may occur when the genetic code table is reprogrammed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2019 / 139126 [Patent Document 2] WO2012 / 026566 [Non-patent literature]

[0006] [Non-Patent Document 1] Shimizu et al., Nat Biotechnol. 2001 Aug; 19(8): 751-755. [Non-patent document 2] Gan et al., Biochem. Biophys. Acta, 2017 Dec; 1861; 3047-3052. [Non-patent document 3] Iwane et al., Method Mol. Biol., 2018, 1728, 17-47. [Non-patent document 4] Passioura et al., J Am. Chem. Soc., 2018, 140, 11551-11555. [Non-Patent Document 5] Passioura et al., Cell Chem. Biol., 2018, 25, 906-915. [Non-patent document 6] Iwane et al., Nat. Chem., 2016, 8, 317-325. [Non-Patent Document 7] Azusa et al., Peptide Science 2009, 2010 Mar; 17-18. [Non-patent document 8] Murakami et al., Nat. Struct. Mol. Biol., 2009 Mar; 16; 353-358. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of the above circumstances, and one of its objects is to provide a composition for translation that can reduce the rate at which unintended amino acids are erroneously translated due to misreading of codons by tRNA, and a method for producing peptides using the composition for translation. [Means for solving the problem]

[0008] The present inventors have found that the above-mentioned problems can be solved by selecting a specific base for the anticodon loop of a tRNA used for translation. The present disclosure is based on this finding and specifically includes the following exemplary embodiments. [1] A composition for translation comprising a first tRNA bound to a first amino acid and a second tRNA bound to a second amino acid, the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, (4) 32U, 33U, 37G, 38U, (5) 32U, 33U, 37A, 38U, or (6) 32C, 33U, 37G, 38A, and the first base of the anticodon of the first tRNA and the second tRNA is different from each other; the second base of the anticodon of the first tRNA and the second tRNA is the same; the third base of the anticodon of the first tRNA and the second tRNA is the same; at least one of the first amino acid and the second amino acid is an unnatural amino acid; where A is adenine, C is cytosine, G is guanine, and U is uracil. The composition. [2] The anticodon of the first tRNA is N 11 N 12 N 13 (where N 11 , N 12 , and N 13 are each independently A, C, G, or U. [3] The anticodon of the second tRNA is N 21 N 22 N 23 (where N 21 , N22 , and N 23 are each independently A, C, G, or U. [4] The composition according to any one of [1] to [3], wherein the tRNA body of the first tRNA is a chimeric tRNA body. [5] The first base of the anticodon of the first tRNA is A or G, and the first base of the anticodon of the second tRNA is C or U, the first base of the anticodon of the first tRNA is C or U, and the first base of the anticodon of the second tRNA is A or G; The composition according to any one of [1] to [4]. [6] The composition according to any one of [1] to [5], wherein the first base of the anticodon of the first tRNA is A or G, and the first base of the anticodon of the second tRNA is C or U. [7] The first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is C, or the first base of the anticodon of the first tRNA is C and the first base of the anticodon of the second tRNA is A; or the first base of the anticodon of the first tRNA is G and the first base of the anticodon of the second tRNA is C; the first base of the anticodon of the first tRNA is C and the first base of the anticodon of the second tRNA is G; the first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is U; the first base of the anticodon of the first tRNA is U and the first base of the anticodon of the second tRNA is A; or the first base of the anticodon of the first tRNA is G and the first base of the anticodon of the second tRNA is U; or the first base of the anticodon of the first tRNA is U, and the first base of the anticodon of the second tRNA is G; The composition according to any one of [1] to [6]. [8] The first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is C, or the first base of the anticodon of the first tRNA is C and the first base of the anticodon of the second tRNA is A; or the first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is U; the first base of the anticodon of the first tRNA is U and the first base of the anticodon of the second tRNA is A; or the first base of the anticodon of the first tRNA is G and the first base of the anticodon of the second tRNA is U; or the first base of the anticodon of the first tRNA is U, and the first base of the anticodon of the second tRNA is G; The composition according to any one of [1] to [7]. [9] The first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is C; or the first base of the anticodon of the first tRNA is C and the first base of the anticodon of the second tRNA is A; or the first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is U; the first base of the anticodon of the first tRNA is G and the first base of the anticodon of the second tRNA is U; or the first base of the anticodon of the first tRNA is G and the first base of the anticodon of the second tRNA is C; The composition according to any one of [1] to [7].

[10] The first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is C; or the first base of the anticodon of the first tRNA is C and the first base of the anticodon of the second tRNA is A; or The first base of the anticodon of the first tRNA is G and the first base of the anticodon of the second tRNA is C, or the first base of the anticodon of the first tRNA is C and the first base of the anticodon of the second tRNA is G; The composition according to any one of [1] to [7].

[11] The first base of the anticodon of the first tRNA is A, and the first base of the anticodon of the second tRNA is C; or the first base of the anticodon of the first tRNA is C and the first base of the anticodon of the second tRNA is A; The composition according to any one of [1] to

[10] .

[12] The first base of the anticodon of the first tRNA is G, and the first base of the anticodon of the second tRNA is C, or the first base of the anticodon of the first tRNA is C and the first base of the anticodon of the second tRNA is G; The composition according to any one of [1] to [7] and

[10] .

[13] The first base of the anticodon of the first tRNA is A, and the first base of the anticodon of the second tRNA is U; or the first base of the anticodon of the first tRNA is U, and the first base of the anticodon of the second tRNA is A; The composition according to any one of [1] to [8].

[14] The first base of the anticodon of the first tRNA is G, and the first base of the anticodon of the second tRNA is U; or the first base of the anticodon of the first tRNA is U, and the first base of the anticodon of the second tRNA is G; The composition according to any one of [1] to [8].

[15] The composition according to any one of [1] to

[11] , wherein the first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is C.

[16] The combination of the second and third letter bases of the anticodons of the first and second tRNAs is (i) G as the second character and G as the third character; (ii) A as the second character and G as the third character; (iii) C as the second character and C as the third character; (iv) G as the second character and C as the third character; (v) A as the second character and C as the third character; (vi) G as the second character and U as the third character; (vii) The second character is G and the third character is A; or (viii) C as the second character and G as the third character; The composition according to any one of [1] to

[15] ,

[17] The combination of the second and third letter bases of the anticodons of the first and second tRNAs is (i) G as the second character and G as the third character; (ii) A as the second character and G as the third character; (iii) C as the second character and C as the third character; (iv) G as the second character and C as the third character; (v) A as the second character and C as the third character; (vi) The second letter is G and the third letter is U; or (vii) G as the second character and A as the third character; The composition according to any one of [1] to

[16] ,

[18] The combination of the second and third letter bases of the anticodons of the first and second tRNAs is (i) G as the second character and G as the third character; (ii) A as the second character and G as the third character; or (iii) C as the second letter and C as the third letter The composition according to any one of [1] to

[17] ,

[19] The composition according to any one of [1] to

[18] , wherein the second base and the third base of the anticodons of the first and second tRNAs are G.

[20] The composition according to any one of [1] to

[18] , wherein the second base of the anticodon of the first and second tRNAs is A and the third base is G.

[21] The composition according to any one of [1] to

[18] , wherein the second base of the anticodon of the first and second tRNAs is C and the third base of the anticodon is C.

[22] The composition according to any one of [1] to

[21] , wherein the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of the first tRNA are not derived from any of the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having at least one base sequence selected from the group consisting of SEQ ID NOs: 275, 294, 295, 296, 302, 303, and 304.

[23] The composition according to any one of [1] to

[22] , wherein the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of the first tRNA are derived from the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having at least one nucleotide sequence selected from the group consisting of (a) SEQ ID NO: 253, (b) SEQ ID NO: 255, and (c) SEQ ID NO: 254.

[24] The composition according to any one of [1] to

[23] , wherein the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of the first tRNA are the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having at least one base sequence selected from the group consisting of: (a) SEQ ID NO: 253, (b) SEQ ID NO: 255, and (c) SEQ ID NO: 254.

[25] The composition according to any one of [1] to

[24] , wherein the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of the first tRNA have 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having a nucleotide sequence according to any one of (a) to (c) of

[24] .

[26] The composition according to any one of [1] to

[25] , wherein the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of the second tRNA are not derived from any of the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having at least one base sequence selected from the group consisting of SEQ ID NOs: 275, 294, 295, 296, 302, 303, and 304.

[27] The composition according to any one of [1] to

[26] , wherein the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of the second tRNA are derived from the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having at least one nucleotide sequence selected from the group consisting of (a) SEQ ID NO: 253, (b) SEQ ID NO: 255, and (c) SEQ ID NO: 254.

[28] The composition according to any one of [1] to

[27] , wherein the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of the second tRNA are the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having at least one nucleotide sequence selected from the group consisting of (a) SEQ ID NO: 253, (b) SEQ ID NO: 255, and (c) SEQ ID NO: 254.

[29] The composition according to any one of [1] to

[28] , wherein the nucleotide sequences of positions 1 to 31 and 39 to 74 (tRNA numbering rules) of the second tRNA have 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the nucleotide sequences of positions 1 to 31 and 39 to 74 (tRNA numbering rules) of a tRNA having a nucleotide sequence according to any one of (a) to (c) of

[28] .

[30] The composition according to any one of [1] to

[29] , wherein the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of the first tRNA and the second tRNA are identical or have a sequence identity of 80% or more, preferably 90% or more, and more preferably 95% or more.

[31] The composition according to any one of [1] to

[30] , wherein the first tRNA and the second tRNA have the same base sequences at positions 1 to 31 and positions 39 to 74 (tRNA numbering rules).

[32] The composition according to any one of [4] to

[31] , wherein the chimeric tRNA body is a chimeric tRNA body in which the combination of bases at positions 32, 33, 37, and 38 (according to the tRNA numbering rules) and the base sequences at other positions are of different origins.

[33] The composition according to any one of [1] to

[32] , wherein the base sequence of positions 32 to 38 (tRNA numbering rules) of the first tRNA is different from the base sequence of positions 32 to 38 (tRNA numbering rules) of a wild-type tRNA of Escherichia coli.

[34] The composition according to any one of [1] to

[33] , wherein the first tRNA is a tRNA having a chimeric anticodon loop.

[35] The composition according to any one of [1] to

[34] , wherein the second amino acid is an unnatural amino acid.

[36] The composition according to any one of [1] to

[35] , wherein the first amino acid is an unnatural amino acid.

[37] The composition according to any one of [1] to

[36] , wherein either the first amino acid or the second amino acid, or both, are N-substituted amino acids.

[38] The composition according to any one of [1] to

[37] , wherein the first tRNA is an artificial tRNA.

[39] The composition according to any one of [1] to

[38] , wherein the second tRNA is an artificial tRNA.

[40] The composition according to any one of [1] to

[39] , wherein the base sequence at positions 75 and 76 (tRNA numbering rules) of the first tRNA is CA.

[41] The composition according to any one of [1] to

[40] , wherein the base sequence at positions 75 and 76 (tRNA numbering rules) of the second tRNA is CA.

[42] The composition according to any one of [1] to

[41] , wherein the first tRNA is a tRNA that does not have a modified base.

[43] The composition according to any one of [1] to

[42] , wherein the second tRNA is a tRNA that does not have a modified base.

[44] The composition according to any one of [1] to

[43] , wherein the first amino acid is bound to the 3' end of the first tRNA.

[45] The composition according to any one of [1] to

[44] , wherein the second amino acid is bound to the 3' end of the second tRNA.

[46] The composition according to any one of [1] to

[45] , wherein the first tRNA is a tRNA having a base sequence consisting of A, U, G, and C.

[47] The composition according to any one of [1] to

[46] , wherein the second tRNA is a tRNA having a base sequence consisting of A, U, G, and C.

[48] ​​The composition according to any one of [1] to

[47] , wherein the first tRNA is a tRNA to which an amino acid is bound outside the translation system.

[49] The composition according to any one of [1] to

[48] , wherein the second tRNA is a tRNA to which an amino acid is bound outside the translation system.

[50] The composition according to any one of [1] to

[49] , wherein the first tRNA and the second tRNA are tRNAs derived from Escherichia coli.

[51] The combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, or (4) 32U, 33U, 37G, 38U, The composition according to any one of [1] to

[50] ,

[52] The combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, or (3) 32A, 33U, 37A, 38U, The composition according to any one of [1] to

[51] ,

[53] The combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is (1) 32U, 33U, 37G, 38A, (3) 32A, 33U, 37A, 38U, or (4) 32U, 33U, 37G, 38U, The composition according to any one of [1] to

[52] ,

[54] The composition according to any one of [1] to

[53] , wherein the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is 32U, 33U, 37G, 38A, or 32U, 33U, 37G, 38U.

[55] The composition according to any one of [1] to

[54] , wherein the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is 32U, 33U, 37G, and 38A.

[56] The composition according to any one of [1] to

[52] , wherein the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is 32A, 33U, 37G, and 38U.

[57] The composition according to any one of [1] to

[53] , wherein the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is 32A, 33U, 37A, and 38U.

[58] The composition according to any one of [1] to

[51] , wherein the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is 32U, 33U, 37G, and 38U.

[59] The composition according to any one of [1] to

[50] , wherein the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is 32U, 33U, 37A, and 38U.

[60] The composition according to any one of [1] to

[50] , wherein the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is 32C, 33U, 37G, and 38A.

[61] The combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the second tRNA is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, (4) 32U, 33U, 37G, 38U, (5) 32U, 33U, 37A, 38U, or (6) 32C, 33U, 37G, 38A, The composition according to any one of [1] to

[60] ,

[62] The combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the second tRNA is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, or (4) 32U, 33U, 37G, 38U, The composition according to any one of [1] to

[61] ,

[63] The composition according to any one of [1] to

[62] , wherein the tRNA body of the second tRNA is a chimeric tRNA body.

[64] The composition according to any one of [1] to

[63] , which is a composition for cell-free translation.

[65] The composition according to any one of [1] to

[64] , which is a composition for reconstituted cell-free translation.

[66] The composition according to any one of [1] to

[65] , which is a composition for cell-free translation reconstituted with a factor derived from Escherichia coli.

[67] The composition according to any one of [1] to

[66] , which comprises a ribosome derived from Escherichia coli.

[68] The composition described in any one of [1] to

[67] , comprising an mRNA having a codon complementary to the anticodon of the second tRNA.

[69] The composition according to any one of [1] to

[68] , comprising an mRNA having a codon complementary to the anticodon of the first tRNA and a codon complementary to the anticodon of the second tRNA, and optionally, the codon complementary to the anticodon of the first tRNA and the codon complementary to the anticodon of the second tRNA may be contained in the same mRNA or different mRNAs.

[70] The composition described in any of [1] to

[69] , which does not contain an aaRS (amino acyl-tRNA synthetase) capable of binding an amino acid to the first tRNA or the second tRNA.

[71] The composition according to any one of [1] to

[70] (excluding a composition in which the anticodon combination of the first tRNA and the second tRNA is GCG and CCG, and a composition in which the anticodon combination of CCG and GCG is GCG, when the first tRNA and the second tRNA have the combination of bases specified in [1](4) above at positions 32, 33, 37, and 38 (tRNA numbering rules), and have a chimeric tRNA body having the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having the base sequence set forth in SEQ ID NO: 255 at positions 1 to 31 and positions 39 to 74 (tRNA numbering rules).

[72] The composition according to any one of [1] to

[71] (however, excluding a composition in which the anticodon combination of the first tRNA and the second tRNA is GCG and CCG, and a composition in which the anticodon combination of the first tRNA and the second tRNA is CCG and GCG, when the first tRNA and the second tRNA have a chimeric tRNA body having a base combination specified in [1](4) at positions 32, 33, 37, and 38 (tRNA numbering rules)).

[73] The composition according to any one of [1] to

[72] (provided that, when the first tRNA and the second tRNA have a chimeric tRNA body having a combination of bases specified in [1](4) at positions 32, 33, 37, and 38 (tRNA numbering rules), the anticodon combination of the first tRNA and the second tRNA is AAG and UAG, AAG and CAG, or UAG and CAG).

[74] The composition according to any one of [1] to

[73] (excluding a composition in which the first tRNA and the second tRNA have a chimeric tRNA body having a combination of bases specified in [1](4) at positions 32, 33, 37, and 38 (tRNA numbering rules), and the second base of the anticodons of the first tRNA and the second tRNA is both C and the third base is both G).

[75] The composition according to any one of [1] to

[74] (excluding a composition in which the first tRNA and the second tRNA have a chimeric tRNA body having a combination of bases specified in [1](4) at positions 32, 33, 37, and 38 (tRNA numbering rules), and the second base of the anticodon of the first tRNA and the second tRNA is A and the third base of the anticodon of the first tRNA and the second tRNA is G).

[76] The composition according to any one of [1] to

[75] (excluding a composition in which the second base of the anticodon of the first tRNA and the second tRNA is both C and the third base of the anticodon of the second tRNA is both G).

[77] The composition according to any one of [1] to

[76] (excluding a composition in which, when the first tRNA has a combination of bases specified in [1](4) above at positions 32, 33, 37, and 38 (tRNA numbering rules), the second base of the first tRNA anticodon is A and the third base is G).

[78] The composition according to any one of [1] to

[77] (excluding compositions in which the anticodons of the first tRNA are UGG and CGG when the first tRNA has the combination of bases specified in [1](1) at positions 32, 33, 37, and 38 (tRNA numbering rules)).

[79] The composition according to any one of [1] to

[78] (excluding compositions in which the anticodons of the first tRNA are AGG and GGG when the first tRNA has the combination of bases specified in [1](2) at positions 32, 33, 37, and 38 (tRNA numbering rules)).

[80] The composition according to any one of [1] to

[79] (excluding compositions in which the anticodons of the first tRNA are GCG and CCG when the first tRNA has a chimeric tRNA body having a combination of bases specified in [1](6) at positions 32, 33, 37, and 38 (tRNA numbering rules)).

[81] The composition according to any one of [1] to

[80] (excluding compositions in which the anticodons of the first tRNA are AGC and GGC when the first tRNA has a chimeric tRNA body having a combination of bases specified in [1](3) at positions 32, 33, 37, and 38 (tRNA numbering rules)).

[82] The composition according to any one of [1] to

[81] , wherein the first tRNA and the second tRNA are elongation tRNAs.

[83] The composition according to any one of

[37] to

[82] , wherein the N-substituted amino acid is an N-methyl amino acid.

[84] The composition according to any one of [1] to

[83] , wherein the amino acid is a translatable amino acid.

[85] The composition described in any one of [1] to

[84] , wherein the second tRNA is a tRNA having a chimeric anticodon loop.

[86] A method for producing a composition for translation according to any one of [1] to

[85] , comprising a step of preparing the first tRNA by binding the first amino acid to tRNA outside of a translation system, and / or a step of preparing the second tRNA by binding the second amino acid to tRNA outside of a translation system.

[87] The method according to

[86] , wherein the step of preparing the first tRNA and / or the second tRNA is carried out by at least one method selected from the group consisting of the pCpA method, the pdCpA method, a method using flexizyme, and aaRS-based method.

[88] The method described in

[86] or

[87] , comprising a step of preparing the first tRNA by in vitro transcription.

[89] The method according to any one of

[86] to

[88] , comprising a step of preparing the second tRNA by in vitro transcription.

[0009] [A1] A method for producing a peptide, comprising translating a nucleic acid using the composition for translation according to any one of [1] to

[85] or a composition for translation produced by the method according to any one of

[86] to

[89] . [A2] A method for reducing misreading of a codon complementary to the anticodon of the second tRNA by the first tRNA, comprising translating a nucleic acid using the composition for translation according to any one of [1] to

[85] or a composition for translation produced by the method according to any one of

[86] to

[89] . [A3] The method according to [A1] or [A2], wherein the nucleic acid is an mRNA having a codon complementary to the anticodon of the second tRNA. [A4] The method according to [A3], wherein the nucleic acid is an mRNA having a codon complementary to the anticodon of the first tRNA, and optionally, the codon complementary to the anticodon of the first tRNA and the codon complementary to the anticodon of the second tRNA may be contained in the same mRNA or different mRNAs. [A5] The method according to any one of [A1] to [A4], which comprises translating a nucleic acid library containing the nucleic acid to obtain a peptide library. [A6] The method according to any one of [A1] to [A5], wherein the codon complementary to the anticodon of the first tRNA is a codon that forms Watson-Crick base pairs with the anticodon at all three bases. [A7] The method according to any one of [A1] to [A6], wherein the codon complementary to the anticodon of the second tRNA is a codon that forms Watson-Crick base pairs with the anticodon at all three bases. [A8] The method according to any one of [A1] to [A5], wherein the third letter of a codon complementary to the anticodon of the first tRNA forms a wobble base pair with the first letter of the anticodon. [A9] The method according to any one of [A1] to [A6] or [A8], wherein the third letter of a codon complementary to the anticodon of the second tRNA forms a wobble base pair with the first letter of the anticodon. [A10] The method according to any one of [A1] to [A9], wherein the nucleic acid is mRNA. [A11] A peptide or peptide library produced by the method according to any one of [A1] to [A10]. [A12] A peptide produced using the composition for translation according to any one of [1] to

[85] or the composition for translation produced by the method according to any one of

[86] to

[89] . [A13] A peptide library produced using the composition for translation according to any one of [1] to

[85] or the composition for translation produced by the method according to any one of

[86] to

[89] . [A14] A method for identifying a peptide that binds to a target molecule, comprising the steps of producing a peptide library using the method described in any one of [A1] to [A10], and contacting the target molecule with the peptide library.

[0010] [B1] A method for reducing misreading of a second codon by a tRNA, the method comprising substituting at least one base selected from the group consisting of positions 32, 33, 37, and 38 (tRNA numbering rules) of a tRNA having an anticodon complementary to a first codon, the first letter of the first codon and the second codon are the same base, the second character of the first codon and the second codon is the same base, The method as described above, wherein the third letters of the first codon and the second codon are different bases. [B2] The combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) after the substitution is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, (4) 32U, 33U, 37G, 38U, (5) 32U, 33U, 37A, 38U, or (6) 32C, 33U, 37G, 38A, wherein A is adenine, C is cytosine, G is guanine, and U is uracil. [B3] The method described in [B1] or [B2], which comprises substituting at least one base selected from the group consisting of positions 32, 37, and 38 (tRNA numbering rules) of a tRNA having an anticodon complementary to the first codon. [B4] The first codon is M1M2X and the second codon is M1M2Y; M1 and M2 are each independently A, C, G, or U; The method according to any one of [B1] to [B3], wherein X and Y are different bases selected from A, C, G, and U. [B5] The method according to [B4], wherein the combination of bases X and Y is any one selected from the following group: (a1) U and G; (a2) G and U; (a3) ​​U and A; (a4) A and U; (a5) C and A; (a6) A and C; (a7) C and G; and (a8) G and C. [B6] The method according to [B4] or [B5], wherein the combination of bases X and Y is any one selected from the following group: (a1) U and G; (a2) G and U; (a3) ​​U and A; (a5) C and A; and (a7) C and G. [B7] The method according to [B5], wherein the combination of bases X and Y is (a1) U and G, or (a2) G and U. [B8] The method according to any one of [B5] to [B7], wherein the combination of bases X and Y is (a1) U and G. [B9] The method according to any one of [B5] to [B7], wherein the combination of bases X and Y is (a2) G and U. [B10] The method according to [B5] or [B6], wherein the combination of bases X and Y is (a3) ​​U and A. [B11] The method according to [B5] or [B6], wherein the combination of bases X and Y is (a5) C and A. [B12] The method according to [B5] or [B6], wherein the combination of bases X and Y is (a7) C and G. [B13] The method according to any one of [B4] to [B12], wherein M1M2 is any one of the base sequences selected from the following group: (b1) CC; (b2) CU; (b3) GG; (b4) GU; (b5) GC; (b6) UC; (b7) CG; and (b8) AC. [B14] The method according to [B13], wherein M1M2 is any one of the base sequences selected from the following group: (b1) CC; (b2) CU; and (b3) GG. [B15] The method described in [B13] or [B14], wherein the base sequence of M1M2 is (b1) CC. [B16] The method according to [B13] or [B14], wherein the base sequence of M1M2 is (b2) CU. [B17] The method described in [B13] or [B14], wherein the base sequence of M1M2 is (b3) GG. [B18] The method according to any one of [B1] to [B17], wherein the first codon and the anticodon of the tRNA form Watson-Crick base pairs at all three bases. [B19] The method according to any one of [B1] to [B17], wherein the third base of the first codon and the first base of the anticodon of the tRNA form a wobble base pair. [B20] The method according to any one of [B1] to [B19], wherein the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (according to the tRNA numbering rules) of the tRNA are derived from the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (according to the tRNA numbering rules) of a tRNA having at least one nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 253, (b) SEQ ID NO: 255, and (c) SEQ ID NO: 254. [B21] The method according to any one of [B1] to [B20], wherein the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (according to the tRNA numbering rules) of the tRNA are the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (according to the tRNA numbering rules) of a tRNA having at least one nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 253, (b) SEQ ID NO: 255, and (c) SEQ ID NO: 254. [B22] The method according to any one of [B1] to [B21], wherein the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (according to the tRNA numbering rules) of the tRNA have 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the nucleotide sequences of positions 1 to 31 and positions 39 to 74 (according to the tRNA numbering rules) of a tRNA having a nucleotide sequence described in any one of (a) to (c) of [B20]. [B23] The method according to any one of [B1] to [B22], which comprises binding an amino acid to the tRNA outside the translation system. [B24] The method according to [B23], wherein the binding of the amino acid to tRNA outside the translation system is carried out by at least one method selected from the group consisting of the pCpA method, the pdCpA method, a method using flexizyme, and a method using aaRS. [B25] The method according to [B23] or [B24], wherein the amino acid is an unnatural amino acid. [B26] The method according to any one of [B23] to [B25], wherein the unnatural amino acid is an N-substituted amino acid. [B27] The method according to any one of [B1] to [B26], further comprising synthesizing the tRNA from a template nucleic acid by in vitro transcription. [B28] The method according to any one of [B1] to [B27], wherein the base at position 37 (according to the tRNA numbering rules) of the tRNA before modification is A. [B29] The method according to any one of [B1] to [B28], wherein the base at position 33 (according to the tRNA numbering rules) of the tRNA before modification is U. [B30] The method according to any one of [B1] to [B29], wherein the base at position 32 (according to the tRNA numbering rules) of the tRNA before modification is C or U. [B31] The method according to any one of [B1] to [B30], wherein the base at position 32 (according to the tRNA numbering rules) of the tRNA before modification is C. [B32] The method according to any one of [B1] to [B30], wherein the base at position 32 (according to the tRNA numbering rules) of the tRNA before modification is U. [B33] The method according to any one of [B1] to [B32], wherein the base at position 38 (according to the tRNA numbering rules) of the tRNA before modification is A or C. [B34] The method according to any one of [B1] to [B33], wherein the base at position 38 (according to the tRNA numbering rules) of the tRNA before modification is A. [B35] The method according to any one of [B1] to [B33], wherein the base at position 38 (according to the tRNA numbering rules) of the tRNA before modification is C. [B36] The combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) after the substitution is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, or (4) 32U, 33U, 37G, 38U, The method according to any one of [B1] to [B35]. [B37] The combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) after the substitution is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, or (3) 32A, 33U, 37A, 38U, A method according to any one of [B1] to [B36]. [B38] The method according to any one of [B1] to [B37] (excluding a method in which the combination of the first codon and the second codon is CGC and CGG, and a method in which the combination of the first codon and the second codon is CGC and CGG, and a method in which the combination of the first codon and the second codon is CGC and CGG, when the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) after the substitution is a combination specified in [B2] (4), and the base sequences at positions 1 to 31 and 39 to 74 (tRNA numbering rules) of the tRNA are the base sequences of positions 1 to 31 and 39 to 74 (tRNA numbering rules) of a tRNA having the base sequence set forth in SEQ ID NO: 255). [B39] The method according to any one of [B1] to [B38], further comprising translating the nucleic acid in a translation system containing a tRNA with a substituted base. [B40] The method according to any one of [B26] to [B39], wherein the N-substituted amino acid is an N-methyl amino acid. [B41] The method according to any one of [B23] to [B40], wherein the amino acid is a translatable amino acid. [Effects of the Invention]

[0011] The present disclosure provides a translation composition containing a tRNA having specific bases at positions 32, 33, 37, and 38 (according to the tRNA numbering rules), and a method for producing a peptide that includes translating a nucleic acid using the tRNA. Use of the composition and method disclosed herein can reduce the rate at which unintended amino acids are erroneously translated during peptide synthesis due to misreading of codons by the tRNA. [Brief explanation of the drawings]

[0012] [Figure 1]This figure shows the percentage of misreadings of the codon CCG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the CCG codon in the presence of tRNAs having cgg and agg as anticodons (see Table 6 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 242) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 241) × 100 [Figure 2] This figure shows the percentage of misreadings of the codon CCU due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the CCU codon in the presence of tRNAs having agg and cgg as anticodons (see Table 7 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 242) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 241) × 100 [Figure 3]This figure shows the percentage of misreadings of the codon GGG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the GGG codon in the presence of tRNAs having ccc and acc as anticodons (see Table 8 for specific translation amounts). The percentage (%) of misread peptides relative to the target product, shown on the vertical axis, was calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243) × 100 [Figure 4] This figure shows the percentage of misreadings of the codon GGU due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the GGU codon in the presence of tRNAs having acc and ccc as anticodons (see Table 9 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243) × 100 [Figure 5]This figure shows the percentage of misreadings of the codon CUG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA containing the CUG codon in the presence of tRNAs containing cag and aag as anticodons (see Table 10 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239) × 100 [Figure 6] This figure shows the percentage of misreadings of the codon CUU due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA containing the CUU codon in the presence of tRNAs having aag and cag as anticodons (see Table 11 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239) × 100 [Figure 7]This figure shows the percentage of misreadings of the codon CUG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (AsnE2). The values ​​in the figure are based on the results of translating mRNA having the CUG codon in the presence of tRNAs having cag and aag as anticodons (see Table 12 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239) × 100 [Figure 8] This figure shows the percentage of misreadings of the codon CUU due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (AsnE2). The values ​​in the figure are based on the results of translating mRNA containing the CUU codon in the presence of tRNAs having aag and cag as anticodons (see Table 13 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239) × 100 [Figure 9]This figure shows the percentage of misreadings of the codon CUG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Asp1). The values ​​in the figure are based on the results of translating mRNA containing the CUG codon in the presence of tRNAs containing cag and aag as anticodons (see Table 14 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239) × 100 [Figure 10] This figure shows the percentage of misreadings of the codon CUU due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Asp1). The values ​​in the figure are based on the results of translating mRNA having the CUU codon in the presence of tRNAs having aag and cag as anticodons (see Table 15 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239) × 100 [Figure 11]This figure shows the percentage of misreadings of the codon GGA due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the GGA codon in the presence of tRNAs having ucc and acc as anticodons (see Table 16 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243) × 100 [Figure 12] This figure shows the percentage of misreadings of the codon GGA due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA containing the GGA codon in the presence of tRNAs having ucc and gcc as anticodons (see Table 17 for specific translation amounts). The percentage (%) of misread peptides relative to the target product, shown on the vertical axis, was calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243) × 100 [Figure 13]This figure shows the percentage of misreadings of the codon GGG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the GGG codon in the presence of tRNAs having ccc and gcc as anticodons (see Table 18 for specific translation amounts). The percentage (%) of misread peptides relative to the target product, shown on the vertical axis, was calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243) × 100 [Figure 14] This figure shows the percentage of misreadings of the codon GGG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the GGG codon in the presence of tRNAs having acc and ccc as anticodons (see Table 21 for specific translation amounts). The percentage (%) of misread peptides relative to the target product, shown on the vertical axis, was calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:Pic2:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 245) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243) × 100 [Figure 15]This figure shows the percentage of misreadings of the codon GGG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the GGG codon in the presence of tRNAs having acc and ccc as anticodons (see Table 22 for specific translation amounts). The percentage (%) of misread peptides relative to the target product, shown on the vertical axis, was calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeG:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 246) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243) × 100 [Figure 16] This figure shows the percentage of misreadings of the codon GGG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the GGG codon in the presence of tRNAs having acc and ccc as anticodons (see Table 23 for specific translation amounts). The percentage (%) of misread peptides relative to the target product, shown on the vertical axis, was calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:dA:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 248) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Leu:Phe:nBuG:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 247) × 100 [Figure 17]This figure shows the percentage of misreadings of the codon CCG due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the CCG codon in the presence of tRNAs having cgg and agg as anticodons (see Table 24 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 242) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeG:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 249) × 100 [Figure 18] This figure shows the percentage of misreadings of the codon CCU due to different combinations of bases at positions 32, 33, 37, and 38 of tRNA (Glu2). The values ​​in the figure are based on the results of translating mRNA having the CCU codon in the presence of tRNAs having agg and cgg as anticodons (see Table 25 for specific translation amounts). The percentage (%) of misread peptides relative to the target product on the vertical axis is calculated using the following formula: Percentage of misread peptides relative to the target product (%) = Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 242) / Amount (μM) of translated peptide corresponding to the sequence BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeG:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 249) × 100 DETAILED DESCRIPTION OF THE INVENTION

[0013] For purposes of interpreting this specification, the following definitions will apply, and wherever applicable, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0014] A "codon" refers to a triplet (a combination of three bases) corresponding to each amino acid when genetic information in a living organism is translated into a protein. In the case of DNA, four bases, adenine (A), guanine (G), cytosine (C), and thymine (T), are used. In the case of mRNA, four bases, adenine (A), guanine (G), cytosine (C), and uracil (U), are used. A table showing the correspondence between each codon and an amino acid is called a genetic code table or codon table. 20 amino acids are assigned to 61 codons, excluding the stop codon (Table 1). The genetic code table shown in Table 1 is also called the standard genetic code table or universal genetic code table because it is commonly used by almost all organisms, both eukaryotes and prokaryotes (eubacteria and archaea). In this disclosure, the genetic code table used in naturally occurring organisms is referred to as a "natural genetic code table" and is distinguished from a genetic code table that has been artificially reprogrammed (in which the correspondence between codons and amino acids has been altered). In the genetic code table, four codons that share the same first and second letters and differ only in the third letter are usually grouped together in one box, and these are called codon boxes. In the present disclosure, a specific codon box may be represented by arranging "M," which means any base selected from A, C, G, and U, after the first and second bases of the codon. For example, a codon box in which Ser is assigned in the natural genetic code table and the first letter of the codon is U and the second letter is C, is represented as "UCM," and a codon box to which Pro is assigned is represented as "CCM." [Table 1]

[0015] In the present disclosure, codons in mRNA may be represented as "M1M2M3," where M1, M2, and M3 represent the first, second, and third bases of the codon, respectively.

[0016] An "anticodon" refers to three consecutive bases on a tRNA that correspond to a codon on an mRNA. Similar to mRNA, anticodons use four types of bases: adenine (A), guanine (G), cytosine (C), and uracil (U). Modified bases obtained by modifying these bases may also be used. The codon is specifically recognized by the anticodon, allowing the genetic information on the mRNA to be read and translated into a protein. The 5'-to-3' codon sequence on the mRNA and the 5'-to-3' anticodon sequence on the tRNA bind complementarily, forming complementary base pairs between the first, second, and third bases of the codon and the third, second, and first bases of the anticodon, respectively. In the present disclosure, a "modified base" refers to a base whose structure is partially different from that of A, C, G, or U.

[0017] In the present disclosure, the anticodon in the first tRNA is "N 11 N 12 N 13 " and the anticodon in the second tRNA is "N 21 N 22 N 23 " where N 11 , N 12 , and N 13 and N 21 , N 22 , and N 23 represent the first, second, and third bases of the anticodon, respectively. According to the tRNA numbering rules described below, N 11 , N 12 , and N 13 and N 21 , N 22 , and N 23are numbered at positions 34, 35, and 36 of the tRNA, respectively.

[0018] In this disclosure, the bases A, C, G, U, and T may be written in lower case, although upper and lower case letters are used interchangeably. For example, GGG and ggg are used interchangeably.

[0019] In this disclosure, thermodynamically stable base pairs are said to be "complementary" to each other. In addition to Watson-Crick base pairs such as adenine and uracil (AU) and guanine and cytosine (GC), non-Watson-Crick wobble base pairs such as guanine and uracil (GU) are also included in "complementary" base pairs in this disclosure. In particular, because there is a spatial wobble between the third letter of the codon and the first letter of the anticodon, it is believed that the formation of such non-Watson-Crick base pairs is also permitted (wobble hypothesis).

[0020] In the present disclosure, a certain relationship between a codon and an anticodon may be referred to as "complementary." A "complementary" relationship is one in which a Watson-Crick base pair is formed between the first letter of the codon and the third letter of the anticodon, and between the second letter of the codon and the second letter of the anticodon, and a Watson-Crick or wobble base pair is formed between the third letter of the codon and the first letter of the anticodon. For example, the anticodons complementary to the codon UCU are AGA and GGA, and the codons complementary to the anticodon GCG are CGC and CGU.

[0021] Messenger RNA (mRNA) is RNA that contains genetic information that can be translated into proteins. Genetic information is encoded in mRNA as codons, each corresponding to one of the 20 amino acids. Protein translation begins with a start codon and ends with a stop codon. In eukaryotes, the start codon is typically AUG, but prokaryotes (eubacteria and archaea) may use other start codons, such as GUG and UUG. AUG encodes methionine (Met), and translation begins with methionine in eukaryotes and archaea. In contrast, in eubacteria, only the start codon AUG corresponds to N-formylmethionine (fMet), so translation begins with formylmethionine. There are three types of stop codons: UAA (ochre), UAG (amber), and UGA (opal). When the stop codon is recognized by a protein called a translation release factor (RF), the peptide chain synthesized up to that point dissociates from the tRNA, and the translation process terminates.

[0022] Transfer RNA (tRNA) is a short RNA of less than 100 bases that mediates peptide synthesis using mRNA as a template. Its secondary structure is cloverleaf-shaped, consisting of three stem-loops (D arm, anticodon arm, and T arm) and one stem (acceptor stem). Some tRNAs may also contain an additional variable loop. The anticodon arm contains a region of three consecutive bases called the anticodon, which recognizes the codon on the mRNA by base pairing with it. The 3' end of the tRNA contains a nucleic acid sequence consisting of cytidine-cytidine-adenosine (CCA sequence), to which an amino acid is added (specifically, an ester bond is formed between the hydroxyl group at the 2nd or 3rd position of the ribose of the adenosine residue and the carboxyl group of the amino acid). A tRNA with an amino acid attached is called an aminoacyl-tRNA. In the present disclosure, aminoacyl-tRNA is also included in the definition of tRNA. Furthermore, as described below, a method is known in which two terminal residues (C and A) are removed from the CCA sequence of tRNA and the resulting residue is used to synthesize aminoacyl-tRNA. Such tRNAs with the CA sequence removed from the 3' end are also included in the definition of tRNA in the present disclosure. In vivo, the addition of amino acids to tRNA is carried out by an enzyme called aminoacyl-tRNA synthetase (aaRS or ARS). Typically, one type of aminoacyl-tRNA synthetase exists for each amino acid, and each aminoacyl-tRNA synthetase specifically recognizes only a specific tRNA as a substrate from among multiple tRNAs, so the correspondence between tRNA and amino acid is strictly controlled.

[0023] Each base in a tRNA is numbered according to the tRNA numbering rule (Sprinzl et al., Nucleic Acids Res (1998) 26: 148-153). In the present disclosure, the numbering of bases in a tRNA follows this rule. For example, the anticodon is numbered at positions 34 to 36, and the CCA sequence at the 3' end is numbered at positions 74 to 76.

[0024] In the present disclosure, the tRNA numbering system (Sprinzl et al., Nucleic Acids Res (1998) 26: 148-153) and the abbreviation of the base (A, C, G, or U) are used to represent the base at a specific position in tRNA. For example, "32U" means that the base at position 32 according to the tRNA numbering system is U (uracil). These are also used to represent the substitution of the base at a specific position in tRNA. For example, "C32U" means the substitution of C (cytosine) with U (uracil) at the base at position 32 according to the tRNA numbering system.

[0025] The "anticodon loop" refers to seven consecutive bases, including the bases 32-38 in the tRNA, i.e., the three consecutive bases of the anticodon, and two bases on the 5' and 3' ends of the anticodon. Four types of bases, adenine (A), guanine (G), cytosine (C), and uracil (U), are used in the anticodon loop. Furthermore, modified bases obtained by modifying these bases may also be used.

[0026] As used herein, the term "tRNA body" refers to the main portion of a tRNA (the main structural portion composed of nucleic acids) excluding the anticodon (positions 34-36). In some embodiments, the term "tRNA body" as used herein refers to positions 1-33 and 37-76 of a tRNA. In other embodiments, the term "tRNA body" as used herein refers to positions 1-33 and 37-74 of a tRNA. A "chimeric tRNA body" refers to a tRNA body in which a portion of the tRNA body is derived from a specific source or type of tRNA, while the remaining portion is derived from a different source or type of tRNA. A chimeric tRNA body does not include a tRNA body derived from only one type of tRNA. A chimeric tRNA body may be derived from two or more types of tRNAs, and may also be derived from three or more types of tRNAs. The chimeric tRNA body of the present disclosure may be a chimeric tRNA body in which the combination of bases at positions 32, 33, 37, and 38 differs from the origin of the base sequences at other positions. An example of a chimeric tRNA body is a tRNA body in which the combination of bases at positions 32, 33, 37, and 38 is derived from tRNA Pro2, and the remaining base sequence is derived from tRNA Glu2. In the present disclosure, the origin of the combination of bases at positions 32, 33, 37, and 38 of a certain tRNA can be determined by referring to the combination of bases at positions 32, 33, 37, and 38 of the base sequence of a tRNA derived from Escherichia coli (SEQ ID NOs: 274 to 319), and determining that the tRNA is derived from a tRNA having the same combination of bases at positions 32, 33, 37, and 38. For example, the combination of bases CUxxxAC at positions 32, 33, 37, and 38 can be determined to be derived from tRNA Glu2 or tRNA Asp1 by referring to SEQ ID NOs: 274 to 319. In this case, a tRNA body in which the base sequence other than positions 32, 33, 37, and 38 is derived from tRNA Glu2 or tRNA Asp1 is not included in the chimeric tRNA body because it is derived from only one type of tRNA.The derived tRNA can be determined according to the sequence information of bases 32 to 38 of the tRNA, using the following criteria: UUxxxAC = Ala1B, AUxxxAU = Ala2, CUxxxGA = Arg3, CUxxxAC = Glu2 or Asp1, UUxxxGU = Leu2, Leu1 or Leu3, UUxxxAA = Phe, Cys, Gly1, Gly3, Leu4, Leu5, Thr1, Thr2 or Thr3, AUxxxGU = Pro2, UUxxxGA = Pro3 or Pro1, CUxxxAA = Ser5, Arg2, Arg4, Arg5, Asn, Gly2, Ile1, Ile2, Lys, Met, Ser1, Ser2, Ser3, Thr4, Trp, Tyr1, Tyr2 or Val1, UUxxxAU = Val2 (a collective term for Val2A and Val2B), Gln1, Gln2, or His (where xxx refers to the three bases of the anticodon). For example, in a tRNA whose base sequence at positions 32 to 38 is UUxxxGU, the combination of bases at positions 32, 33, 37, and 38 is determined to be derived from tRNALeu2, tRNALeu1, or tRNALeu3.

[0027] A certain base, a certain combination of bases, or a certain base sequence is "derived from" a certain source means that the base, a certain combination of bases, or a base sequence, or a sequence highly similar to the base, a certain combination of bases, or a base sequence, is isolated from a certain source. For example, when the base, a certain combination of bases, or a base sequence constituting a certain tRNA is isolated from a certain type of tRNA, the base, a certain combination of bases, or a base sequence is said to be "derived from" the certain type of tRNA.

[0028] In the present disclosure, tRNAs may be represented as follows: "tRNA Xxx" or "tRNA(Xxx)": refers to a tRNA body with a specific base sequence identified by Xxx, such as tRNA Glu2 or tRNA(Glu2), tRNA Ser5 or tRNA(Ser5), tRNA AsnE2 or tRNA(AsnE2), or tRNA Asp1 or tRNA(Asp1). "tRNA(Xxx)nnn": A full-length tRNA with a specific tRNA body identified by Xxx and an anticodon sequence of nnn. Examples include tRNA(Glu2)uga and tRNA(AsnE2)uga. "tRNA(Xxx+Yyy)": This refers to a chimeric tRNA body that has a specific base sequence identified by Xxx other than positions 32, 33, 37, and 38, and a specific base combination identified by Yyy at positions 32, 33, 37, and 38. Examples include tRNA(Glu2+Ser5) and tRNA(AsnE2+Phe). "tRNA(Xxx+Yyy)nnn": A full-length tRNA that has a specific base sequence identified by Xxx at positions other than 32, 33, 37, and 38, and a chimeric tRNA body with a specific base combination identified by Yyy at positions 32, 33, 37, and 38, and whose anticodon sequence is nnn. Examples include tRNA(Glu2+Ser5)uga and tRNA(AsnE2+Phe)uga. The names of the above tRNAs may be suffixed with "-CA" to indicate that the CA sequence at the 3' end of the tRNA has been removed. Examples include tRNA(Glu2)-CA, tRNA(Glu2)uga-CA, tRNA(Glu2+Ser5)-CA, and tRNA(Glu2+Ser5)uga-CA.

[0029] In the present disclosure, specific combinations of bases at positions 32, 33, 37, and 38 in a tRNA may be represented by the name of the tRNA from which they are derived. In the present disclosure, the base sequence at positions 32 to 38 is represented by XXxxxXX, and the combinations of bases at positions 32, 33, 37, and 38 of a tRNA are named as follows: Ala1B sequence = UUxxxAC, Ala2 sequence = AUxxxAU, Arg3 sequence = CUxxxGA, Glu2 sequence = CUxxxAC, Leu2 sequence = UUxxxGU, Phe sequence = UUxxxAA, Pro2 sequence = AUxxxGU, Pro3 sequence = UUxxxGA, Ser5 sequence = CUxxxAA, Val2 sequence = UUxxxAU.

[0030] An "initiator tRNA" is a specific tRNA used at the start of mRNA translation. The initiator tRNA, bound to an initiator amino acid, is introduced into the ribosome via the catalysis of a translation initiation factor (IF) and binds to the start codon on the mRNA, thereby initiating translation. Since AUG, the codon for methionine, is generally used as the start codon, the initiator tRNA has an anticodon corresponding to AUG, and is bound to methionine (formylmethionine in prokaryotes) as the start amino acid. An example of an initiator tRNA is tRNA fMet (SEQ ID NO: 283, 284).

[0031] "Elongator tRNA" refers to a tRNA used in the peptide chain elongation reaction in the translation process. In peptide synthesis, the elongation reaction of the peptide chain proceeds when the elongator tRNA bound to an amino acid is sequentially transported to the ribosome by the GTP-conjugated translation elongation factor (EF) EF-Tu / eEF-1. Examples of elongator tRNAs include tRNAs corresponding to various amino acids (SEQ ID NOs: 274-282, 285-319).

[0032] In the present disclosure, a "translation system" is defined as a composition for translating a peptide (sometimes referred to as a "composition for translation" in the present disclosure). A typical translation system includes, but is not limited to, components such as ribosomes, translation factors, tRNA, amino acids, aminoacyl-tRNA synthetases (aaRS), and factors necessary for peptide translation reactions, such as ATP and GTP. Major types of translation systems include translation systems that utilize living cells and translation systems that utilize cell extracts (cell-free translation systems (used synonymously with "composition for cell-free translation" in the present disclosure)). Known examples of translation systems that utilize living cells include systems in which desired aminoacyl-tRNA and mRNA are introduced into living cells, such as Xenopus oocytes or mammalian cells, by microinjection or lipofection to perform peptide translation (Nowak et al., Science (1995) 268: 439-442). Examples of cell-free translation systems include those using extracts from Escherichia coli (Chen et al., Methods Enzymol (1983) 101: 674-690), yeast (Gasior et al., J Biol Chem (1979) 254: 3965-3969), wheat germ (Erickson et al., Methods Enzymol (1983) 96: 38-50), rabbit reticulocytes (Jackson et al., Methods Enzymol (1983) 96: 50-74), HeLa cells (Barton et al., Methods Enzymol (1996) 275: 35-57), and insect cells (Swerdel et al., Comp Biochem Physiol B (1989) 93: 803-806). Such a translation system can be prepared appropriately by methods known to those skilled in the art or methods equivalent thereto. Cell-free translation systems also include translation systems constructed by isolating and purifying factors necessary for peptide translation and reconstituting them (reconstituted cell-free translation systems) (Shimizu et al., Nat Biotech (2001) 19: 751-755).Although not limited thereto, a reconstituted cell-free translation system may typically contain ribosomes, amino acids, tRNA, aminoacyl-tRNA synthetase (aaRS), translation initiation factors (e.g., IF1, IF2, IF3), translation elongation factors (e.g., EF-Tu, EF-Ts, EF-G), translation termination factors (e.g., RF1, RF2, RF3), ribosome recycling factors (RRF), NTPs as an energy source, an energy regeneration system, and other factors necessary for translation. When a transcription reaction from DNA is also performed, an RNA polymerase may also be contained. The various factors contained in the cell-free translation system can be isolated and purified by methods well known to those skilled in the art, and a reconstituted cell-free translation system can be appropriately constructed using them. Alternatively, commercially available reconstituted cell-free translation systems such as PUREfrex® from Gene Frontier and PURExpress® from New England BioLabs can also be used. In the case of a reconstituted cell-free translation system, a desired translation system can be constructed by reconstituting only the necessary components among the components of the translation system.

[0033] By incorporating a specific combination of amino acid, tRNA, and aminoacyl-tRNA synthetase into a translation system, aminoacyl-tRNA is synthesized within the translation system and used in peptide translation. Alternatively, aminoacyl-tRNA prepared outside the translation system can be used directly as a component of the translation system (sometimes referred to as the "precharge method" in this disclosure). Examples of precharge methods include a method in which an amino acid is attached to tRNA outside the translation system using an aaRS, the pdCpA method, the pCpA method, and a method using an artificial RNA catalyst (flexizyme). In particular, when amino acids that are difficult to aminoacylate with an aaRS, such as some unnatural amino acids, are used in translation, a precharge method using a tRNA that has been previously aminoacylated with an unnatural amino acid, such as the pdCpA method, the pCpA method, or a method using flexizyme, is preferred.

[0034] Translation is initiated by adding mRNA to a translation system. mRNA typically contains a sequence encoding the peptide of interest and may also contain sequences to increase the efficiency of the translation reaction (e.g., Shine-Dalgarno (SD) sequence in prokaryotes, Kozac sequence in eukaryotes, etc.). Pre-transcribed mRNA can be added directly to the system, or alternatively, a promoter-containing template DNA and an appropriate RNA polymerase (e.g., T7 promoter and T7 RNA polymerase) can be added to the system, allowing mRNA to be transcribed from the template DNA.

[0035] "Codon misreading" refers to the translational incorporation of an unintended amino acid when an aminoacyl-tRNA with a specific anticodon recognizes a codon that is not complementary to the anticodon. For example, an aminoacyl-tRNA with the anticodon AGG, which is complementary to the codon CCU, mistakenly recognizes the codon CCG, resulting in the unintended translational incorporation of an amino acid acylated by the tRNA. In the natural genetic code, Pro is assigned to both the codons CCG and CCU, so the presence or absence of such a misreading does not affect the amino acid that is translated. However, this codon misreading can be problematic when reprogramming the genetic code to assign different amino acids to the codons CCG and CCU.

[0036] In this disclosure, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms or unsaturated carbon-carbon bonds in the backbone, and has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. The carbon chain length n ranges from 1 to 20, and examples of alkyl include C1-C 10Examples include alkyl, C1-C6 alkyl, and C1-C3 alkyl, and specific examples include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, t-butyl, sec-butyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1,2-dimethylpropyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, isopentyl, and neopentyl.

[0037] In the present disclosure, "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. Examples of cycloalkyl include C3-C 10 Examples include cycloalkyl, specifically cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, and the like.

[0038] In the present disclosure, "alkenyl" refers to a monovalent group having at least one double bond (two adjacent SP2 carbon atoms). Depending on the configuration of the double bond and any substituents, the geometry of the double bond can be in an entgegen (E) or zusammen (Z) configuration, a cis or trans configuration. Included are straight-chain or branched alkenyls, including straight-chains containing internal olefins. Alkenyls include, for example, C2-C 10 Alkenyl, C2-C6 alkenyl, etc., are exemplified, and specific examples include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, hexenyl, etc.

[0039] In the present disclosure, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). It includes straight-chain or branched-chain alkynyl, including internal alkylene. Examples of alkynyl include, for example, C2-C 10 alkynyl, C2-C6 alkynyl, etc., and specific examples thereof include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, 3-methyl-(5-phenyl)-4-pentynyl, etc.

[0040] In the present disclosure, "aryl" refers to a monovalent aromatic hydrocarbon ring. Examples of aryl include C6-C 10 Examples include aryl, specifically phenyl, naphthyl (for example, 1-naphthyl, 2-naphthyl), and the like.

[0041] In the present disclosure, "heteroaryl" refers to a monovalent aromatic ring group containing a heteroatom among the atoms constituting the ring, and may be partially saturated. The ring may be a single ring or two condensed rings (e.g., a bicyclic heteroaryl condensed with benzene or a single ring heteroaryl). The number of atoms constituting the ring is, for example, 5-10 (5-membered to 10-membered heteroaryl). The number of heteroatoms contained among the atoms constituting the ring is, for example, 1-5. Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl.

[0042] In the present disclosure, "arylalkyl (aralkyl)" refers to a group containing both aryl and alkyl, for example, a group in which at least one hydrogen atom of the alkyl is substituted with an aryl. Examples of aralkyl include C5-C 10 Examples include aryl C1-C6 alkyl, and specific examples include benzyl.

[0043] In this disclosure, "alkylene" refers to a divalent group derived from the aforementioned "alkyl" by further removing one optional hydrogen atom, and may be straight-chain or branched. Examples of straight-chain alkylene include C2-C6 straight-chain alkylene and C4-C5 straight-chain alkylene, specifically, -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, etc. Examples of branched alkylene include C2-C6 branched alkylene and C4-C5 branched alkylene, specifically, -CH(CH3)CH2-, -C(CH3)2-, -CH(CH3)CH2CH2-, -C(CH3)2CH2-, -CH2CH(CH3)CH2-, -CH2C(CH3)2-, -CH2CH2CH(CH3)-, etc.

[0044] In the present disclosure, "alkenylene" refers to a divalent group derived from the "alkenyl" defined above by removing one optional hydrogen atom, and may be straight-chain or branched. Depending on the configuration of the double bond and substituents (if any), it may be in an entgegen (E) or zusammen (Z) configuration, a cis or trans configuration. Examples of straight-chain alkenylene include C2-C6 straight-chain alkenylene and C4-C5 straight-chain alkenylene, and specific examples include -CH=CH-, -CH=CHCH2-, -CH2CH=CH-, -CH=CHCH2CH2-, -CH2CH=CHCH2-, -CH2CH2CH=CH-, -CH2CH2CH=CH-, -CH2CH=CHCH2CH2-, -CH2CH2CH=CHCH2-, -CH2CH2CH=CH-, -CH2CH2CH=CHCH2-, -CH2CH2CH=CHCH2-, and -CH2CH2CH2CH=CH-.

[0045] In the present disclosure, "arylene" refers to a divalent group derived from the aryl by further removing one arbitrary hydrogen atom. The ring may be a single ring or a fused ring. The number of atoms constituting the ring is not particularly limited, but is, for example, 6 to 10 (C6-C 10 Specific examples of arylene include phenylene and naphthylene.

[0046] In the present disclosure, the term "heteroarylene" refers to a divalent group derived from the heteroaryl by further removing one arbitrary hydrogen atom. The ring may be a single ring or a fused ring. The number of atoms constituting the ring is not particularly limited, but is, for example, 5 to 10 (5- to 10-membered heteroarylene). Specific examples of heteroarylene include pyrrolediyl, imidazolediyl, pyrazolediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazolediyl, triazinediyl, isoxazolediyl, oxazolediyl, oxadiazolediyl, isothiazolediyl, thiazolidinyl, thiadiazolediyl, furandiyl, and thiophenediyl.

[0047] <Compositions, Kits, and Methods> In one aspect, the present disclosure relates to a translation composition and a translation kit comprising a first tRNA bound to a first amino acid and a second tRNA bound to a second amino acid. In another aspect, the present disclosure relates to a method for translating a nucleic acid using a first tRNA bound to a first amino acid and a second tRNA bound to a second amino acid. By using the composition, kit, and method, it is possible to reduce or prevent the erroneous translation of an unintended amino acid due to misreading of a codon by tRNA. Therefore, in one aspect, the present disclosure relates to a method for producing a peptide while reducing or preventing misreading of a codon by tRNA, as well as a composition, kit, etc. for use in the method.

[0048] In addition to the tRNA in the present disclosure, the compositions in the present disclosure can include buffers and substances commonly used in nucleic acid translation. In one aspect, the tRNA in the present disclosure can be packaged in advance together with various substances commonly used in peptide translation and supplied as a kit. In a further aspect, the various substances included in the kit in the present disclosure can be in powder or liquid form according to their usage modes. These can be stored in appropriate containers and used in a timely manner.

[0049] <tRNA and tRNA body> In some embodiments, the combination of bases at positions 32, 33, 37, and 38 of the tRNA of the present disclosure is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, (4) 32U, 33U, 37G, 38U, (5) 32U, 33U, 37A, 38U, or (6) 32C, 33U, 37G, 38A, and may be. In some embodiments, the tRNA of the present disclosure may be a tRNA in which positions 32, 33, 37, and 38 are a combination of bases selected from the group consisting of the above (1)-(4); the above (1)-(3); or, the above (1), (3) and (4), or may be a tRNA which is a combination of bases of the above (1); the above (2); the above (3); the above (4); the above (5); or, the above (6). In some embodiments, the bases at positions 32, 33, 37 and 38 of the tRNA of the present disclosure are not modified bases.

[0050] In the present disclosure, the anticodon of the first tRNA of the present disclosure is represented as N 11 N 12 N 13 and the anticodon of the second tRNA of the present disclosure may be represented as N 21 N 22 N 23 and may be represented as. The above N 11 , N12 , and N 13 and N 21 N 22 N 23 may each independently be A, C, G, or U. The first, second, and third letters of the anticodon of a tRNA of the present disclosure may each independently be A, C, G, or U. The second and third letter base sequences of the anticodon of a tRNA of the present disclosure may be CC, GC, AC, GU, CG, GG, AG, or GA, or may be GG, AG, or CC. In some embodiments, the second and third letter base sequences of the anticodon are not CG. In some embodiments, a tRNA of the present disclosure does not have a modified base in the anticodon, or the first letter of the anticodon may be a modified base, or the nucleoside of the first letter may have a modification as described below.

[0051] In some embodiments, the tRNA of the present disclosure is a tRNA derived from a prokaryote or a tRNA derived from a eukaryote. The tRNA may be produced by modifying a tRNA derived from a prokaryote or a tRNA derived from a eukaryote, or the tRNA produced by modification may have the highest base sequence identity with a tRNA derived from a prokaryote or a tRNA derived from a eukaryote. Eukaryotes are further classified into animals, plants, fungi, and protists. The tRNA of the present disclosure may be, for example, a tRNA derived from humans. Prokaryotes are further classified into eubacteria and archaea. Examples of eubacteria include Escherichia coli, Bacillus subtilis, lactic acid bacteria, and Desulfitobacterium hafniense. Examples of archaea include extreme halophiles, thermophiles, and methanogens (e.g., Methanosarcina mazei, Methanosarcina barkeri, and Methanocaldococcus jannaschii). The tRNA of the present disclosure may be, for example, a tRNA derived from Escherichia coli, Desulfitobacterium hafniense, or Methanosarcina mazei.

[0052] In some embodiments, a tRNA of the disclosure may differ from the base sequence of a reference tRNA by one or more bases, including 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 bases, including 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer bases. In some embodiments, a tRNA of the disclosure may have 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence identity compared to the base sequence of a reference tRNA. In this disclosure, "percent (%) sequence identity" for a given base sequence is defined as the percentage of bases in a candidate sequence that are identical to bases in a reference base sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment of base sequences for purposes of determining percent sequence identity can be accomplished by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0053] In some embodiments, the nucleotide sequence of the anticodon loop (positions 32 to 38) of a tRNA of the present disclosure may differ from that of a reference tRNA. In certain embodiments, a tRNA of the present disclosure may have a chimeric anticodon loop. In the present disclosure, a "chimeric anticodon loop" refers to an anticodon loop in which the nucleotides at positions 32, 33, 37, and 38 and the anticodon nucleotide sequence are derived from different tRNAs. For example, whether an anticodon loop containing the anticodon UGA corresponds to a chimeric anticodon loop can be determined as follows. First, tRNA Ser1 (SEQ ID NO: 306) derived from Escherichia coli is identified as a tRNA having the anticodon UGA, and it can be determined that the anticodon UGA is derived from tRNA Ser1 (the nucleotide sequences of tRNAs set forth in SEQ ID NOs: 274 to 319 may be referenced when determining the origin of the anticodon). Here, the nucleotide sequence of the anticodon loop of the tRNA Ser1 is CUugaAA. Therefore, when the bases at positions 32, 33, 37, and 38 contained in an anticodon loop are a combination of bases other than those in the Ser5 sequence (32C, 33U, 37A, and 38A), the anticodon loop is determined to be a chimeric anticodon loop. In one embodiment, the anticodon loop of a tRNA of the present disclosure may be different from the anticodon loop (positions 32 to 38 according to the tRNA numbering rules) contained in a tRNA having a base sequence set forth in any of SEQ ID NOs: 274 to 282, 285 to 304, and 306 to 319. In some embodiments, a tRNA body of the disclosure may differ from the base sequence of a reference tRNA body by one or more bases, including 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 bases, including 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer bases.In some embodiments, a tRNA body of the disclosure may have 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence identity compared to the base sequence of a reference tRNA body. In some embodiments, a tRNA of the disclosure may differ from the base of a reference tRNA body at at least one base selected from the group consisting of positions 32, 33, 37, and 38. In some embodiments, a tRNA body of the disclosure may differ from the base of a reference tRNA body at at least one base selected from the group consisting of positions 32, 33, 37, and 38. In some embodiments, a tRNA of the disclosure is characterized as a non-naturally occurring tRNA. In certain embodiments, the tRNA body of the present disclosure may be a tRNA body that is not derived from a tRNA having the nucleotide sequence set forth in SEQ ID NO: 275, and / or may be a tRNA body that is not derived from any of the tRNAs having the nucleotide sequences set forth in SEQ ID NOs: 294, 295, and 296, and / or may be a tRNA body that is not derived from any of the tRNAs having the nucleotide sequences set forth in SEQ ID NOs: 302, 303, and 304. In certain embodiments, when positions 32, 33, 37, and 38 of a tRNA body of the present disclosure are Leu2 sequences, the tRNA body may not be derived from any of the tRNAs having the nucleotide sequences set forth in SEQ ID NOs: 294, 295, and 296; when positions 32, 33, 37, and 38 are Ala2 sequences, the tRNA body may not be derived from any of the tRNAs having the nucleotide sequence set forth in SEQ ID NO: 275; and when positions 32, 33, 37, and 38 are Pro2 or Pro3 sequences, the tRNA body may not be derived from any of the tRNAs having the nucleotide sequences set forth in SEQ ID NOs: 302, 303, and 304.

[0054] The reference tRNA and reference tRNA body in the present disclosure may each be a naturally occurring tRNA derived from any organism (e.g., Escherichia coli) or its body, or a non-naturally occurring tRNA whose sequence is artificially synthesized and different from that of a naturally occurring tRNA or its body, or a tRNA (artificial tRNA) whose sequence is artificially synthesized from that of a naturally occurring tRNA or its body, or a tRNA chimera formed by artificially combining tRNAs of different origins or its body.

[0055] The reference tRNA or reference tRNA body in the present disclosure can be appropriately selected from tRNAs or tRNA bodies having any desired base sequence. In some embodiments, the reference tRNA or reference tRNA body may be at least one tRNA or a body thereof selected from the group consisting of tRNA Ala, tRNA Arg, tRNA Asn, tRNA Asp, tRNA Cys, tRNA Gln, tRNA Glu, tRNA Gly, tRNA His, tRNA Ile, tRNA Leu, tRNA Lys, tRNA Met, tRNA Phe, tRNA Pro, tRNA Ser, tRNA Thr, tRNA Trp, tRNA Tyr, tRNA Val, and tRNA Sec (selenocysteine) (SEQ ID NOS: 274 to 282, 285 to 319), and tRNA Glu2, tRNA AsnE2, and tRNA Asp1 (SEQ ID NOS: 322 to 324). Other examples of tRNA fMet (SEQ ID NOs: 283, 284), tRNA Pyl (pyrrolysine), tRNA AsnE2 (see Ohta, A.; Murakami, H.; Higashimura, E.; Suga, H. Chem. Biol. 2007, 14, 1315-1322), and their bodies may also be used as references. Also, tRNA Pro1E2, a tRNA chimera in which the T stem of tRNA Glu2 is grafted onto tRNA Pro1 with further mutations (see WO2019 / 077887), and their bodies may also be used as references. In certain embodiments, the tRNA or tRNA body of the present disclosure may be at least one tRNA or its body selected from the group consisting of tRNA Glu2, tRNA Asp1, and tRNA AsnE2. Exemplary base sequences of bases 1 to 74 of several tRNA bodies are shown in SEQ ID NOs: 253 to 255, 320, and 321. The reference tRNA bodies exemplified here may also be used as the sequences from which portions of the chimeric tRNA bodies of the present disclosure other than the anticodon loop are derived.

[0056] In some embodiments, the difference between a tRNA or tRNA body of the present disclosure and a reference tRNA or reference tRNA body may be generated by modifying a portion of the sequence based on sequence information (e.g., nucleotide sequence information) of the reference tRNA or reference tRNA body. In this case, once the sequence information of a tRNA or tRNA body of the present disclosure is obtained, it is possible to prepare a tRNA or tRNA body of the present disclosure without the need for the reference sequence information. In the present disclosure, "modification" means introducing at least one, or two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more types of modification selected from the following group into the base sequence (existing sequence) of an existing tRNA or tRNA body: (i) addition (adding any new base to the existing sequence), (ii) deletion (deleting any nucleotide from the existing sequence), (iii) substitution (replacing any base in the existing sequence with another any base), (iv) insertion (adding any new nucleotide between any two nucleotides in the existing sequence), or (v) modification (changing a part of the structure of any nucleoside in the existing sequence (e.g., the base moiety or sugar moiety) to a different structure). Modifications may be made to any structure of the tRNA or tRNA body (e.g., the D arm, anticodon arm, T arm, acceptor stem, variable loop, etc.). In certain embodiments, the modification of a tRNA or tRNA body of the present disclosure is performed on at least one, two or more, three or more, or four bases selected from the group consisting of positions 32, 33, 37, and 38 of the tRNA, or the group consisting of positions 32, 37, and 38. In some embodiments, the tRNA and tRNA body of the present disclosure may not actually be modified based on a reference tRNA or reference tRNA body. tRNAs and tRNA bodies of the present disclosure also include tRNAs and tRNA bodies having base sequences obtained when such modifications are made to a reference tRNA or reference tRNA body.

[0057] Modifications in the present disclosure include, for example, substituting bases so that the combination of bases at positions 32, 33, 37, and 38 of the tRNA is the same as the combination of bases at the corresponding positions of the tRNA of the present disclosure. The base before substitution is not particularly limited, but for example, the base at position 32 before substitution may be C or U, the base at position 37 before substitution may be A, the base at position 38 before substitution may be A or C, and / or the base at position 33 before substitution may be U. In some embodiments, if the base at position 32, 33, 37, or 38 is already the desired base, substitution is not necessary. For example, if the base at position 33 of the tRNA before substitution is U and the desired base at position 33 is also U, substitution of the base at position 33 is not necessary.

[0058] In some embodiments, the tRNA of the present disclosure may have a chimeric tRNA body. Examples of chimeric tRNA bodies include those derived from tRNA bodies in which the anticodon loop portion (i.e., positions 32, 33, 37, and 38) is different from the remaining portions. Examples of the base sequence of positions 1 to 74 of a chimeric tRNA body include the following:

[0059] [Table 2]

[0060] In some embodiments, the base sequence at positions 32 to 38 of a tRNA of the present disclosure may differ from the base sequence at positions 32 to 38 of a wild-type tRNA in E. coli or a naturally occurring tRNA. Without limitation, in some embodiments, when a tRNA of the present disclosure has a chimeric tRNA body with a base combination of 32U, 33U, 37G, and 38A, tRNAs whose anticodons are UGG and / or CGG may be excluded from the tRNA of the present disclosure. In some embodiments, when a tRNA of the present disclosure has a chimeric tRNA body with a base combination of 32A, 33U, 37G, and 38U, tRNAs whose anticodons are AGG and / or GGG may be excluded from the tRNA of the present disclosure. In some embodiments, when a tRNA of the present disclosure has a chimeric tRNA body with a base combination of 32A, 33U, 37A, and 38U, tRNAs whose anticodons are AGC and / or GGC may be excluded from the tRNA of the present disclosure. In some embodiments, when a tRNA of the present disclosure has a chimeric tRNA body with a base combination of 32C, 33U, 37G, and 38A, tRNAs whose anticodon is UCG and / or CCG may be excluded from the tRNA of the present disclosure. In some embodiments, when a tRNA of the present disclosure has a chimeric tRNA body with a base combination of 32U, 33U, 37G, and 38U, tRNAs whose anticodon has an A as the second base and a G as the third base may be excluded from the tRNA of the present disclosure.

[0061] In some embodiments, the base sequence other than the anticodon loop of the tRNA of the present disclosure is not particularly limited and may be selected from among tRNA Ala, tRNA Pro, and tRNA Leu. Here, the base sequence other than the anticodon loop may be the base sequences of positions 1 to 31 and 39 to 76 of the tRNA.

[0062] In some embodiments, the base sequences of positions 1 to 31 and 39 to 74 of a tRNA of the present disclosure may be derived from, but are not limited to, the base sequences of positions 1 to 31 and 39 to 74 of a tRNA having at least one base sequence selected from the group consisting of (a) SEQ ID NO: 253, (b) SEQ ID NO: 255, and (c) SEQ ID NO: 254. In a specific embodiment, the base sequences of positions 1 to 31 and 39 to 74 of a tRNA of the present disclosure may be the base sequences of positions 1 to 31 and 39 to 74 of a tRNA having at least one base sequence selected from the group consisting of (a) to (c). In some embodiments, the nucleotide sequences of positions 1 to 31 and positions 39 to 74 of a tRNA of the present disclosure may have sequence identity of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more to the nucleotide sequences of positions 1 to 31 and positions 39 to 74 of a tRNA having the nucleotide sequence of any one of (a) to (c) above.

[0063] In some embodiments, the base at position 75 of a tRNA of the present disclosure may be C, and the base at position 76 may be A. Positions 75 and 76 of a tRNA of the present disclosure may be pCpA (a dinucleotide containing cytidine and adenosine) or pdCpA (a dinucleotide containing deoxycytidine and adenosine). In some embodiments, a tRNA of the present disclosure may be a tRNA having an amino acid bound to its 3' end, more specifically to the adenosine residue at the 3' end, more specifically to the adenosine residue at position 76, which is the 3' end.

[0064] In the present disclosure, examples of modifications include modifications to the first base or nucleoside of the anticodon of tRNA (e.g., substitution with lysidine, a lysidine derivative, agmatidine, or an agmatidine derivative). Here, a lysidine derivative refers to a molecule prepared by modifying a portion of the lysidine structure (e.g., the base portion), which, when used as part of an anticodon, has the same codon discrimination ability (ability to form a complementary base pair) as lysidine. Furthermore, an agmatidine derivative refers to a molecule prepared by modifying a portion of the agmatidine structure (e.g., the base portion), which, when used as part of an anticodon, has the same codon discrimination ability (ability to form a complementary base pair) as agmatidine. In the present disclosure, a base that has undergone the modifications exemplified here or other modifications may be referred to as a "modified base." In the present disclosure, "L" in a base sequence or nucleic acid sequence refers to lysidine.

[0065] Lysidine in natural tRNA is synthesized by the action of an enzyme called tRNA Ile-lysidine synthetase (TilS). TilS specifically recognizes tRNA corresponding to isoleucine (tRNA Ile2) as a substrate and has the activity of modifying (converting) cytidine (C) in the first letter (N1) of the anticodon to lysidine (k2C). The lysidine in the tRNA of the present disclosure may be synthesized via TilS or may be synthesized without the intervention of TilS.

[0066] In some embodiments, the tRNA of the present disclosure may be a tRNA that does not contain modified bases. In this disclosure, tRNA prepared by in vitro transcription may be referred to as "transcribed tRNA." The tRNA of the present disclosure may be a transcribed tRNA or a transcribed tRNA that does not contain modified bases. In this disclosure, the term "artificial tRNA" may be used to distinguish it from naturally occurring tRNA. The tRNA of the present disclosure may be an artificial tRNA or an artificial tRNA that does not contain modified bases or modified nucleosides. The method for preparing tRNA that does not contain modified bases, tRNA that does not contain modified nucleosides, transcribed tRNA, and artificial tRNA is not particularly limited. For example, tRNA may be prepared by synthesizing tRNA from template DNA by in vitro transcription using an RNA polymerase such as T7 RNA polymerase, and purifying the RNA as needed. An RNeasy kit (Qiagen) or the like can be used to purify the RNA. In some embodiments, the bases of the tRNA of the present disclosure may consist of A, C, G, and U. In some embodiments, the tRNA of the present disclosure may have bases consisting of A, C, G, and U.

[0067] In some embodiments, an amino acid may be bound to the tRNA of the present disclosure. The amino acid is usually bound to the 3'-terminus of the tRNA, more specifically, to the adenosine residue of the CCA sequence at the 3'-terminus. In some embodiments, the adenosine residue at the 3'-terminus may be at position 76 according to the tRNA numbering rules. The specific type of amino acid bound to the tRNA can be appropriately selected from the amino acids listed below, and examples include unnatural amino acids.

[0068] In the present disclosure, amino acids include α-amino acids, β-amino acids, γ-amino acids, etc. The three-dimensional structures include both L-amino acids and D-amino acids. Furthermore, the amino acids in the present disclosure include natural amino acids and unnatural amino acids. Natural amino acids consist of the following 20 types of α-amino acids: glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), histidine (His), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), cysteine ​​(Cys), methionine (Met), lysine (Lys), arginine (Arg), and proline (Pro). Natural amino acids are usually L-amino acids.

[0069] In the present disclosure, unnatural amino acids refer to all amino acids other than the 20 naturally occurring α-amino acids. Examples of unnatural amino acids include β-amino acids, γ-amino acids, D-amino acids, α-amino acids with side chains different from those of naturally occurring amino acids, α,α-disubstituted amino acids, amino acids with a substituent in the main chain amino group (sometimes referred to as "N-substituted amino acids" in the present disclosure), and hydroxycarboxylic acids (hydroxy acids). Examples of N-substituted amino acids include, but are not limited to, N-methyl amino acids, N-ethyl amino acids, N-propyl amino acids, and N-butyl amino acids. The side chains of unnatural amino acids may contain, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, and the like, in addition to hydrogen atoms. In the case of α,α-disubstituted amino acids, the two side chains may form a ring. Furthermore, these side chains may contain one or more substituents. In certain embodiments, the substituents can be selected from any functional group including a halogen atom, an O atom, a S atom, a N atom, a B atom, a Si atom, or a P atom. For example, in the present disclosure, "C1-C6 alkyl having a halogen atom as a substituent" means "C1-C6 alkyl" in which at least one hydrogen atom in the alkyl is substituted with a halogen atom, and specifically includes, for example, trifluoromethyl, difluoromethyl, fluoromethyl, pentafluoroethyl, tetrafluoroethyl, trifluoroethyl, difluoroethyl, fluoroethyl, trichloromethyl, dichloromethyl, chloromethyl, pentachloroethyl, tetrachloroethyl, trichloroethyl, dichloroethyl, chloroethyl, etc. 10 "Aryl C1-C6 alkyl" refers to a "C5-C" alkyl group in which at least one hydrogen atom in the aryl and / or alkyl is replaced by a substituent. 10"Aryl C1-C6 alkyl" refers to "having two or more substituents." Furthermore, "having two or more substituents" also includes having a functional group (e.g., a functional group containing an S atom) as a substituent, and that functional group further having another substituent (e.g., a substituent such as amino or halogen). Specific examples of unnatural amino acids can be found in WO2013 / 100132 and WO2018 / 143145.

[0070] The amino group in the main chain of an unnatural amino acid may be an unsubstituted amino group (-NH group) or a substituted amino group (-NHR group). Here, R represents alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl, which may have a substituent. Alternatively, as in proline, the carbon chain bonded to the N atom of the amino group in the main chain and the carbon atom at the α-position may form a ring. The substituent can be selected from any functional group containing a halogen atom, O atom, S atom, N atom, B atom, Si atom, or P atom. Examples of alkyl substitution of an amino group include N-methylation, N-ethylation, N-propylation, and N-butylation, and examples of aralkyl substitution include N-benzylation. Specific examples of N-methylamino acids include N-methylalanine, N-methylglycine, N-methylphenylalanine, N-methyltyrosine, N-methyl-3-chlorophenylalanine, N-methyl-4-chlorophenylalanine, N-methyl-4-methoxyphenylalanine, N-methyl-4-thiazolealanine, N-methylhistidine, N-methylserine, and N-methylaspartic acid.

[0071] Examples of substituents containing halogen atoms include fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I), and the like.

[0072] Examples of substituents containing an O atom include hydroxyl (-OH), oxy (-OR), carbonyl (-C=OR), carboxyl (-COH), oxycarbonyl (-C=O-OR), carbonyloxy (-OC=OR), thiocarbonyl (-C=O-SR), carbonylthio (-SC=OR), aminocarbonyl (-C=O-NHR), carbonylamino (-NH-C=OR), oxycarbonylamino (-NH-C=O-OR), sulfonylamino (-NH-SO-R), aminosulfonyl (-SO-NHR), sulfamoylamino (-NH-SO-NHR), thiocarboxyl (-C(=O)-SH), carboxylcarbonyl (-C(=O)-COH), and the like.

[0073] Examples of oxy (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, and the like.

[0074] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0075] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0076] Examples of carbonyloxy (-OC=OR) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, aralkylcarbonyloxy, and the like.

[0077] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0078] Examples of carbonylthio (-SC=OR) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0079] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. Furthermore, the H atom bonded to the N atom in -C=O-NHR may be substituted with a substituent selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl.

[0080] Examples of carbonylamino (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. Furthermore, the H atom bonded to the N atom in -NH-C=OR may be substituted with a substituent selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl.

[0081] Examples of oxycarbonylamino (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. Furthermore, the H atom bonded to the N atom in -NH-C=O-OR may be substituted with a substituent selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl.

[0082] Examples of sulfonylamino (-NH-SO-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. Furthermore, the H atom bonded to the N atom in -NH-SO-R may be substituted with a substituent selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl.

[0083] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. Furthermore, the H atom bonded to the N atom in -SO2-NHR may be substituted with a substituent selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl.

[0084] Examples of sulfamoylamino (-NH-SO-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, and aralkylsulfamoylamino. Furthermore, at least one of the two H atoms bonded to the N atom in -NH-SO-NHR may be substituted with a substituent selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl. When both H atoms are substituted, the substituents may be independently selected, and these two substituents may form a ring.

[0085] Examples of substituents containing an S atom include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-S(O)2-R), sulfo (-SO3H), and the like.

[0086] Examples of thio (-SR) include alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0087] Examples of sulfinyl (-S=OR) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, and the like.

[0088] Examples of sulfonyl (-S(O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0089] Examples of substituents containing an N atom include azido (-N3), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R), tertiary amino (-NR(R')), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R''), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R''), aminocarbonylamino (-NR-CO-NR'R''), and the like.

[0090] Examples of secondary amino (-NH-R) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, aralkylamino, and the like.

[0091] The two substituents R and R' on the N atom of the tertiary amino (-NR(R')) can be independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl. Examples of tertiary amino include alkyl(aralkyl)amino. These two substituents may form a ring.

[0092] The three substituents R, R', and R'' on the N atom of the substituted amidino (-C(=NR)-NR'R'') can be independently selected from the group consisting of a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl. Examples of the substituted amidino include alkyl(aralkyl)(aryl)amidino. These substituents may join together to form a ring.

[0093] The four substituents R, R', R'', and R''' on the N atom of the substituted guanidino (-NR-C(=NR''')-NR'R'') can be independently selected from the group consisting of a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl. These substituents may join together to form a ring.

[0094] The three substituents R, R', and R'' on the N atom of aminocarbonylamino (-NR-CO-NR'R'') can be independently selected from the group consisting of a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl. These substituents may be joined together to form a ring.

[0095] Examples of the substituent containing a B atom include boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')). The two substituents R and R' on the B atom can be independently selected from the group consisting of a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl. These substituents may join together to form a ring.

[0096] Hydroxycarboxylic acids in the present disclosure include α-hydroxycarboxylic acids, β-hydroxycarboxylic acids, γ-hydroxycarboxylic acids, etc. Similar to amino acids, the α-carbon atom of a hydroxycarboxylic acid may have a side chain other than a hydrogen atom attached thereto. The stereostructure may include both L- and D-forms. The structure of the side chain can be defined in the same manner as the side chains of the above-mentioned natural amino acids or unnatural amino acids. Examples of hydroxycarboxylic acids include hydroxyacetic acid, lactic acid, and phenyllactic acid.

[0097] An amino acid in the present disclosure may be a translatable amino acid. In the present disclosure, a "translatable" amino acid refers to an amino acid that can be incorporated into a peptide by translational synthesis (e.g., using a translation system described in the present disclosure). Whether an amino acid is translatable can be confirmed by a translational synthesis experiment using a tRNA bound to the amino acid. A reconstituted cell-free translation system may be used in the translational synthesis experiment (see, for example, WO2013100132).

[0098] In some embodiments, examples of the amino acids in the present disclosure include Pic2 ((2S)-piperidine-2-carboxylic acid), dA ((2R)-2-aminopropanoic acid), MeHph ((2S)-2-(methylamino)-4-phenyl-butanoic acid), SPh2Cl ((2S)-2-amino-3-(2-chlorophenoxy)propanoic acid), MeG (2-(methylamino)acetic acid), nBuG (2-(butylamino)acetic acid), and the like.

[0099] The unnatural amino acids in the present disclosure can be prepared by conventionally known chemical synthesis methods, the synthesis methods described in the examples below, or synthesis methods analogous thereto.

[0100] <Preparation of tRNA> tRNA can be synthesized, for example, by preparing DNA encoding a desired tRNA gene, arranging an appropriate promoter such as T7, T3, or SP6 upstream thereof, and performing a transcription reaction using an RNA polymerase adapted to each promoter with the DNA as a template. tRNA can also be prepared by purification from biological materials. For example, an extract can be prepared from a material containing tRNA such as cells, and tRNA can be recovered by adding a probe containing a sequence complementary to the base sequence of tRNA thereto. At this time, an expression vector capable of expressing the desired tRNA can be prepared, and preparation can also be performed using cells transformed with the expression vector as a material. Usually, tRNA synthesized by in vitro transcription contains only the four typical bases, adenine, guanine, cytosine, and uracil. On the other hand, tRNA synthesized intracellularly may also contain modified bases obtained by modifying them. Modified bases in natural tRNA (such as lysidine) are thought to be specifically introduced into the tRNA by the action of an enzyme (such as TilS) for performing the modification after the tRNA is synthesized by transcription.

[0101] Aminoacyl-tRNAs can also be prepared by chemical and / or biological synthesis. For example, aminoacyl-tRNAs can be synthesized by attaching an amino acid to a tRNA using an aminoacyl-tRNA synthetase (ARS). The amino acid can be a natural or unnatural amino acid, as long as it can serve as a substrate for the ARS. Alternatively, a natural amino acid can be attached to the tRNA and then chemically modified. Many reports have also shown that the activity of an ARS with unnatural amino acids has been enhanced by introducing amino acid mutations into the ARS (see, for example, WO2006 / 135096, WO2007 / 061136, WO2007 / 103307, WO2008 / 001947, WO2010 / 141851, WO2015 / 120287, etc.). An amino acid can also be attached to a tRNA using such a mutant ARS. In addition to the method using ARS, aminoacyl-tRNA can be synthesized by, for example, removing the CA sequence from the 3' end of tRNA and then attaching aminoacylated pdCpA (a dinucleotide containing deoxycytidine and adenosine as nucleosides) to the tRNA using RNA ligase (pdCpA method; Hecht et al., J Biol Chem (1978) 253: 4517-4520). A method using pCpA (a dinucleotide containing cytidine and adenosine as nucleosides) instead of pdCpA is also known (pCpA method; Wang et al., ACS Chem Biol (2015) 10: 2187-2192).Alternatively, aminoacyl-tRNA can be synthesized by attaching a non-natural amino acid, which has been activated by esterification, to tRNA using the artificial RNA catalyst flexizyme (WO2007 / 066627, WO2012 / 026566, H. Murakami et al., Chemistry & Biology, Vol. 10, 2003, 655-662; H. Murakami et al., Chemistry & Biology, Vol. 10, 2003, 1077-1084; H. Murakami et al., Nature Methods 3, 2006, 357-359; N. Niwa et al., Bioorganic & Medicinal Chemistry Letters 19, 2009, 3892-3894). Flexizyme is an artificial RNA catalyst that can attach an amino acid or hydroxy acid to tRNA. Flexizyme in the present disclosure includes the original Flexizyme (Fx), as well as modified versions thereof, such as dinitrobenzyl Flexizyme (dFx), enhanced Flexizyme (eFx), and aminoflexizyme (aFx).

[0102] In one aspect, the present disclosure provides a set of tRNAs suitable for peptide translation. The set of tRNAs includes multiple different types of tRNAs, and multiple different types of amino acids can be translated from these tRNAs. In one aspect, the present disclosure provides a translation composition including multiple different types of tRNAs suitable for peptide translation. In another aspect, the present disclosure provides a method for producing the translation composition. In another aspect, the present disclosure provides a method for producing a peptide, comprising providing multiple different types of tRNAs suitable for peptide translation. In one aspect, the multiple different types of tRNAs include a tRNA of the present disclosure. The following description relates to these set of tRNAs suitable for peptide translation, translation compositions, methods for producing translation compositions, methods for reducing codon misreading, and methods for producing peptides.

[0103] In some embodiments, the set of tRNAs of the present disclosure may include the above-described tRNA of the present disclosure (sometimes referred to as a "first tRNA" in the present disclosure) and a second tRNA. The second tRNA may be any tRNA, and may be the above-described tRNA of the present disclosure, independent of the first tRNA.

[0104] In some embodiments, the first base of the anticodon of a first tRNA and a second tRNA included in a set of tRNAs of the present disclosure may be different from each other. The second base of the anticodon of the first tRNA and the second tRNA may be the same, and the third base of the anticodon of the first tRNA and the second tRNA may be the same. In some embodiments, a codon complementary to the anticodon of the first tRNA and a codon complementary to the anticodon of the second tRNA may be present in the same codon box. In some embodiments, at least two types of amino acids can be translated from one codon box by using the first tRNA and the second tRNA of the present disclosure. In some embodiments, the first base of the anticodon of the first tRNA may be A or G, and the first base of the anticodon of the second tRNA may be C or U. In some embodiments, the first base of each anticodon of the first tRNA and the second tRNA may be exemplified by the following combinations: (A, C); (C, A); (G, C); (C, G); (A, U); (U, A); (G, U); (U, G). Here, (A, C) indicates that the first base of the anticodon of the first tRNA is A and the first base of the anticodon of the second tRNA is C.

[0105] In some embodiments, the tRNA bodies of the first tRNA and the second tRNA included in the set of tRNAs of the present disclosure may be different or identical to each other. In some embodiments, the nucleotide sequences of positions 1 to 31 and positions 39 to 74 of the first tRNA and the second tRNA may be identical or have 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence identity.

[0106] In some embodiments, the first tRNA and the second tRNA included in the set of tRNAs of the present disclosure may have different amino acids bound to each other. In the present disclosure, an amino acid bound to the first tRNA may be referred to as the first amino acid, and an amino acid bound to the second tRNA may be referred to as the second amino acid. In certain embodiments, at least one selected from the first amino acid and the second amino acid of the present disclosure may be an unnatural amino acid. That is, at least one or both of the set of tRNAs of the present disclosure may have an unnatural amino acid bound to them. In some embodiments, one or both of the first tRNA and the second tRNA may be tRNAs to which an unnatural amino acid is bound outside the translation system.

[0107] In some embodiments, but not limited to, when either or both of a first tRNA and a second tRNA have a chimeric tRNA body having a base combination of 32U, 33U, 37G, and 38U, a set of tRNAs including the first tRNA and the second tRNA having at least one anticodon combination selected from the group consisting of the following (L1) to (L4) may be excluded from the set of tRNAs of the present disclosure: (L1) GCG and CCG, and CCG and GCG; (L2) AAG and UAG, AAG and CAG, and UAG and CAG; (L3) the second base of the anticodon is always C and the third base of the anticodon is always G; and (L4) The second base of the anticodon is always A and the third base is always G.

[0108] The translation composition of the present disclosure is not limited as long as it contains the tRNA of the present disclosure, but may also contain components necessary for translation and may contain the same components as the translation system of the present disclosure. The translation composition of the present disclosure may be a cell-free translation system or a reconstituted cell-free translation system. While not intended to be limiting, in some embodiments, the translation composition of the present disclosure may be a cell-free translation system reconstituted with factors derived from E. coli, and may include ribosomes, translation initiation factors, translation termination factors, translation elongation factors, amino acids, aminoacyl-tRNA synthetases (aaRSs), and the like. In some embodiments, the translation composition of the present disclosure may contain ribosomes derived from E. coli. In some embodiments, the tRNA of the present disclosure may be a tRNA derived from E. coli.

[0109] In some embodiments, a translation composition of the present disclosure may contain a complete set of tRNAs of the present disclosure. A translation composition of the present disclosure may contain 1, 2, 3, 4, 5, 6, 7, or 8 sets of a complete set of tRNAs of the present disclosure, or 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more sets, or 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer sets. In further embodiments, a translation composition of the present disclosure may contain tRNAs other than those described above.

[0110] In some embodiments, the set of tRNAs in the present disclosure contained in the translation composition may be tRNAs orthogonal to the aaRS when the translation composition contains an aaRS. A tRNA orthogonal to the aaRS refers to a tRNA that is not aminoacylated by the aaRS present in the translation composition but can be incorporated into the ribosome to translate an amino acid. Examples of such tRNAs include tRNA Glu2, tRNA AsnE2, tRNA Asp1, and tRNA Pro1E2, or tRNAs derived therefrom. If necessary, an aaRS that recognizes these tRNAs is excluded from the translation composition. In some embodiments, the translation composition of the present disclosure does not need to contain an aaRS capable of binding an amino acid to any of the set of tRNAs.

[0111] In some embodiments, the translation composition and translation kit of the present disclosure can reduce codon misreading. The translation composition and translation kit of the present disclosure can reduce the rate at which unintended amino acids are introduced into a template mRNA due to codon misreading. The translation composition and translation kit of the present disclosure can reduce the rate at which a codon contained in the template mRNA is translated by a tRNA having an anticodon that is not complementary to the codon. In some embodiments, the translation composition and translation kit of the present disclosure can reduce codon misreading by a tRNA in which the second and third letters of the anticodon are complementary to the second and first letters of the codon, respectively, and are capable of forming a Watson-Crick base pair, and in which the first letter of the anticodon is not complementary to the third letter of the codon. In some embodiments, this reduction in misreading allows the translation composition and translation kit of the present disclosure to assign two or more amino acids to the same codon box. In certain embodiments, it allows two or more unnatural amino acids to be assigned to the same codon box. For example, by reducing misreading of the codon CCG by a tRNA with the anticodon AGG, it becomes possible to assign an amino acid to the codon CCG while assigning a different amino acid to the codon complementary to the anticodon AGG (e.g., CCU), i.e., to assign two different amino acids to the same codon box (in this case, the CCM box). In this way, by reducing the frequency of incorrect amino acid translation due to codon misreading, which can occur when two or more amino acids are assigned to the same codon box, it is possible to increase the number of amino acids assigned to the genetic code table while achieving accurate peptide translation.

[0112] In some embodiments, the present disclosure allows for the modification of the properties of tRNA itself, providing greater convenience than methods such as adjusting the amount of aminoacyl-tRNA contained in a translation composition or translation kit or terminating the translation reaction midway. While not intending to be bound by any particular theory, attempts to increase the amount of aminoacyl-tRNA in a translation system to prevent codon misreading due to aminoacyl-tRNA depletion, for example, may result in misreading in the opposite direction. The translation composition and translation kit of the present disclosure are also useful in that they enable accurate peptide translation without increasing the amount of aminoacyl-tRNA in the translation system. In certain embodiments, the translation composition and translation kit of the present disclosure can be used in conjunction with techniques such as adjusting the amount of aminoacyl-tRNA or terminating the translation reaction midway to achieve a greater reduction in codon misreading. In one embodiment, in the translation composition and translation kit of the present disclosure, multiple tRNAs that bind different amino acids assigned to different codons in the same codon box are in an independent relationship that prevents misreading, i.e., an orthogonal relationship. In the translation systems of naturally occurring organisms, a strict correspondence between codons and amino acids is inherently established, and the addition of a non-orthogonal tRNA disrupts this correspondence, potentially resulting in a fatal impact on the function of the translation system. Therefore, in one embodiment of the present disclosure, the establishment of orthogonality between the multiple types of tRNAs can be an important feature.

[0113] In some embodiments, the translation composition and translation kit of the present disclosure enable multiple amino acids, particularly multiple amino acids including unnatural amino acids, to be assigned to a codon box to which only one amino acid is assigned in the natural genetic code, such as UCM, CUM, CCM, CGM, ACM, GUM, GCM, or GGM, while reducing codon misreading. The method of assigning amino acids is not particularly limited, and for example, a first amino acid may be assigned to the M1M2U or M1M2C codon and a second amino acid may be assigned to the M1M2A or M1M2G codon, or a first amino acid may be assigned to the M1M2U codon and a second amino acid may be assigned to the M1M2G codon.

[0114] In some embodiments, the translation composition and translation kit of the present disclosure may include at least one mRNA having a codon complementary to the anticodon of the second tRNA and / or at least one mRNA having a codon complementary to the anticodon of the first tRNA. In certain embodiments, the two codons may be present on the same mRNA or on different mRNAs. In some embodiments, the translation composition of the present disclosure may include multiple mRNAs with different sequences, or may include an mRNA library.

[0115] In some embodiments, the method for producing the composition for translation and the kit for translation of the present disclosure may include preparing the first and / or second tRNA by in vitro transcription. In some embodiments, the method for producing the composition for translation and the kit for translation of the present disclosure may include preparing the tRNA of the present disclosure by binding an amino acid to the tRNA outside of a translation system. In some embodiments, the method for producing the composition for translation and the kit for translation of the present disclosure may include the method for preparing the tRNA of the present disclosure described above. In some embodiments, the amino acid may be an unnatural amino acid.

[0116] In some embodiments, the method for producing a peptide of the present disclosure may include translating a template nucleic acid using the translation composition or translation kit of the present disclosure. The translation method is not limited to, but includes cell-free translation (in vitro translation), for example, translation using a reconstituted cell-free translation system, and more specifically, translation using a reconstituted cell-free translation system derived from Escherichia coli.

[0117] In some embodiments, a method for producing a peptide of the present disclosure may include translating an mRNA having a codon complementary to the anticodon of a second tRNA and / or an mRNA having a codon complementary to the anticodon of a first tRNA using a translation composition or translation kit of the present disclosure. In some embodiments, the two codons may be contained in the same mRNA or different mRNAs. In some embodiments, a method for producing a peptide of the present disclosure may include translating multiple types of nucleic acids having different sequences using a translation composition of the present disclosure, translating a nucleic acid library, or translating an mRNA library. The mRNA may encode a peptide having a desired or random amino acid sequence. Adding mRNA to a translation system of the present disclosure allows translation of the mRNA into a peptide. On the other hand, if the translation system contains an RNA polymerase for transcribing DNA into mRNA, adding DNA to the translation system of the present disclosure allows transcription of the DNA into mRNA and translation of the mRNA into a peptide to be performed simultaneously.

[0118] In some embodiments, the codon complementary to the anticodon may be a codon that forms a Watson-Crick base pair with all three bases of the anticodon, or a codon in which the third letter of the codon forms a wobble base pair with the first letter of the anticodon.

[0119] In some embodiments, the method for producing a peptide of the present disclosure may include assigning multiple amino acids to at least one, two or more, three or more, four or more, five or more, or six or more codon boxes selected from the group consisting of UCM, CUM, CCM, CGM, ACM, GUM, GCM, and GGM. Even in such cases, accurate peptide translation may be possible.

[0120] In some embodiments, the method for producing a peptide of the present disclosure can reduce misreading of a codon while assigning different types of amino acids to codons that exist in the same codon box and whose third letters are at least one combination selected from the group consisting of (i) to (iv) below: (i) U and G, (ii) C and G, (iii) U and A, and (iv) C and A. In some embodiments, the translation method of the present disclosure can reduce misreading of a codon while assigning different types of amino acids to codons that are UCM3, CUM3, CCM3, CGM3, ACM3, GUM3, GCM3, or GGM3 codons, where M3 is at least one combination selected from the group consisting of (i) to (iv).

[0121] In the present disclosure, the reduction of codon misreading by tRNA may be evaluated by translating one type of template mRNA using multiple tRNAs with different anticodons to which different amino acids are bound. The multiple tRNAs may include a tRNA with an anticodon complementary to a specific codon to be translated, and a tRNA with an anticodon complementary to a codon present in the same codon box as the codon but different from the anticodon.

[0122] For example, to evaluate misreading of the codon CCG by a tRNA with the anticodon AGG, a template mRNA containing the codon CCG is translated using a translation system containing a tRNA with the anticodon AGG and the amino acid AA1 bound thereto, and a tRNA with the anticodon CGG and the amino acid AA2 bound thereto. In this case, AA2, which is translationally introduced by the tRNA with the anticodon CGG, which is complementary to the codon CCG, is the target amino acid, and AA1, which is translationally introduced by the tRNA with the anticodon AGG, which is not complementary to the codon CCG, is the translated amino acid resulting from misreading. The proportion of such translation products can be used as an indicator to evaluate the reduction in misreading.

[0123] The template mRNA used for the evaluation may be selected from MR-1 to MR-7 described in the Examples, or other mRNA may be used depending on the codon to be evaluated. A prokaryotic-derived reconstituted cell-free protein synthesis system (e.g., the PURE system) may be used as the translation system for translating the template mRNA. Furthermore, the "translation condition 1" in the present disclosure may be used as the translation condition. More detailed translation and evaluation methods are described in the Examples.

[0124] In the present disclosure, the reduction of codon misreading by tRNA can be evaluated by the percentage (%) of misread peptides relative to the target molecule. The percentage is calculated using the following formula. If the percentage is lower than that of a control tRNA in which the base combinations at positions 32, 33, 37, and 38 are not modified, the tRNA being evaluated is determined to have the effect of reducing codon misreading. The reduction in the percentage is not particularly limited, and the tRNA of the present disclosure may show a reduction in the percentage of 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more compared to the control. In the present disclosure, the peptide obtained when a codon is correctly read may be referred to as the translation product or target molecule when a codon is correctly read.

number

[0125] The amount of translated peptide may be determined by the following method. Specifically, the translation product solution obtained after the translation reaction is diluted and analyzed using an LC-FLR-MS instrument. The dilution ratio is, for example, 10 times. The retention time of the target translated peptide is identified from the obtained MS data, and the fluorescence peak at the corresponding retention time is quantified to evaluate the amount of translated peptide. Quantification is performed by creating a calibration curve using a standard and calculating the content by relative quantification. LCT-67 or LCT-12 may be used as the standard. The sequence of LCT-67 is represented by BdpF:Thr:Phe:Ile:Ile:Gly:Phe:Ile:Ile:Ile:Pro:Ile:Gly (sequence number: 237), and the sequence of LCT-12 is represented by BdpF:Thr:Ile:Phe:Pro:Gly:Phe:Ile:Ile:Thr:Thr:Gly:Thr:Gly:Thr:Gly:Thr:Gly:Ala (sequence number: 238).

[0126] In one aspect, the present disclosure provides peptides and peptide libraries produced using the translation composition of the present disclosure. The peptides of the present disclosure include peptides obtained by post-translational chemical modification, and also peptide-nucleic acid complexes to which nucleic acids are linked.

[0127] Examples of post-translational chemical modifications include cyclization of linear peptides. Examples of bonds that can be used to form cyclic moieties include peptide bonds formed between amino and carboxyl groups. Other bonds that can be used include amide bonds, disulfide bonds, ether bonds, thioether bonds, ester bonds, thioester bonds, carbon-carbon bonds, alkyl bonds, alkenyl bonds, phosphonate ether bonds, azo bonds, amine bonds, C═NC bonds, lactam bridges, carbamoyl bonds, urea bonds, thiourea bonds, thioamide bonds, sulfinyl bonds, sulfonyl bonds, triazole bonds, and benzoxazole bonds, which are formed by combining appropriate functional groups. Carbon-carbon bonds can be formed by transition metal-catalyzed reactions such as the Suzuki reaction, Heck reaction, and Sonogashira reaction. In one embodiment, the peptides of the present disclosure contain at least one pair of functional groups capable of forming the above bonds within the molecule. The cyclic portion may be formed by carrying out a separate reaction to bond the above-mentioned functional groups together after a linear peptide is produced using the translation system of the present disclosure. For the synthesis of peptides having a cyclic portion, WO2013 / 100132, WO2012 / 026566, WO2012 / 033154, WO2012 / 074130, WO2015 / 030014, WO2018 / 052002, Comb Chem High Throughput Screen (2010) 13: 75-87, Nat Chem Biol (2009) 5: 502-507, Nat Chem Biol (2009) 5: 888-90, Bioconjug Chem (2007) 18: 469-476, ChemBioChem (2009) 10: 787-798, Chem Commun (Camb) (2011) 47: 9946-9958, etc. can also be referenced.

[0128] A method for identifying peptides that can bind to target molecules from a peptide library (display library) using peptide-nucleic acid complexes is known. A display library is a library characterized by the association of phenotype and genotype by forming a complex between peptides and the nucleic acids that encode them. Examples of major display libraries include mRNA display (Roberts and Szostak, Proc Natl Acad Sci USA (1997) 94: 12297-12302), in vitro virus display (Nemoto et al., FEBS Lett (1997) 414: 405-408), cDNA display (Yamaguchi et al., Nucleic Acids Res (2009) 37: e108), ribosome display (Mattheakis et al., Proc Natl Acad Sci USA (1994) 91: 9022-9026), covalent display (Reiersen et al., Nucleic Acids Res (2005) 33: e10), and CIS display (Odegrip et al., Proc Natl Acad Sci USA (2004) 101: Examples of display libraries include libraries prepared using in vitro compartmentalization methods (Tawfik and Griffiths, Nat Biotechnol (1998) 16: 652-656).

[0129] In one aspect, the present disclosure provides a method for identifying a peptide having binding activity toward a target molecule, the method comprising contacting the target molecule with a peptide library described herein. The target molecule is not particularly limited and can be appropriately selected from, for example, low molecular weight compounds, high molecular weight compounds, nucleic acids, peptides, proteins, sugars, lipids, and the like. The target molecule may be an extracellular molecule or an intracellular molecule. Alternatively, it may be a molecule present in the cell membrane, in which case the target may be any of the extracellular domain, transmembrane domain, and intracellular domain. In the step of contacting the peptide library with the target molecule, the target molecule is typically immobilized on a solid support (e.g., a microtiter plate or microbeads). Subsequently, peptides that do not bind to the target molecule are removed, and only peptides that bind to the target molecule are recovered, thereby selectively enriching peptides having binding activity toward the target molecule (panning method). When the peptide library used is a nucleic acid display library, the recovered peptides are bound to nucleic acids encoding their genetic information, and the nucleic acid and amino acid sequences encoding the recovered peptides can be easily identified by isolating and analyzing them. Furthermore, based on the obtained nucleic acid sequence or amino acid sequence, the identified peptides can be individually produced by chemical synthesis or genetic recombination techniques.

[0130] In one aspect, the present disclosure provides a method for reducing codon misreading by a tRNA, and compositions and kits for reducing codon misreading by a tRNA. Such compositions and kits may include a tRNA of the present disclosure. The method may also include obtaining a tRNA of the present disclosure by modifying the tRNA. Specifically, the method for reducing misreading of a second codon by a tRNA includes substituting at least one base selected from the group consisting of bases 32, 33, 37, and 38 of a tRNA having an anticodon complementary to a first codon, wherein the tRNA after the substitution is a tRNA of the present disclosure, and the first letter of the first codon and the second codon may be the same base, the second letter of the first codon and the second codon may be the same base, and the third letter of the first codon and the second codon may be different bases.

[0131] In some embodiments, in the method for reducing codon misreading of the present disclosure, the first codon is M1M2X and the second codon is M1M2Y, wherein M1 and M2 are each independently A, C, G, or U, and X and Y may be different bases selected from A, C, G, and U. The combination of bases of X and Y may be any one selected from the group consisting of the following (a1) to (a8): (a1) U and G; (a2) G and U; (a3) ​​U and A; (a4) A and U; (a5) C and A; (a6) A and C; (a7) C and G; and (a8) G and C. In the present disclosure, the combination of bases of X and Y may be any one selected from the group consisting of M 31 and M 32 The term "X" means that the X is a base M 31 and Y is a base M 32In one embodiment, the combination of bases of X and Y may be any one selected from the group consisting of (a1) to (a3), (a5) and (a7), or the group consisting of (a1) and (a2). In some embodiments, M1M2 may be any one of the base sequences selected from the group consisting of the following (b1) to (b8): (b1) CC; (b2) CU; (b3) GG; (b4) GU; (b5) GC; (b6) UC; (b7) CG; and (b8) AC. In one embodiment, M1M2 may be any one of the base sequences selected from the group consisting of (b1) to (b3).

[0132] In some embodiments, in the method of reducing codon misreading of the present disclosure, the first codon and the anticodon of the tRNA may form Watson-Crick base pairs at all three bases, or the third base of the first codon and the first base of the anticodon of the tRNA may form a wobble base pair.

[0133] In some embodiments, the method for reducing codon misreading of the present disclosure may be a method for reducing misreading of a codon complementary to the anticodon of a second tRNA of the present disclosure by a first tRNA of the present disclosure. In some embodiments, the method for reducing codon misreading of the present disclosure may include the method for preparing a tRNA of the present disclosure described above. [Example]

[0134] The present invention will be further illustrated by, but not limited to, the following examples. In the examples, the following abbreviations are used: AA Ammonium Acetate CH2CN cyanomethyl group DBU 1,8-diazabicyclo[5.4.0]-7-undecene DCM dichloromethane DIC N,N-diisopropylcarbodiimide DIPEA N,N-Diisopropylethylamine DMF Dimethylformamide DMSO dimethyl sulfoxide FA formic acid Fmoc 9-fluorenylmethyloxycarbonyl group F-Pnaz 4-(2-(4-fluorophenyl)acetamido)benzyloxycarbonyl group: TIFF0007744244000004.tif25170HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol MeCN acetonitrile NMP N-methyl-2-pyrrolidone TEA Triethylamine TFA trifluoroacetic acid TFE 2,2,2-trifluoroethanol THF tetrahydrofuran

[0135] Furthermore, the following abbreviations were used in the present examples: Gly or G (glycine), Ile or I (isoleucine), Leu or L (leucine), Phe or F (phenylalanine), Pro or P (proline), and Thr or T (threonine). In addition, the abbreviations listed in Table 3 were used in the present disclosure. In the amino acid sequences in the present disclosure, BdpFL-Phe may be abbreviated as "BdpF."

[0136] [Table 3]

[0137] The LCMS analysis conditions are shown in Table 4 below.

[0138] [Table 4]

[0139] Example 1. Synthesis of aminoacyl pCpA Aminoacyl pCpAs (SS14, SS15, SS16, SS45) were synthesized according to the following scheme. TIFF0007744244000007.tif184170

[0140] Synthesis of (S)-1-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)piperidine-2-carboxylic acid (compound SS17, F-Pnaz-Pic2-OH) TIFF0007744244000008.tif23170

[0141] Under a nitrogen atmosphere, a mixture of (S)-piperidine-2-carboxylic acid (42.6 mg, 0.33 mmol) and (4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl carbonate (compound ts11) (140 mg, 0.44 mmol) synthesized by the method described in patent document (WO2018143145A1) was added to DMF (330 μL) at room temperature. After stirring at room temperature for 5 minutes, triethylamine (105.6 μL, 2.25 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for 30 minutes and then purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to give (S)-1-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)piperidine-2-carboxylic acid (compound SS17, F-Pnaz-Pic2-OH) (92 mg, 67%). LCMS(ESI) m / z = 413 (M−H)- Retention time: 0.70 minutes (Analysis conditions SQDFA05_01)

[0142] (S)-Piperidine-1,2-dicarboxylic acid 1-(4-(2-(4-fluorophenyl)acetamido)benzyl) 2-(cyanomethyl) (compound SS18, F-Pnaz-Pic2-OCH 2 Synthesis of CN TIFF0007744244000009.tif34170

[0143] Under a nitrogen atmosphere, ((S)-1-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)piperidine-2-carboxylic acid (compound SS17, F-Pnaz-Pic2-OH) (30 mg, 0.072 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (20.23 μL, 0.116 mmol) were dissolved in acetonitrile (90 μL), and 2-bromoacetonitrile (5.34 μL, 0.080 mmol) was added at 0° C., followed by stirring at room temperature for 2 hours. The reaction mixture was concentrated to give the crude product (S)-piperidine-1,2-dicarboxylate 1-(4-(2-(4-fluorophenyl)acetamido)benzyl) 2-(cyanomethyl) (Compound SS18, F-Pnaz-Pic-OCHCN) was obtained. The obtained crude product was dissolved in acetonitrile (2.00 mL) and used directly in the next step. LCMS(ESI) m / z = 452 (M−H) Retention time: 0.79 minutes (Analysis conditions SQDFA05_01)

[0144] Synthesis of (2S)-piperidine-1,2-dicarboxylic acid 1-(4-(2-(4-fluorophenyl)acetamido)benzyl) 2-((2R,3S,4R,5R)-2-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl) (compound SS14, F-Pnaz-Pic2-pCpA) TIFF0007744244000010.tif234170

[0145] In buffer solution A (40 mL) was dissolved ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl dihydrogenphosphate (compound pc01) (113 mg, 0.156 mmol), which had been synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297-310), and 1-(4-(2-(4-fluorophenyl)acetamido)benzyl (S)-piperidine-1,2-dicarboxylate was added. A solution of 2-(cyanomethyl) (compound SS18, F-Pnaz-Pic2-OCH2CN) (35.4 mg, 0.078 mmol) in acetonitrile (2.00 mL) was added and stirred at room temperature for 150 minutes. The reaction solution was cooled to 0°C, and then trifluoroacetic acid (2.00 mL) was added. After stirring at 0°C for 45 minutes, the reaction solution was purified by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (compound SS14, F-Pnaz-Pic2-pCpA) (6.0 mg, 7.3%). LCMS(ESI) m / z = 1047.5 (M−H) Retention time: 0.50 minutes (Analysis conditions SQDFA05_01)

[0146] Buffer A was prepared as follows. Acetic acid was added to an aqueous solution of N,N,N-trimethylhexadecan-1-aminium chloride (6.40 g, 20 mmol) and imidazole (6.81 g, 100 mmol) to obtain Buffer A (1 L) with a pH of 8 and a concentration of 20 mM N,N,N-trimethylhexadecan-1-aminium and 100 mM imidazole.

[0147] Synthesis of O-(2-chlorophenyl)-N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-L-serine (compound SS19, F-Pnaz-SPh2Cl-OH) TIFF0007744244000011.tif36170

[0148] Under a nitrogen atmosphere, DMSO (15 mL) and triethylamine (0.95 g, 9.42 mmol) were added to a mixture of O-(2-chlorophenyl)-L-serine (compound aa63) (1.25 g, 5.80 mmol) synthesized by the method described in patent document (WO2018225864) and carbonate-(4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl (compound ts11) (2 g, 4.71 mmol) synthesized by the method described in patent document (WO2018143145A1) at room temperature. The reaction mixture was stirred at room temperature for 16 hours and then purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to give O-(2-chlorophenyl)-N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-L-serine (compound SS19, F-Pnaz-SPh2Cl-OH) (1.8 g, 73%). LCMS(ESI) m / z = 523 (M+Na)+ Retention time: 1.26 minutes (Analysis conditions SMD method 1)

[0149] Cyanomethyl O-(2-chlorophenyl)-N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-L-serinate (compound SS20, F-Pnaz-SPh2Cl-OCH 2 Synthesis of CN TIFF0007744244000012.tif36170

[0150] Under a nitrogen atmosphere, O-(2-chlorophenyl)-N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-L-serine (compound SS19, F-Pnaz-SPhCl-OH) (800 mg, 1.60 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (0.412 g, 3.19 mmol) were dissolved in DCM (15 mL), and 2-bromoacetonitrile (760 mg, 6.34 mmol) was added at room temperature, followed by stirring at room temperature for 16 hours. The reaction mixture was concentrated and purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to give cyanomethyl O-(2-chlorophenyl)-N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-L-serinate (compound SS20, F-Pnaz-SPhCl-OCHCN) (220 mg, 26%). The resulting product was dissolved in acetonitrile (5 mL) and used in the next step. LCMS(ESI) m / z = 562 (M+Na)+ Retention time: 1.15 minutes (Analysis conditions SMD method 2)

[0151] Synthesis of (2R,3S,4R,5R)-2-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl O-(2-chlorophenyl)-N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-L-serinate (Compound SS15, F-Pnaz-SPh2Cl-pCpA) TIFF0007744244000013.tif234170

[0152] In Buffer A (100 mL), ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl dihydrogen phosphate (Compound pc01) (400 mg, 0.55 mmol) was dissolved, and cyanomethyl A solution of O-(2-chlorophenyl)-N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-L-serinate (compound SS20, F-Pnaz-SPh2Cl-OCH2CN) (220 mg, 0.41 mmol) in acetonitrile (5 mL) was added dropwise over 15 minutes using a syringe pump, followed by stirring at room temperature for 5 minutes. Subsequently, trifluoroacetic acid (2.3 mL) was added to the reaction mixture. The reaction mixture was lyophilized and then purified by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to yield the title compound (compound SS15, F-Pnaz-SPh2Cl-pCpA) (20.7 mg, 2%). LCMS(ESI) m / z = 1133.4 (M−H)- Retention time: 0.55 minutes (Analysis conditions SQDFA05_01)

[0153] Synthesis of (S)-2-(methylamino)-4-phenylbutanoic acid (compound SS21, MeHph-OH) TIFF0007744244000014.tif31170

[0154] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (compound aa11) (150 mg, 0.361 mmol) synthesized by the method described in patent document (WO2018225864) DCM (903 μL), water (903 μL) and piperidine (178 μL, 1.805 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 30 minutes and then purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain ((S)-2-(methylamino)-4-phenylbutanoic acid (compound SS21, MeHph-OH) (55 mg, 79%). LCMS(ESI) m / z = 192 (M−H)- Retention time: 0.15 minutes (Analysis conditions SQDFA05_02)

[0155] Synthesis of (S)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (compound SS22, F-Pnaz-MeHph-OH) TIFF0007744244000015.tif36170

[0156] Under a nitrogen atmosphere, DMSO (727 μL) was added to a mixture of ((S)-2-(methylamino)-4-phenylbutanoic acid (compound SS21, MeHph-OH) (35.1 mg, 0.182 mmol) and (4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl carbonate (compound ts11) (85 mg, 0.20 mmol) synthesized by the method described in patent document (WO2018143145A1) at room temperature. Triethylamine (76 μL) , 0.545 mmol) was added at 50° C. The reaction mixture was stirred at 40° C. for 16 hours and then purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain (S)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (compound SS22, F-Pnaz-MeHph-OH) (80 mg, 92%). LCMS(ESI) m / z = 477 (M−H)- Retention time: 0.85 minutes (Analysis conditions SQDFA05_02)

[0157] (S)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)(methyl)amino)-4-phenylbutanoate cyanomethyl ester (compound SS23, F-Pnaz-MeHph-OCH 2 Synthesis of CN TIFF0007744244000016.tif36170

[0158] Under a nitrogen atmosphere, acetonitrile (533 μL) was added to a mixture of (S)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (compound SS22, F-Pnaz-MeHph-OH) (77 mg, 0.16 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (31 μL, 0.176 mmol) at room temperature. 2-Bromoacetonitrile (86 μL, 1.280 mmol) was then added at room temperature, and the reaction mixture was stirred at 40° C. for 1 hour. The reaction mixture was concentrated to obtain the crude product, cyanomethyl (S)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)(methyl)amino)-4-phenylbutanoate (compound SS23, F-Pnaz-MeHph-OCHCN). The obtained crude product was dissolved in acetonitrile (5.00 mL) and used as it was in the next step. LCMS(ESI) m / z = 516 (M−H)- Retention time: 0.92 minutes (Analysis conditions SQDFA05_02)

[0159] Synthesis of (2S)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (2R,3S,4R,5R)-2-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl (compound SS16, F-Pnaz-MeHph-pCpA) TIFF0007744244000017.tif234170

[0160] In Buffer A (100 mL) was added dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl (S)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)(methyl)amino)-4-phenylbutanoate cyanomethyl (Compound pc01) (127 mg, 0.176 mmol) was dissolved in the solution, and an acetonitrile solution (5.00 mL) of (S)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)(methyl)amino)-4-phenylbutanoate cyanomethyl (Compound SS23, F-Pnaz-MeHph-OCHCN) (83 mg, 0.16 mmol) was added, followed by stirring at room temperature for 1 hour. The reaction solution was cooled to 0°C, and then trifluoroacetic acid (5.00 mL) was added. After stirring the reaction mixture at 0°C for 1 hour, the mixture was purified by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid solution / 0.05% trifluoroacetic acid-acetonitrile solution) and then further purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid-acetonitrile solution) to obtain the title compound (Compound SS16, F-Pnaz-MeHph-pCpA) (26 mg, 14.6%). LCMS(ESI) m / z = 1111.5 (M−H)- Retention time: 0.64 minutes (Analysis conditions SQDFA05_02)

[0161] Synthesis of N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-N-methylglycine (compound SS46, F-Pnaz-MeG-OH) TIFF0007744244000018.tif22170

[0162] Under a nitrogen atmosphere, a mixture of sarcosine (483 mg, 5.42 mmol), 4-(4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl carbonate (compound ts11) (2.0 g, 4.71 mmol) synthesized by the method described in patent document (WO2018143145A1) was added at room temperature with DMSO (15 mL) and triethylamine (953.4 mg, 9.42 mmol). The reaction mixture was stirred at room temperature for 16 hours and then purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-N-methylglycine (compound SS46, F-Pnaz-MeG-OH) (1.4 g, 79%). LCMS(ESI) m / z = 397 (M+Na)+ Retention time: 0.88 minutes (Analysis conditions SMD method 3)

[0163] Cyanomethyl N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-N-methylglycinate (compound SS47, F-Pnaz-MeG-OCH 2 Synthesis of CN TIFF0007744244000019.tif22170

[0164] Under a nitrogen atmosphere, N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-N-methylglycine (compound SS46, F-Pnaz-MeG-OH) (1.38 g, 3.69 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (0.95 g, 7.38 mmol) were dissolved in DMF (28 mL), and 2-bromoacetonitrile (1.74 g, 14.75 mmol) was added at room temperature. The mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated and purified by normal-phase silica gel column chromatography (ethyl acetate / petroleum ether) to give cyanomethyl N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-N-methylglycinate (compound SS47, F-Pnaz-MeG-OCHCN) (1.2 g, 79%). LCMS(ESI) m / z = 436 (M+Na)+ Retention time: 0.70 minutes (Analysis conditions SMD method 4)

[0165] Synthesis of (2R,3S,4R,5R)-2-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-N-methylglycinate (compound SS45, F-Pnaz-MeG-pCpA) TIFF0007744244000020.tif234170

[0166] In buffer solution A (100 mL) was dissolved (422 mg, 0.58 mmol) of ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl dihydrogenphosphate (compound pc01) synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297-310), and the resulting solution was treated with cyanomethyl A solution of N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-N-methylglycinate (compound SS47, F-Pnaz-MeG-OCHCN) (120.7 mg, 0.29 mmol) in acetonitrile (5 mL) was added dropwise over 15 minutes using a syringe pump, and the mixture was stirred at room temperature for 5 hours. Trifluoroacetic acid (2.3 mL) was added to the reaction mixture, and the reaction mixture was lyophilized. The mixture was then purified by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to give the title compound (compound SS45, F-Pnaz-MeG-pCpA) (76.7 mg, 26%). LCMS(ESI) m / z = 1007.5 (M−H)- Retention time: 0.48 minutes (Analysis conditions SQDFA05_02)

[0167] Example 2. Synthesis of BdpFL-Phe-pCpA (MT01) Synthesis of (3-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)propanoyl)-L-phenylalanine (compound MT02, BdpFL-Phe-OH) TIFF0007744244000021.tif38170

[0168] Under a nitrogen atmosphere, a solution of 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-4-ium (200 mg, 0.685 mmol) and 1-hydroxypyrrolidine-2,5-dione (87 mg, 0.753 mmol) in NMP (4.5 mL) was added with DIC (0.128 mL, 0.822 mmol) at room temperature and stirred overnight at 40 °C. After returning to room temperature, L-phenylalanine (113 mg, 0.685 mmol) and TEA (0.191 mL, 1.369 mmol) were added to the reaction mixture and stirred overnight at 40 °C. The reaction mixture was purified by reverse-phase column chromatography (0.1% FA MeCN / HO) to obtain (3-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)propanoyl)-L-phenylalanine (compound MT02, BdpFL-Phe-OH) (102 mg, 34% yield). LCMS(ESI) m / z = 438.3 (M−H)- Retention time: 0.78 minutes (Analysis conditions SQDFA05_02)

[0169] (3-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)propanoyl)-L-phenylalanine cyanomethyl ester (compound MT03, BdpFL-Phe-OCH 2 Synthesis of CN TIFF0007744244000022.tif40170

[0170] Under a nitrogen atmosphere, (3-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)propanoyl)-L-phenylalanine (50 mg, 0.114 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (31.0 μL, 0.177 mmol) were dissolved in acetonitrile (500 μL), and 2-bromoacetonitrile (12 μL, 0.177 mmol) was added at 0°C. The mixture was then stirred at 40°C for 3 hours. The reaction mixture was concentrated to give (3-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)propanoyl)-L-phenylalanine cyanomethyl ester (compound MT03, BdpFL-Phe-OCH2CN) as a crude product, which was used directly in the next step. LCMS(ESI) m / z = 477.3(MH)- Retention time: 0.86 minutes (Analysis conditions SQDFA05_01)

[0171] 3-(3-(((2S)-1-(((2R,3S,4R,5R)-2-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl)oxy)-1-oxo-3-phenylpropan-2-yl)amino)-3-oxopropyl)-5,5-difluoro-7,9-dimethyl-5H-5λ4-dipyrrolo Synthesis of [1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium (compound MT01, BdpFL-Phe-pCpA) TIFF0007744244000023.tif134170

[0172] In Buffer A (11.3 mL), ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl dihydrogen phosphate (Compound pc01) (33.2 mg, 0.046 mmol) was dissolved, and (3-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborin-3-yl)propanoyl)-L-phenylalanine A solution (0.13 mL) of the cyanomethyl ester (compound MT03, BdpFL-Phe-OCHCN) (11 mg, 0.023 mmol) in acetonitrile was added, followed by stirring at room temperature for 45 minutes. TFA (0.56 mL) was added to the reaction mixture at 0°C, and the mixture was stirred for 5 minutes, followed by stirring at room temperature for 10 minutes. The reaction mixture was purified by reverse-phase silica gel column chromatography (0.05% TFA MeCN / HO) to obtain the title compound (compound MT01, BdpFL-Phe-pCpA) (2.1 mg, 8.5% yield). LCMS(ESI) m / z = 1072.5 (M−H)- Retention time: 0.56 minutes (Analysis conditions SQDFA05_02)

[0173] Synthesis of lysidine-diphosphate for introducing a lysidine unit into the 3' end of tRNA fragments by ligation We improved the synthesis of lysidine diphosphate, which is used to introduce a lysidine unit to the 3' end of tRNA fragments by ligation. Specifically, lysidine-diphosphate (SS04, pLp) was synthesized according to the following scheme. TIFF0007744244000024.tif234170

[0174] Synthesis of ((3aR,4R,12R,12aR)-2,2-dimethyl-3a,4,12,12a-tetrahydro-5H,8H-4,12-epoxy[1,3]dioxolo[4,5-e]pyrimido[2,1-b][1,3]oxazocin-8-ylidene)benzyl carbamate (compound SS24) TIFF0007744244000025.tif44170

[0175] Under a nitrogen atmosphere, DCM (17.2 mL) was added to a mixture of the known compound 2',3'-O-isopropylidene-4-N-(benzyloxycarbonyl)-cytidine (718.2 mg, 1.72 mmol) and triphenylphosphine (474 ​​mg, 1.81 mmol) at room temperature (Antiviral Chemistry & Chemotherapy, 2003, 14(4), 183-194). The mixture was cooled in an ice bath, and then diisopropyl azodicarboxylate (385 μL, 1.98 mmol) was added. The mixture was then warmed to room temperature and stirred at room temperature for 1.5 hours. The reaction mixture was concentrated, and toluene (20 mL) was added. The resulting precipitate was collected by filtration. The resulting solid was washed three times with toluene to give ((3aR,4R,12R,12aR)-2,2-dimethyl-3a,4,12,12a-tetrahydro-5H,8H-4,12-epoxy[1,3]dioxolo[4,5-e]pyrimido[2,1-b][1,3]oxazocin-8-ylidene)benzyl carbamate (compound SS24) (525.7 mg, 76%). LCMS(ESI) m / z = 400.3 (M+H)+ Retention time: 0.48 minutes (Analysis conditions SQDFA05_02)

[0176] Synthesis of benzyl (2S)-6-[[1-[(3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate;2,2,2-trifluoroacetic acid (compound SS25) TIFF0007744244000026.tif83170

[0177] Under a nitrogen atmosphere, a mixture of ((3aR,4R,12R,12aR)-2,2-dimethyl-3a,4,12,12a-tetrahydro-5H,8H-4,12-epoxy[1,3]dioxolo[4,5-e]pyrimido[2,1-b][1,3]oxazocin-8-ylidene)benzylcarbamate (Compound SS24) (300 mg, 0.75 mmol) and lithium chloride (159 mg, 3.76 mmol) was added to THF (7.5 mL) at room temperature and cooled in an ice bath. To this mixture was added a mixture of benzyl ((benzyloxy)carbonyl)-L-lysinate benzenesulfonate (813 mg, 2.01 mmol) and DBU (673 μL, 4.51 mmol) in THF (7.5 mL) under ice bath conditions, and the reaction mixture was stirred at 0 °C for 30 minutes. DMSO was added to the reaction mixture in an ice bath, and the mixture was warmed to room temperature. The reaction mixture was concentrated to remove THF. The residue was purified by reverse-phase silica gel column chromatography (0.05% TFA aqueous solution / 0.05% TFA acetonitrile solution) to give benzyl (2S)-6-[[1-[(3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate; 2,2,2-trifluoroacetic acid (compound SS25) (726.6 mg) in quantitative yield. LCMS(ESI) m / z = 768.6 (M−H) Retention time: 0.74 minutes (Analysis conditions SQDFA05_02)

[0178] Synthesis of benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate;2,2,2-trifluoroacetic acid (compound SS26) TIFF0007744244000027.tif83170

[0179] Benzyl (2S)-6-[[1-[(3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate; 2,2,2-trifluoroacetic acid (compound SS25) (281.1 mg, 0.318 mmol) was dissolved in a mixture of TFA (4.24 mL) and ultrapure water (2.12 mL) while cooling in an ice bath, and the mixture was stirred at room temperature for 50 minutes. Toluene and acetonitrile were added, and the reaction solution was concentrated. This operation was repeated several times to distill off water and TFA, and the crude product, benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate; 2,2,2-trifluoroacetic acid (Compound SS26) (272.6 mg), was obtained. The resulting crude product, benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate; 2,2,2-trifluoroacetic acid (Compound SS26), was used directly in the next step. LCMS(ESI) m / z = 728.5 (M−H) Retention time: 0.69 minutes (Analysis conditions SQDFA05_02)

[0180] Synthesis of benzyl (2S)-6-[[1-[(4aR,6R,7R,7aS)-2,2-ditert-butyl-7-hydroxy-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxacillin-6-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate;2,2,2-trifluoroacetic acid (Compound SS27) TIFF0007744244000028.tif83170

[0181] Under a nitrogen atmosphere, the crude product obtained in the previous step, benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate; 2,2,2-trifluoroacetic acid (Compound SS26) (258 mg, 0.306 mmol) was dissolved in DMF (3.06 mL), and the mixture was cooled in an ice bath. After that, di-tert-butylsilyl bis(trifluoromethanesulfonate) (396 μL, 1.22 mmol) was added and the mixture was stirred in an ice bath for 2 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture in an ice bath, and the resulting mixture was purified by reverse-phase silica gel column chromatography (0.05% aqueous TFA / 0.05% TFA acetonitrile solution) to give benzyl (2S)-6-[[1-[(4aR,6R,7R,7aS)-2,2-ditert-butyl-7-hydroxy-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxacillin-6-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate; 2,2,2-trifluoroacetic acid (Compound SS27) (234.0 mg, 78%, 2 steps). LCMS(ESI) m / z = 868.8 (M−H) Retention time: 0.88 minutes (Analysis conditions SQDFA05_02)

[0182] Synthesis of benzyl (2S)-6-[[1-[(4aR,6R,7R,7aR)-2,2-ditert-butyl-7-tetrahydropyran-2-yloxy-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxacillin-6-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate;2,2,2-trifluoroacetic acid (Compound SS28) TIFF0007744244000029.tif83170

[0183] Under a nitrogen atmosphere, benzyl (2S)-6-[[1-[(4aR,6R,7R,7aS)-2,2-ditert-butyl-7-hydroxy-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxacillin-6-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate; 2,2,2-trifluoroacetic acid (Compound SS27) (30 mg, 0.03 mmol) and TFA (6.98 μL, 0.09 mmol) were dissolved in DCM (610 μL) at room temperature, and 3,4-dihydro-2H-pyran (83 μL, 0.915 mmol) was added. The reaction mixture was stirred at room temperature for 13 hours, and then toluene was added. The reaction mixture was concentrated to obtain the crude product, benzyl (2S)-6-[[1-[(4aR,6R,7R,7aR)-2,2-ditert-butyl-7-tetrahydropyran-2-yloxy-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxacillin-6-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate; 2,2,2-trifluoroacetic acid (compound SS28), as a mixture of diastereomers derived from the asymmetric carbon on the THP protection. The resulting crude product, benzyl (2S)-6-[[1-[(4aR,6R,7R,7aR)-2,2-ditert-butyl-7-tetrahydropyran-2-yloxy-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxacillin-6-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate; 2,2,2-trifluoroacetic acid (Compound SS28), was used directly in the next step. LCMS(ESI) m / z = 952.8 (M−H) Retention time: 3.17 minutes, 3.38 minutes (Analysis conditions SQDAA50long)

[0184] Synthesis of benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-tetrahydropyran-2-yloxy-tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate (compound SS29) TIFF0007744244000030.tif83170

[0185] Under a nitrogen atmosphere, the crude product obtained in the previous step, benzyl (2S)-6-[[1-[(4aR,6R,7R,7aR)-2,2-ditert-butyl-7-tetrahydropyran-2-yloxy-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl]-4-(benzyloxycarbonylamino)pyrimidin-2-ylidene]amino]-2-(benzyloxycarbonylamino)hexanoate;2,2,2-trifluoroacetic acid (compound SS28), was dissolved in THF (610 μL) at room temperature, and tetrabutylammonium fluoride (~1 mol / L tetrahydrofuran solution) (305 μL, ~0.305 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. DMSO was added to the reaction mixture, and the mixture was concentrated to remove the THF. The residue was purified by reverse-phase silica gel column chromatography (10 mM AA aqueous solution / 10 mM AA acetonitrile solution) to give benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-tetrahydropyran-2-yloxy-tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate (compound SS29) (21.51 mg, 87%, 2 steps) as a mixture of diastereomers derived from the asymmetric carbon on the THP protection. LCMS(ESI) m / z = 812.7 (M−H) Retention time: 1.74 minutes (Analysis conditions SQDAA50long)

[0186] Synthesis of benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4R,5R)-4-dibenzyloxyphosphoryloxy-5-(dibenzyloxyphosphoryloxymethyl)-3-tetrahydropyran-2-yloxy-tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate (Compound SS30) TIFF0007744244000031.tif170170

[0187] Under a nitrogen atmosphere, benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-tetrahydropyran-2-yloxy-tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate (compound SS29) (21.51 mg, 0.026 mmol) and 1H-tetrazole (22.22 mg, 0.317 mmol) were dissolved in acetonitrile (1.06 mL) at room temperature, and dibenzyl N,N-diisopropylphosphoramidite (53.2 μL, 0.159 mmol) was added and the mixture was stirred at room temperature for 1 hour. Dess-Martin periodinane (135 mg, 0.317 mmol) was added and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was then purified by reverse-phase silica gel column chromatography (10 mM AA aqueous solution / 10 mM AA acetonitrile solution) to quantitatively obtain benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4R,5R)-4-dibenzyloxyphosphoryloxy-5-(dibenzyloxyphosphoryloxymethyl)-3-tetrahydropyran-2-yloxy-tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate (compound SS30) (36.59 mg, 2 steps) as a mixture of diastereomers derived from the asymmetric carbon of the THP protection. LCMS(ESI) m / z = 1332.8 (M−H)- Retention time: 3.08 minutes, 3.11 minutes (Analysis conditions SQDAA50long)

[0188] Synthesis of (2S)-2-amino-6-[[4-amino-1-[(2R,3R,4S,5R)-3-hydroxy-4-phosphonooxy-5-(phosphonooxymethyl)tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoic acid (compound SS04, pLp) TIFF0007744244000032.tif151170

[0189] Benzyl (2S)-2-(benzyloxycarbonylamino)-6-[[4-(benzyloxycarbonylamino)-1-[(2R,3R,4R,5R)-4-dibenzyloxyphosphoryloxy-5-(dibenzyloxyphosphoryloxymethyl)-3-tetrahydropyran-2-yloxy-tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoate (Compound SS30) (36.59 mg, 0.027 mmol) was dissolved in a mixed solvent of methanol (649 μL) and ultrapure water (152 μL) at room temperature, and palladium / carbon (Pd 10%) (5.84 mg, 5.48 μmol) was added under a nitrogen atmosphere. The mixture was stirred at room temperature for 18 hours under a hydrogen atmosphere. The reaction mixture was filtered through Celite and washed several times with ultrapure water. To the resulting filtrate (24.66 mL), 1 mol / L hydrochloric acid (2.74 mL, 2.74 mmol) was added and the mixture was allowed to stand at room temperature for 1 hour. The reaction mixture was filtered through Celite and washed several times with ultrapure water. The filtrate was lyophilized, and the resulting powder was dissolved again in ultrapure water (1.52 mL). The mixture was centrifuged and the supernatant was collected to obtain an aqueous solution of (2S)-2-amino-6-[[4-amino-1-[(2R,3R,4S,5R)-3-hydroxy-4-phosphonooxy-5-(phosphonooxymethyl)tetrahydrofuran-2-yl]pyrimidin-2-ylidene]amino]hexanoic acid (compound SS04, pLp) (1.37 mL, 17.47 mM, 87%, 2 steps). LCMS(ESI) m / z = 530.1 (M−H) Retention time: 1.60 minutes (Analysis conditions LTQTEA / HFIP05_02)

[0190] Example 3 Example 3-1. Synthesis of peptide (LCT-67) having BdpFL at the N-terminus TIFF0007744244000033.tif76170

[0191] Peptide elongation was carried out on a peptide synthesizer using 2-chlorotriethyl resin (100 mg) loaded with Fmoc-Gly-OH. The Fmoc amino acids were Fmoc-Gly-OH, Fmoc-Thr(THP)-OH (aa01), Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-MePhe-OH, and Fmoc-Pro-OH (synthesized by the method described in patent document (WO2018225864)). The amino acid abbreviations are described elsewhere in this specification. Peptide elongation was carried out according to the Fmoc peptide synthesis method (WO2013100132B2). After peptide elongation, the N-terminal Fmoc group was removed on the peptide synthesizer, and the resin was washed with DCM. The resin was added with TFE / DCM (1:1, v / v, 2 mL) and shaken for 1 hour to cleave the peptide from the resin. After completion of the reaction, the solution in the tube was filtered through a synthesis column to remove the resin, which was then washed twice with TFE / DCM (1:1, v / v, 1 mL). All extracts were combined, DMF (2 mL) was added, and the mixture was concentrated under reduced pressure. The resulting residue was dissolved in NMP (1 mL) and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid N-succinimidyl ester (5 mg, 0.013 mmol) was added at room temperature. After stirring for 19 hours, the reaction mixture was subjected to reverse-phase silica gel column chromatography (0.1% FA MeCN / HO), and the fraction containing the intermediate was concentrated under reduced pressure. The resulting residue was dissolved in 5% TFA in DCM (2 mL) and stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase silica gel column chromatography (0.1% FA MeCN / HO) to obtain the title compound (LCT-67) (13 mg). The amino acid sequence of LCT-67 is shown in SEQ ID NO: 237. LCMS(ESI) m / z = 1751.2(MH)- Retention time: 0.97 minutes (Analysis conditions SQDFA05_02)

[0192] Example 3-2 Synthesis of a peptide (LCT-12) having BdpFL at the N-terminus TIFF0007744244000034.tif234170

[0193] Peptide elongation was carried out on a peptide synthesizer using 2-chlorotriethyl resin (100 mg) loaded with Fmoc-Ala-OH. The Fmoc amino acids were Fmoc-Gly-OH, Fmoc-Thr(THP)-OH (aa01), Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Pro-OH (amino acid abbreviations are described elsewhere in this specification). Peptide elongation was carried out according to the Fmoc peptide synthesis method (WO2013100132B2). After peptide elongation, the N-terminal Fmoc group was removed on the peptide synthesizer, and the resin was washed with DCM. The resin was added with TFE / DCM (1:1, v / v, 2 mL) and shaken for 1 hour to cleave the peptide from the resin. After the reaction was complete, the solution in the tube was filtered through a synthesis column to remove the resin, which was then washed twice with TFE / DCM (1:1, v / v, 1 mL). All extracts were combined, DMF (2 mL) was added, and the mixture was concentrated under reduced pressure. The resulting residue was dissolved in NMP (0.5 mL), and one-quarter of the solution (125 μL) was used in the next reaction. 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid N-succinimidyl ester (140 μL), prepared at 76.5 mM, was added to the NMP solution of the peptide at room temperature, stirred overnight at 40°C, and then concentrated under reduced pressure. The resulting residue was dissolved in 0.05 M tetramethylammonium hydrogen sulfate in HFIP (1.2 ml, 0.060 mmol) and stirred at room temperature for 2 hours. The reaction mixture was purified by reverse-phase silica gel column chromatography (0.1% FA MeCN / HO) to obtain the title compound (LCT-12) (0.3 mg). The amino acid sequence of LCT-12 is shown in SEQ ID NO: 238. LCMS(ESI) m / z = 1972.9 (M−H) Retention time: 0.74 minutes (Analysis conditions SQDFA05_01)

[0194] Example 4 Synthesis of aminoacyl-tRNA Example 4-1 Preparation of tRNA tRNAs (TR-1 to TR-107) were synthesized from template DNAs (TD-1 to TD-107) by in vitro transcription using T7 RNA polymerase, and purified using an RNeasy kit (Qiagen).

[0195] Template DNA SEQ ID NO: 1 (TD-1) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaagAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0196] Template DNA SEQ ID NO: 2 (TD-2) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaagACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0197] Template DNA SEQ ID NO: 3 (TD-3) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaagAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0198] Template DNA SEQ ID NO: 4 (TD-4) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaagACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0199] Template DNA SEQ ID NO: 5 (TD-5) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaagGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0200] Template DNA SEQ ID NO: 6 (TD-6) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaagATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0201] Template DNA SEQ ID NO: 7 (TD-7) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaagGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0202] Template DNA SEQ ID NO: 8 (TD-8) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaagGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0203] Template DNA SEQ ID NO: 9 (TD-9) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATaagGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0204] Template DNA SEQ ID NO: 10 (TD-10) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATaagATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0205] Template DNA SEQ ID NO: 11 (TD-11) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcagAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0206] Template DNA SEQ ID NO: 12 (TD-12) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcagACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0207] Template DNA SEQ ID NO: 13 (TD-13) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcagAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0208] Template DNA SEQ ID NO: 14 (TD-14) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcagACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0209] Template DNA SEQ ID NO: 15 (TD-15) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcagGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0210] Template DNA SEQ ID NO: 16 (TD-16) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcagATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0211] Template DNA SEQ ID NO: 17 (TD-17) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcagGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0212] Template DNA SEQ ID NO: 18 (TD-18) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcagGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0213] Template DNA SEQ ID NO: 19 (TD-19) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATcagGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0214] Template DNA SEQ ID NO: 20 (TD-20) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATcagATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0215] Template DNA SEQ ID NO: 21 (TD-21) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaggAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0216] Template DNA SEQ ID NO: 22 (TD-22) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaggACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0217] Template DNA SEQ ID NO: 23 (TD-23) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaggAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0218] Template DNA SEQ ID NO: 24 (TD-24) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaggACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0219] Template DNA SEQ ID NO: 25 (TD-25) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaggGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0220] Template DNA SEQ ID NO: 26 (TD-26) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaggATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0221] Template DNA SEQ ID NO: 27 (TD-27) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaggGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0222] Template DNA SEQ ID NO: 28 (TD-28) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaggGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0223] Template DNA SEQ ID NO: 29 (TD-29) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATaggGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0224] Template DNA SEQ ID NO: 30 (TD-30) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATaggATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0225] Template DNA SEQ ID NO: 31 (TD-31) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcggAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0226] Template DNA SEQ ID NO: 32 (TD-32) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcggACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0227] Template DNA SEQ ID NO: 33 (TD-33) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcggAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0228] Template DNA SEQ ID NO: 34 (TD-34) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcggACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0229] Template DNA SEQ ID NO: 35 (TD-35) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcggGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0230] Template DNA SEQ ID NO: 36 (TD-36) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcggATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0231] Template DNA SEQ ID NO: 37 (TD-37) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcggGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0232] Template DNA SEQ ID NO: 38 (TD-38) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcggGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0233] Template DNA SEQ ID NO: 39 (TD-39) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATcggGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0234] Template DNA SEQ ID NO: 40 (TD-40) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATcggATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0235] Template DNA SEQ ID NO: 41 (TD-41) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaccAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0236] Template DNA SEQ ID NO: 42 (TD-42) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaccACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0237] Template DNA SEQ ID NO: 43 (TD-43) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaccAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0238] Template DNA SEQ ID NO: 44 (TD-44) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaccACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0239] Template DNA SEQ ID NO: 45 (TD-45) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTaccGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0240] Template DNA SEQ ID NO: 46 (TD-46) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaccATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0241] Template DNA SEQ ID NO: 47 (TD-47) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaccGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0242] Template DNA SEQ ID NO: 48 (TD-48) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTaccGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0243] Template DNA SEQ ID NO: 49 (TD-49) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATaccGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0244] Template DNA SEQ ID NO: 50 (TD-50) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATaccATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0245] Template DNA SEQ ID NO: 51 (TD-51) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcccAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0246] Template DNA SEQ ID NO: 52 (TD-52) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcccACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0247] Template DNA SEQ ID NO: 53 (TD-53) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcccAAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0248] Template DNA SEQ ID NO: 54 (TD-54) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcccACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0249] Template DNA SEQ ID NO: 55 (TD-55) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTcccGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0250] Template DNA SEQ ID NO: 56 (TD-56) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcccATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0251] Template DNA SEQ ID NO: 57 (TD-57) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcccGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0252] Template DNA SEQ ID NO: 58 (TD-58) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTcccGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0253] Template DNA SEQ ID NO: 59 (TD-59) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATcccGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0254] Template DNA SEQ ID NO: 60 (TD-60) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCATcccATGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0255] Template DNA SEQ ID NO: 61 (TD-61) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCCTaagAAGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0256] Template DNA SEQ ID NO: 62 (TD-62) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTaagACGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0257] Template DNA SEQ ID NO: 63 (TD-63) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTaagAAGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0258] Template DNA SEQ ID NO: 64 (TD-64) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCCTaagACGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0259] Template DNA SEQ ID NO: 65 (TD-65) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCCTaagGAGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0260] Template DNA SEQ ID NO: 66 (TD-66) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTaagATGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0261] Template DNA SEQ ID NO: 67 (TD-67) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTaagGTGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0262] Template DNA SEQ ID NO: 68 (TD-68) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTaagGAGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0263] Template DNA SEQ ID NO: 69 (TD-69) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCATaagGTGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0264] Template DNA SEQ ID NO: 70 (TD-70) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCATaagATGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0265] Template DNA SEQ ID NO: 71 (TD-71) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCCTcagAAGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0266] Template DNA SEQ ID NO: 72 (TD-72) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTcagACGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0267] Template DNA SEQ ID NO: 73 (TD-73) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTcagAAGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0268] Template DNA SEQ ID NO: 74 (TD-74) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCCTcagACGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0269] Template DNA SEQ ID NO: 75 (TD-75) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCCTcagGAGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0270] Template DNA SEQ ID NO: 76 (TD-76) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTcagATGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0271] Template DNA SEQ ID NO: 77 (TD-77) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTcagGTGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0272] Template DNA SEQ ID NO: 78 (TD-78) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTcagGAGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0273] Template DNA SEQ ID NO: 79 (TD-79) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCATcagGTGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0274] Template DNA SEQ ID NO: 80 (TD-80) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCATcagATGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0275] Template DNA SEQ ID NO: 81 (TD-81) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGACTaagAATCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0276] Template DNA SEQ ID NO: 82 (TD-82) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTaagACTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0277] Template DNA SEQ ID NO: 83 (TD-83) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTaagAATCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0278] Template DNA SEQ ID NO: 84 (TD-84) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGACTaagACTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0279] Template DNA SEQ ID NO: 85 (TD-85) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGACTaagGATCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0280] Template DNA SEQ ID NO: 86 (TD-86) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTaagATTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0281] Template DNA SEQ ID NO: 87 (TD-87) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTaagGTTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0282] Template DNA SEQ ID NO: 88 (TD-88) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTaagGATCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0283] Template DNA SEQ ID NO: 89 (TD-89) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGAATaagGTTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0284] Template DNA SEQ ID NO: 90 (TD-90) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGAATaagATTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0285] Template DNA SEQ ID NO: 91 (TD-91) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGACTcagAATCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0286] Template DNA SEQ ID NO: 92 (TD-92) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTcagACTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0287] Template DNA SEQ ID NO: 93 (TD-93) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTcagAATCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0288] Template DNA SEQ ID NO: 94 (TD-94) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGACTcagACTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0289] Template DNA SEQ ID NO: 95 (TD-95) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGACTcagGATCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0290] Template DNA SEQ ID NO: 96 (TD-96) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTcagATTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0291] Template DNA SEQ ID NO: 97 (TD-97) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTcagGTTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0292] Template DNA SEQ ID NO: 98 (TD-98) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTcagGATCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0293] Template DNA SEQ ID NO: 99 (TD-99) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGAATcagGTTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0294] Template DNA SEQ ID NO: 100 (TD-100) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGAATcagATTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0295] Template DNA SEQ ID NO: 101 (TD-101) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTgccACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0296] Template DNA SEQ ID NO: 102 (TD-102) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTgccGTGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0297] Template DNA SEQ ID NO: 103 (TD-103) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTgccGAGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0298] Template DNA SEQ ID NO: 104 (TD-104) DNA sequence: GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTtccACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGC

[0299] Template DNA SEQ ID NO: 105 (TD-105) DNA sequence: GGCGTAATACGACTCACTATAGGCGGGGTGGAGCAGCCTGGTAGCTCGTCGGGCTcatAACCCGAAGATCGTCGGTTCAAATCCGGCCCCGCAAC

[0300] Template DNA SEQ ID NO: 106 (TD-106) DNA sequence: GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCTTtagGTGCAGGGGGTCGCGGGTTCGAGTCCCGTCCGTTCCGC

[0301] Template DNA SEQ ID NO: 107 (TD-107) DNA sequence: GGCGTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGATTtagGTTCCGTATGTCACTGGTTCGAGTCCAGTCAGAGCCGC

[0302] tRNA SEQ ID NO: 108 (TR-1) tRNA(Glu2+Ser5)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaagAAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0303] tRNA SEQ ID NO: 109 (TR-2) tRNA(Glu2+Ala1B)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaagACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0304] tRNA SEQ ID NO: 110 (TR-3) tRNA(Glu2+Phe)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaagAAGGCGGUAACAGGGGGUUCGAAUCCCCUAGGGGACGC

[0305] tRNA SEQ ID NO:111(TR-4) tRNA(Glu2)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaagACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0306] tRNA SEQ ID NO:112(TR-5) tRNA(Glu2+Arg3)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaagGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0307] tRNA SEQ ID NO:113(TR-6) tRNA(Glu2+Val2)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaagAUGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0308] tRNA SEQ ID NO:114(TR-7) tRNA(Glu2+Leu2)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaagGUGGCGGUAACAGGGGGUUCGAAUCCCCUAGGGGACGC

[0309] tRNA SEQ ID NO:115(TR-8) tRNA(Glu2+Pro3)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaagGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0310] tRNA SEQ ID NO: 116 (TR-9) tRNA(Glu2+Pro2)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUaagGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0311] tRNA SEQ ID NO: 117 (TR-10) tRNA(Glu2+Ala2)aag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUaagAUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0312] tRNA SEQ ID NO: 118 (TR-11) tRNA(Glu2+Ser5)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUcagAAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0313] tRNA SEQ ID NO: 119 (TR-12) tRNA(Glu2+Ala1B)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcagACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0314] tRNA SEQ ID NO: 120 (TR-13) tRNA(Glu2+Phe)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcagAAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0315] tRNA SEQ ID NO: 121 (TR-14) tRNA(Glu2)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUcagACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0316] tRNA SEQ ID NO: 122 (TR-15) tRNA(Glu2+Arg3)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUcagGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0317] tRNA SEQ ID NO: 123 (TR-16) tRNA(Glu2+Val2)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcagAUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0318] tRNA SEQ ID NO: 124 (TR-17) tRNA(Glu2+Leu2)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcagGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0319] tRNA SEQ ID NO: 125 (TR-18) tRNA(Glu2+Pro3)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcagGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0320] tRNA SEQ ID NO: 126 (TR-19) tRNA(Glu2+Pro2)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUcagGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0321] tRNA SEQ ID NO:127(TR-20) tRNA(Glu2+Ala2)cag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUcagAUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0322] tRNA SEQ ID NO:128(TR-21) tRNA(Glu2+Ser5)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaggAAGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0323] tRNA SEQ ID NO:129(TR-22) tRNA(Glu2+Ala1B)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaggACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0324] tRNA SEQ ID NO:130(TR-23) tRNA(Glu2+Phe)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaggAAGGCGGUAACAGGGGGUUCGAAUCCCCUAGGGGACGC

[0325] tRNA SEQ ID NO:131(TR-24) tRNA(Glu2)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaggACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0326] tRNA SEQ ID NO: 132 (TR-25) tRNA(Glu2+Arg3)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaggGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0327] tRNA SEQ ID NO: 133 (TR-26) tRNA(Glu2+Val2)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaggAUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0328] tRNA SEQ ID NO: 134 (TR-27) tRNA(Glu2+Leu2)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaggGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0329] tRNA SEQ ID NO: 135 (TR-28) tRNA(Glu2+Pro3)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaggGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0330] tRNA SEQ ID NO: 136 (TR-29) tRNA(Glu2+Pro2)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUaggGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0331] tRNA SEQ ID NO: 137 (TR-30) tRNA(Glu2+Ala2)agg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUaggAUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0332] tRNA SEQ ID NO: 138 (TR-31) tRNA(Glu2+Ser5)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUcggAAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0333] tRNA SEQ ID NO: 139 (TR-32) tRNA(Glu2+Ala1B)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcggACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0334] tRNA SEQ ID NO: 140 (TR-33) tRNA(Glu2+Phe)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcggAAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0335] tRNA SEQ ID NO: 141 (TR-34) tRNA(Glu2)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUcggACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0336] tRNA SEQ ID NO: 142 (TR-35) tRNA(Glu2+Arg3)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUcggGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0337] tRNA SEQ ID NO: 143 (TR-36) tRNA(Glu2+Val2)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcggAUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0338] tRNA SEQ ID NO: 144 (TR-37) tRNA(Glu2+Leu2)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcggGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0339] tRNA SEQ ID NO: 145 (TR-38) tRNA(Glu2+Pro3)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcggGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0340] tRNA SEQ ID NO: 146 (TR-39) tRNA(Glu2+Pro2)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUcggGUGGCGGUAACAGGGGGUUCGAAUCCCCUAGGGGACGC

[0341] tRNA SEQ ID NO:147(TR-40) tRNA(Glu2+Ala2)cgg-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUcggAUGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0342] tRNA SEQ ID NO:148(TR-41) tRNA(Glu2+Ser5)acc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaccAAGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0343] tRNA SEQ ID NO:149(TR-42) tRNA(Glu2+Ala1B)acc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaccACGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0344] tRNA SEQ ID NO:150(TR-43) tRNA(Glu2+Phe)acc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaccAAGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0345] tRNA SEQ ID NO:151(TR-44) tRNA(Glu2)acc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaccACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0346] tRNA SEQ ID NO:152(TR-45) tRNA(Glu2+Arg3)acc-CA RNA sequence: GUCCCUUCGUCUAGAGGCCCAGGACACCGCCCUaccGAGGCGUAACAGGGGUUCGAAUCCCUAGGGGACGC

[0347] tRNA SEQ ID NO:153(TR-46) tRNA(Glu2+Val2)acc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaccAUGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0348] tRNA SEQ ID NO:154(TR-47) tRNA(Glu2+Leu2)acc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaccGUGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0349] tRNA SEQ ID NO:155(TR-48) tRNA(Glu2+Pro3)acc-CA RNA sequence: GUCCCUUCGUCUAGAGGCCCAGGACACCGCCUUaccGAGGCGUAACAGGGGUUCGAAUCCCUAGGGGACGC

[0350] tRNA SEQ ID NO:156(TR-49) tRNA(Glu2+Pro2)acc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUaccGUGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0351] tRNA SEQ ID NO:157(TR-50) tRNA(Glu2+Ala2)acc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUaccAUGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0352] tRNA SEQ ID NO:158(TR-51) tRNA(Glu2+Ser5)ccc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUcccAAGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0353] tRNA SEQ ID NO:159(TR-52) tRNA(Glu2+Ala1B)ccc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUCccACGGCGGUAACAGGGGGUUCGAAUCCCCUAGGGGACGC

[0354] tRNA SEQ ID NO:160(TR-53) tRNA(Glu2+Phe)ccc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcccAAGGCGGUAAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0355] tRNA SEQ ID NO:161(TR-54) tRNA(Glu2)ccc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUCcccACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0356] tRNA SEQ ID NO:162(TR-55) tRNA(Glu2+Arg3)ccc-CA RNA sequence: GUCCCUUCGUCUAGAGGCCCAGGACACCGCCCUcccGAGGCGUAACAGGGGUUCGAAUCCCUAGGGGACGC

[0357] tRNA SEQ ID NO:163(TR-56) tRNA(Glu2+Val2)ccc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUCccAUGGCGGUAACAGGGGGUUCGAAUCCCCUAGGGGACGC

[0358] tRNA SEQ ID NO:164(TR-57) tRNA(Glu2+Leu2)ccc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcccGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0359] tRNA SEQ ID NO:165(TR-58) tRNA(Glu2+Pro3)ccc-CA RNA sequence: GUCCCUUCGUCUAGAGGCCCAGGACACCGCCUUcccGAGGCGUAACAGGGGUUCGAAUCCCUAGGGGACGC

[0360] tRNA SEQ ID NO:166(TR-59) tRNA(Glu2+Pro2)ccc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUcccGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0361] tRNA SEQ ID NO: 167 (TR-60) tRNA(Glu2+Ala2)ccc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCAUcccAUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0362] tRNA SEQ ID NO: 168 (TR-61) tRNA(Asp1+Ser5)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCCUaagAAGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0363] tRNA SEQ ID NO: 169 (TR-62) tRNA(Asp1+Ala1B)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUaagACGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0364] tRNA SEQ ID NO: 170 (TR-63) tRNA(Asp1+Phe)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUaagAAGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0365] tRNA SEQ ID NO: 171 (TR-64) tRNA(Asp1)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCCUaagACGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0366] tRNA SEQ ID NO: 172 (TR-65) tRNA(Asp1+Arg3)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCCUaagGAGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0367] tRNA SEQ ID NO: 173 (TR-66) tRNA(Asp1+Val2)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUaagAUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0368] tRNA SEQ ID NO: 174 (TR-67) tRNA(Asp1+Leu2)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUaagGUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0369] tRNA SEQ ID NO: 175 (TR-68) tRNA(Asp1+Pro3)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUaagGAGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0370] tRNA SEQ ID NO: 176 (TR-69) tRNA(Asp1+Pro2)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCAUaagGUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0371] tRNA SEQ ID NO: 177 (TR-70) tRNA(Asp1+Ala2)aag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCAUaagAUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0372] tRNA SEQ ID NO: 178 (TR-71) tRNA(Asp1+Ser5)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCCUcagAAGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0373] tRNA SEQ ID NO: 179 (TR-72) tRNA(Asp1+Ala1B)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUcagACGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0374] tRNA SEQ ID NO: 180 (TR-73) tRNA(Asp1+Ph2)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUcagAAGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0375] tRNA SEQ ID NO: 181 (TR-74) tRNA(Asp1)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCCUcagACGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0376] tRNA SEQ ID NO: 182 (TR-75) tRNA(Asp1+Arg3)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCCUcagGAGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0377] tRNA SEQ ID NO: 183 (TR-76) tRNA(Asp1+Val2)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUcagAUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0378] tRNA SEQ ID NO: 184 (TR-77) tRNA(Asp1+Leu2)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUcagGUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0379] tRNA SEQ ID NO: 185 (TR-78) tRNA(Asp1+Pro3)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUcagGAGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0380] tRNA SEQ ID NO: 186 (TR-79) tRNA(Asp1+Pro2)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCAUcagGUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0381] tRNA SEQ ID NO: 187 (TR-80) tRNA(Asp1+Ala2)cag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCAUcagAUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0382] tRNA SEQ ID NO: 188 (TR-81) tRNA(AsnE2)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGACUaagAAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0383] tRNA SEQ ID NO: 189 (TR-82) tRNA(AsnE2+Ala1B)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUaagACUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0384] tRNA SEQ ID NO: 190 (TR-83) tRNA(AsnE2+Phe)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUaagAAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0385] tRNA SEQ ID NO: 191 (TR-84) tRNA(AsnE2+Glu2)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGACUaagACUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0386] tRNA SEQ ID NO: 192 (TR-85) tRNA(AsnE2+Arg3)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGACUaagGAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0387] tRNA SEQ ID NO: 193 (TR-86) tRNA(AsnE2+Val2)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUaagAUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0388] tRNA SEQ ID NO: 194 (TR-87) tRNA(AsnE2+Leu2)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUaagGUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0389] tRNA SEQ ID NO: 195 (TR-88) tRNA(AsnE2+Pro3)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUaagGAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0390] tRNA SEQ ID NO: 196 (TR-89) tRNA(AsnE2+Pro2)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAAUaagGUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0391] tRNA SEQ ID NO: 197 (TR-90) tRNA(AsnE2+Ala2)aag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAAUaagAUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0392] tRNA SEQ ID NO: 198 (TR-91) tRNA(AsnE2)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGACUcagAAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0393] tRNA SEQ ID NO: 199 (TR-92) tRNA(AsnE2+Ala1B)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUcagACUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0394] tRNA SEQ ID NO: 200 (TR-93) tRNA(AsnE2+Phe)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUcagAAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0395] tRNA SEQ ID NO: 201 (TR-94) tRNA(AsnE2+Glu2)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGACUcagACUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0396] tRNA SEQ ID NO: 202 (TR-95) tRNA(AsnE2+Arg3)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGACUcagGAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0397] tRNA SEQ ID NO: 203 (TR-96) tRNA(AsnE2+Val2)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUcagAUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0398] tRNA SEQ ID NO: 204 (TR-97) tRNA(AsnE2+Leu2)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUcagGUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0399] tRNA SEQ ID NO: 205 (TR-98) tRNA(AsnE2+Pro3)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUcagGAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0400] tRNA SEQ ID NO: 206 (TR-99) tRNA(AsnE2+Pro2)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAAUcagGUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0401] tRNA SEQ ID NO: 207 (TR-100) tRNA(AsnE2+Ala2)cag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAAUcagAUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0402] tRNA SEQ ID NO: 208 (TR-101) tRNA(Glu2)gcc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUgccACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0403] tRNA SEQ ID NO: 209 (TR-102) tRNA(Glu2+Leu2)gcc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUgccGUGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0404] tRNA SEQ ID NO: 210 (TR-103) tRNA(Glu2+Pro3)gcc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCUUgccGAGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0405] tRNA SEQ ID NO: 211 (TR-104) tRNA(Glu2)ucc-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUuccACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC

[0406] tRNA SEQ ID NO: 212 (TR-105) tRNA(fMet)cau-CA RNA sequence: GGCGGGGUGGAGCAGCCUGGUAGCUCGUCGGGCUcauAACCCGAAGAUCGUCGGUUCAAAUCCGGCCCCGCAAC

[0407] tRNA SEQ ID NO: 213 (TR-106) tRNA(Asp1+Leu2)_uag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUUuagGUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0408] tRNA SEQ ID NO: 214 (TR-107) tRNA(AsnE2+Leu2)_uag-CA RNA sequence: GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUUuagGUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0409] Example 4-2 Preparation of tRNA-CA by ligation reaction Various tRNA-CAs were prepared by ligating the tRNA5' fragment, pLp, and tRNA3' fragment using the procedure described below. Chemically synthesized tRNA5' fragment and tRNA3' fragment (Gene Design Inc.) were used. The sequences of each tRNA fragment and its full length are shown below. FR-1 and FR-2 were used as the tRNA5' fragment and tRNA3' fragment, respectively, to prepare TR-108, and FR-3 and FR-4 were used to prepare TR-109.

[0410] SEQ ID NO: 217 (FR-1) tRNA(Asp1)5' RNA sequence GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUU

[0411] SEQ ID NO: 218 (FR-2) tRNA(Asp1)3'ag RNA sequence AGGUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC tRNA SEQ ID NO: 215 (TR-108) tRNA(Asp1+Leu2)Lag-CA RNA sequence: GGAGCGGUAGUUCAGUCGGUUAGAAUACCUGCUULagGUGCAGGGGGUCGCGGGUUCGAGUCCCGUCCGUUCCGC

[0412] SEQ ID NO: 219 (FR-3) tRNA(AsnE2) 5' RNA sequence GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUU

[0413] SEQ ID NO: 220 (FR-4) tRNA(AsnE2)3'ag RNA sequence AGGUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0414] tRNA SEQ ID NO: 216 (TR-109) tRNA(AsnE2+Leu2)Lag-CA RNA sequence GGCUCUGUAGUUCAGUCGGUAGAACGGCGGAUULagGUUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGC

[0415] Ligation of the tRNA fragment 5' with pLp was carried out by incubating the reaction solution containing 50 mM HEPES-KOH (pH 7.5), 20 mM MgCl2, 1 mM ATP, 0.125–0.25 mM pLp, 25 μM tRNA 5' fragment, 0.6 U / μL T4 RNA ligase (New England Biolabs), and 10% DMSO overnight at 15°C. The ligation product was extracted with phenol and chloroform and recovered by ethanol precipitation.

[0416] To prevent unreacted tRNA5' fragments from being carried over to the next ligation reaction, the ribose at the 3' end of the tRNA5' fragment was cleaved with sodium periodate (NaIO4). Specifically, the cleavage reaction was carried out by incubating the mixture on ice in the dark for 30 minutes in the presence of 10 μM ligation product and 10 mM sodium periodate. After the reaction, 1 / 10 the volume of 100 mM glucose was added and the mixture was incubated on ice in the dark for 30 minutes to decompose the excess sodium periodate. The reaction product was recovered by ethanol precipitation.

[0417] The ligation product after periodate treatment was treated with T4 PNK (T4 polynucleotide kinase) to phosphorylate the 5' end and dephosphorylate the 3' end. The reaction solution, consisting of 10 μM periodate-treated ligation product, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM DTT, 300 μM ATP, and 0.5 U / μL T4 PNK (TaKaRa), was left at 37°C for 30 to 60 minutes. The reaction product was extracted with phenol and chloroform and recovered by ethanol precipitation.

[0418] The PNK-treated reaction product was ligated with the tRNA 3' fragment. First, a solution consisting of 10 μM PNK-treated reaction product, 10 μM tRNA 3' fragment, 50 mM HEPES-KOH (pH 7.5), and 15 mM MgCl2 was heated at 65°C for 7 minutes and then allowed to stand at room temperature for 30 minutes to 1 hour to allow annealing between the PNK-treated reaction product and the tRNA 3' fragment. Next, T4 PNK treatment was performed to phosphorylate the 5' end of the tRNA 3' fragment. T4 PNK treatment was performed by adding DTT (final concentration 3.5 mM), ATP (final concentration 300 μM), and T4 PNK (final concentration 0.5 U / μL) to the annealing solution and allowing it to stand at 37°C for 30 minutes. Next, T4 RNA ligase (New England Biolabs) was added to this solution to a final concentration of 0.9 U / μL, and the mixture was left to stand at 37°C for 30 to 40 minutes to carry out the ligation reaction. The ligation product was extracted with phenol-chloroform and recovered by ethanol precipitation.

[0419] The tRNA-CA prepared by the ligation method was purified by high-performance reverse-phase chromatography (HPLC) (15 mM TEA and 400 mM HFIP in water / 15 mM TEA and 400 mM HFIP in methanol), and then confirmed to have the desired length by denaturing urea 10% polyacrylamide gel electrophoresis.

[0420] RNase T 1 Analysis of tRNA fragments cleaved by The tRNA-CA prepared by the ligation reaction was fragmented with RNase and analyzed to confirm that the lysidine (L) introduced by pLp had been introduced into the desired site.

[0421] A reaction solution containing 10 μM tRNA-CA, 5 U / μL RNase T1 (Epicentre or ThermoFisher Scientific), and 10 mM ammonium acetate (pH 5.3) was left at 37°C for 1 hour to specifically cleave the RNA at the 3' end of the G base, and the RNA fragments containing lysidine (L) introduced by pLp were analyzed.

[0422] CUU L AGp LCMS(ESI) m / z = 1020 ((M-2H) / 2)- Retention time: 3.84 minutes (Analysis conditions LTQTEA / HFIP05_03) Because the molecular weight of the fragment (CUUAGp) expected when pLp is not ligated and the fragment (UUCAGp) derived from another part of the RNA are the same, we also analyzed unfragmented RNA (TR-108). LCMS(ESI) m / z = 1109 ((M-22H) / 22)- Retention time: 3.92 minutes (Analysis conditions LTQTEA / HFIP05_01) By comparing the mass chromatograms of RNA expected when pLp was not ligated with those of RNA expected when uridine was used instead of lysidine, it was confirmed that ligation of most of the pLp had progressed.

[0423] AUU L AGp LCMS(ESI) m / z = 1032 ((M-2H) / 2)- Retention time: 4.16 minutes (Analysis conditions LTQTEA / HFIP05_03) By comparing the mass chromatograms of the fragment (AUUAGp) expected when pLp was not ligated with the mass chromatogram of the fragment (AUUUAGp) expected when uridine was used instead of lysidine, it was confirmed that ligation of most of the pLp had progressed.

[0424] Preparation of elongated aminoacyl-tRNA using aminoacyl-pCpA A reaction mixture containing 25 μM transcribed tRNA(Glu2+Ser5)aag-CA(TR-1), 50 mM HEPES-KOH pH 7.5, 20 mM MgCl2, 1 mM ATP, 0.6 Unit / μL T4 RNA ligase (New England Biolabs), and 0.25 mM aminoacyl pCpA (SS15 in DMSO) was diluted with nuclease-free water and subjected to ligation for 45 minutes at 15°C. Before adding T4 RNA ligase and aminoacyl pCpA, the reaction mixture was heated at 95°C for 2 minutes and then left at room temperature for 5 minutes to allow tRNA refolding.

[0425] Sodium acetate was added to the ligation reaction mixture to a concentration of 0.3 M, followed by phenol-chloroform extraction to prepare the elongated aminoacyl-tRNA (AAtR-1). AAtR-1 was recovered by ethanol precipitation and dissolved in 1 mM aqueous sodium acetate before being added to the translation mixture.

[0426] Similarly, aminoacyl-pCpA (SS15) was ligated to the transcribed tRNAs (TR-2 to TR-103, TR-106 to TR-109) as described above, followed by phenol-chloroform extraction and ethanol precipitation to prepare elongated aminoacyl-tRNAs (AAtR-2 to AAtR-103, AAtR-132, AAtR-133, AAtR-136, and AAtR-137). These aminoacyl-tRNAs were dissolved in 1 mM sodium acetate solution before being added to the translation mixture.

[0427] Similarly, aminoacyl-pCpA (SS16) was used to prepare elongated aminoacyl-tRNAs (AAtR-104 to AAtR-114) for the transcribed tRNAs (TR-4, TR-14, TR-24, TR-34, TR-44, TR-54, TR-64, TR-74, TR-81, TR-91, and TR-104) by ligation, phenol-chloroform extraction, and ethanol precipitation as described above. These aminoacyl-tRNAs were dissolved in 1 mM sodium acetate solution before being added to the translation mixture.

[0428] Similarly, aminoacyl-tRNAs (AAtR-115 to AAtR-118) were prepared by ligation of aminoacyl-pCpA (SS14) for transcribed tRNAs (TR-44, TR-47, TR-48, and TR-50) as described above, followed by phenol-chloroform extraction and ethanol precipitation. These aminoacyl-tRNAs were dissolved in 1 mM sodium acetate solution before being added to the translation mixture.

[0429] Similarly, aminoacyl-tRNAs (AAtR-119 to AAtR-122, AAtR-129, and AAtR-130) were prepared by ligation of aminoacyl-pCpA (SS45) for transcribed tRNAs (TR-44, TR-47, TR-48, TR-50, TR-24, and TR-34) as described above, followed by phenol-chloroform extraction and ethanol precipitation. These aminoacyl-tRNAs were dissolved in 1 mM sodium acetate solution before being added to the translation mixture.

[0430] Similarly, aminoacyl-pCpA (compound TS24, synthesized by the method described in patent document WO2018143145A1) was ligated to transcribed tRNAs (TR-44, TR-47, TR-48, TR-50, TR-77, and TR-97) using the same method, followed by phenol-chloroform extraction and ethanol precipitation to prepare elongated aminoacyl-tRNAs (AAtR-123 to AAtR-126, AAtR-134, and AAtR-138). These aminoacyl-tRNAs were dissolved in 1 mM aqueous sodium acetate before being added to the translation mixture.

[0431] Similarly, aminoacyl-pCpA (compound ts14, synthesized by the method described in patent document WO2018143145A1) was ligated to transcribed tRNAs (TR-54, TR-67, and TR-87) using the same method, followed by phenol-chloroform extraction and ethanol precipitation to prepare elongated aminoacyl-tRNAs (AAtR-127, AAtR-131, and AAtR-135). These aminoacyl-tRNAs were dissolved in 1 mM aqueous sodium acetate before being added to the translation mixture. <h2 style=";text-align:left;direction:ltr">

[0432] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-1 SPh2Cl-tRNA(Glu2+Ser5)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000035.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0433] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-2SPh2Cl-tRNA(Glu2+Ala1B)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000036.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0434] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-3 SPh2Cl-tRNA(Glu2+Phe)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000037.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0435] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-4 SPh2Cl-tRNA(Glu2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000038.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0436] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-5 SPh2Cl-tRNA(Glu2+Arg3)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000039.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0437] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-6 SPh2Cl-tRNA(Glu2+Val2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000040.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0438] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-7 SPh2Cl-tRNA(Glu2+Leu2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000041.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0439] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-8 SPh2Cl-tRNA(Glu2+Pro3)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000042.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0440] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-9 SPh2Cl-tRNA(Glu2+Pro2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000043.tif31170

[0441] AAtR-10 SPh2Cl-tRNA(Glu2+Ala2)aag TIFF0007744244000044.tif31170

[0442] AAtR-11 SPh2Cl-tRNA(Glu2+Ser5)cag TIFF0007744244000045.tif31170

[0443] AAtR-12 SPh2Cl-tRNA(Glu2+Ala1B)cag TIFF0007744244000046.tif31170

[0444] AAtR-13 SPh2Cl-tRNA(Glu2+Phe)cag TIFF0007744244000047.tif31170

[0445] AAtR-14 SPh2Cl-tRNA(Glu2)cag TIFF0007744244000048.tif31170

[0446] AAtR-15 SPh2Cl-tRNA(Glu2+Arg3)cag TIFF0007744244000049.tif31170

[0447] AAtR-16 SPh2Cl-tRNA(Glu2+Val2)cag TIFF0007744244000050.tif31170

[0448] AAtR-17 SPh2Cl-tRNA(Glu2+Leu2)cag TIFF0007744244000051.tif31170

[0449] AAtR-18 SPh2Cl-tRNA(Glu2+Pro3)cag TIFF0007744244000052.tif31170

[0450] AAtR-19 SPh2Cl-tRNA(Glu2+Pro2)cag TIFF0007744244000053.tif31170

[0451] AAtR-20 SPh2Cl-tRNA(Glu2+Ala2)cag TIFF0007744244000054.tif31170

[0452] AAtR-21 SPh2Cl-tRNA(Glu2+Ser5)agg TIFF0007744244000055.tif31170

[0453] AAtR-22 SPh2Cl-tRNA(Glu2+Ala1B)agg TIFF0007744244000056.tif31170

[0454] AAtR-23 SPh2Cl-tRNA(Glu2+Phe)agg TIFF0007744244000057.tif31170

[0455] AAtR-24 SPh2Cl-tRNA(Glu2)agg TIFF0007744244000058.tif31170

[0456] AAtR-25 SPh2Cl-tRNA(Glu2+Arg3)agg TIFF0007744244000059.tif31170

[0457] AAtR-26 SPh2Cl-tRNA(Glu2+Val2)agg TIFF0007744244000060.tif31170

[0458] AAtR-27 SPh2Cl-tRNA(Glu2+Leu2)agg TIFF0007744244000061.tif31170

[0459] AAtR-28 SPh2Cl-tRNA(Glu2+Pro3)agg TIFF0007744244000062.tif31170

[0460] AAtR-29 SPh2Cl-tRNA(Glu2+Pro2)agg TIFF0007744244000063.tif31170

[0461] AAtR-30 SPh2Cl-tRNA(Glu2+Ala2)agg TIFF0007744244000064.tif31170

[0462] AAtR-31 SPh2Cl-tRNA(Glu2+Ser5)cgg TIFF0007744244000065.tif31170

[0463] AAtR-32 SPh2Cl-tRNA(Glu2+Ala1B)cgg TIFF0007744244000066.tif31170

[0464] AAtR-33 SPh2Cl-tRNA(Glu2+Phe)cgg TIFF0007744244000067.tif31170

[0465] AAtR-34 SPh2Cl-tRNA(Glu2)cgg TIFF0007744244000068.tif31170

[0466] AAtR-35 SPh2Cl-tRNA(Glu2+Arg3)cgg TIFF0007744244000069.tif31170

[0467] AAtR-36 SPh2Cl-tRNA(Glu2+Val2)cgg TIFF0007744244000070.tif31170

[0468] AAtR-37 SPh2Cl-tRNA(Glu2+Leu2)cgg TIFF0007744244000071.tif31170

[0469] AAtR-38 SPh2Cl-tRNA(Glu2+Pro3)cgg TIFF0007744244000072.tif31170

[0470] AAtR-39 SPh2Cl-tRNA(Glu2+Pro2)cgg TIFF0007744244000073.tif31170

[0471] AAtR-40 SPh2Cl-tRNA(Glu2+Ala2)cgg TIFF0007744244000074.tif31170

[0472] AAtR-41 SPh2Cl-tRNA(Glu2+Ser5)acc TIFF0007744244000075.tif31170

[0473] AAtR-42 SPh2Cl-tRNA(Glu2+Ala1B)acc TIFF0007744244000076.tif31170

[0474] AAtR-43 SPh2Cl-tRNA(Glu2+Phe)acc TIFF0007744244000077.tif31170

[0475] AAtR-44 SPh2Cl-tRNA(Glu2)acc TIFF0007744244000078.tif31170

[0476] AAtR-45 SPh2Cl-tRNA(Glu2+Arg3)acc TIFF0007744244000079.tif31170

[0477] AAtR-46 SPh2Cl-tRNA(Glu2+Val2)acc TIFF0007744244000080.tif31170

[0478] AAtR-47 SPh2Cl-tRNA(Glu2+Leu2)acc TIFF0007744244000081.tif31170

[0479] AAtR-48 SPh2Cl-tRNA(Glu2+Pro3)acc TIFF0007744244000082.tif31170

[0480] AAtR-49 SPh2Cl-tRNA(Glu2+Pro2)acc TIFF0007744244000083.tif31170

[0481] AAtR-50 SPh2Cl-tRNA(Glu2+Ala2)acc TIFF0007744244000084.tif31170

[0482] AAtR-51 SPh2Cl-tRNA(Glu2+Ser5)ccc TIFF0007744244000085.tif31170

[0483] AAtR-52 SPh2Cl-tRNA(Glu2+Ala1B)ccc TIFF0007744244000086.tif31170

[0484] AAtR-53 SPh2Cl-tRNA(Glu2+Phe)ccc TIFF0007744244000087.tif31170

[0485] AAtR-54 SPh2Cl-tRNA(Glu2)ccc TIFF0007744244000088.tif31170

[0486] AAtR-55 SPh2Cl-tRNA(Glu2+Arg3)ccc TIFF0007744244000089.tif31170

[0487] AAtR-56 SPh2Cl-tRNA(Glu2+Val2)ccc TIFF0007744244000090.tif31170

[0488] AAtR-57 SPh2Cl-tRNA(Glu2+Leu2)ccc TIFF0007744244000091.tif31170

[0489] AAtR-58 SPh2Cl-tRNA(Glu2+Pro3)ccc TIFF0007744244000092.tif31170

[0490] AAtR-59 SPh2Cl-tRNA(Glu2+Pro2)ccc TIFF0007744244000093.tif31170

[0491] AAtR-60 SPh2Cl-tRNA(Glu2+Ala2)ccc TIFF0007744244000094.tif31170

[0492] AAtR-61 SPh2Cl-tRNA(Asp1+Ser5)aag TIFF0007744244000095.tif31170 <h2 style=";text-align:left;direction:ltr">

[0493] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-62 SPh2Cl-tRNA(Asp1+Ala1B)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000096.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0494] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-63 SPh2Cl-tRNA(Asp1+Phe)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000097.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0495] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-64 SPh2Cl-tRNA(Asp1)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000098.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0496] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-65 SPh2Cl-tRNA(Asp1+Arg3)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000099.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0497] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-66 SPh2Cl-tRNA(Asp1+Val2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000100.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0498] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-67 SPh2Cl-tRNA(Asp1+Leu2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000101.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0499] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-68 SPh2Cl-tRNA(Asp1+Pro3)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000102.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0500] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-69 SPh2Cl-tRNA(Asp1+Pro2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000103.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0501] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-70 SPh2Cl-tRNA(Asp1+Ala2)aag<h2 style=";text-align:left;direction:ltr"> TIFF0007744244000104.tif31170

[0502] AAtR-71 SPh2Cl-tRNA(Asp1+Ser5)cag TIFF0007744244000105.tif31170

[0503] AAtR-72 SPh2Cl-tRNA(Asp1+Ala1B)cag TIFF0007744244000106.tif31170

[0504] AAtR-73 SPh2Cl-tRNA(Asp1+Phe)cag TIFF0007744244000107.tif31170

[0505] AAtR-74 SPh2Cl-tRNA(Asp1)cag TIFF0007744244000108.tif31170

[0506] AAtR-75 SPh2Cl-tRNA(Asp1+Arg3)cag TIFF0007744244000109.tif31170

[0507] AAtR-76 SPh2Cl-tRNA(Asp1+Val2)cag TIFF0007744244000110.tif31170

[0508] AAtR-77 SPh2Cl-tRNA(Asp1+Leu2)cag TIFF0007744244000111.tif31170

[0509] AAtR-78 SPh2Cl-tRNA(Asp1+Pro3)cag TIFF0007744244000112.tif31170

[0510] <h2 style=";text-align:left;direction:ltr">AAtR-79 SPh2Cl-tRNA(Asp1+Pro2)cag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000113.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0511] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-80 SPh2Cl-tRNA(Asp1+Ala2)cag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000114.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0512] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-81 SPh2Cl-tRNA(AsnE2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000115.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0513] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-82 SPh2Cl-tRNA(AsnE2+Ala1B)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000116.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0514] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-83 SPh2Cl-tRNA(AsnE2+Phe)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000117.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0515] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-84 SPh2Cl-tRNA(AsnE2+Glu2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000118.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0516] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-85 SPh2Cl-tRNA(AsnE2+Arg3)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000119.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0517] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-86 SPh2Cl-tRNA(AsnE2+Val2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000120.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0518] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-87 SPh2Cl-tRNA(AsnE2+Leu2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000121.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0519] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-88 SPh2Cl-tRNA(AsnE2+Pro3)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000122.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0520] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-89 SPh2Cl-tRNA(AsnE2+Pro2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000123.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0521] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-90 SPh2Cl-tRNA(AsnE2+Ala2)aag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000124.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0522] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-91 SPh2Cl-tRNA(AsnE2)cag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000125.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0523] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-92 SPh2Cl-tRNA(AsnE2+Ala1B)cag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000126.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0524] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-93 SPh2Cl-tRNA(AsnE2+Phe)cag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000127.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0525] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-94 SPh2Cl-tRNA(AsnE2+Glu2)cag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000128.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0526] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-95 SPh2Cl-tRNA(AsnE2+Arg3)cag<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIFF0007744244000129.tif31170<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0527] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAtR-96 SPh2Cl-tRNA(AsnE2+Val2)cag<h2 style=";text-align:left;direction:ltr"> TIFF0007744244000130.tif31170

[0528] AAtR-97 SPh2Cl-tRNA(AsnE2+Leu2)cag TIFF0007744244000131.tif31170

[0529] AAtR-98 SPh2Cl-tRNA(AsnE2+Pro3)cag TIFF0007744244000132.tif31170

[0530] AAtR-99 SPh2Cl-tRNA(AsnE2+Pro2)cag TIFF0007744244000133.tif31170

[0531] AAtR-100 SPh2Cl-tRNA(AsnE2+Ala2)cag TIFF0007744244000134.tif31170

[0532] AAtR-101 SPh2Cl-tRNA(Glu2)gcc TIFF0007744244000135.tif31170

[0533] AAtR-102 SPh2Cl-tRNA(Glu2+Leu2)gcc TIFF0007744244000136.tif31170

[0534] AAtR-103 SPh2Cl-tRNA(Glu2+Pro3)gcc TIFF0007744244000137.tif31170

[0535] AAtR-104 MeHph-tRNA(Glu2)aag TIFF0007744244000138.tif31170

[0536] AAtR-105 MeHph-tRNA(Glu2)cag TIFF0007744244000139.tif31170

[0537] AAtR-106 MeHph-tRNA(Glu2)agg TIFF0007744244000140.tif31170

[0538] AAtR-107 MeHph-tRNA(Glu2)cgg TIFF0007744244000141.tif31170

[0539] AAtR-108 MeHph-tRNA(Glu2)acc TIFF0007744244000142.tif31170

[0540] AAtR-109 MeHph-tRNA(Glu2)ccc TIFF0007744244000143.tif31170

[0541] AAtR-110 MeHph-tRNA(Asp1)aag TIFF0007744244000144.tif31170

[0542] AAtR-111 MeHph-tRNA(Asp1)cag TIFF0007744244000145.tif31170

[0543] AAtR-112 MeHph-tRNA(AsnE2)aag TIFF0007744244000146.tif31170

[0544] AAtR-113 MeHph-tRNA(AsnE2)cag TIFF0007744244000147.tif31170

[0545] AAtR-114 MeHph-tRNA(Glu2)ucc TIFF0007744244000148.tif31170

[0546] AAtR-115 Pic2- tRNA(Glu2)acc TIFF0007744244000149.tif26170

[0547] AAtR-116 Pic2- tRNA(Glu2+Leu2)acc TIFF0007744244000150.tif26170

[0548] AAtR-117 Pic2- tRNA(Glu2+Pro3)acc TIFF0007744244000151.tif26170

[0549] AAtR-118 Pic2- tRNA(Glu2+Ala2)acc TIFF0007744244000152.tif26170

[0550] AAtR-119 MeG- tRNA(Glu2)acc TIFF0007744244000153.tif20170

[0551] AAtR-120 MeG- tRNA(Glu2+Leu2)acc TIFF0007744244000154.tif20170

[0552] AAtR-121 MeG- tRNA(Glu2+Pro3)acc TIFF0007744244000155.tif20170

[0553] AAtR-122 MeG- tRNA(Glu2+Ala2)acc TIFF0007744244000156.tif20170

[0554] AAtR-123 dA- tRNA(Glu2)acc TIFF0007744244000157.tif20170

[0555] AAtR-124 dA- tRNA(Glu2+Leu2)acc TIFF0007744244000158.tif20170

[0556] AAtR-125 dA- tRNA(Glu2+Pro3)acc TIFF0007744244000159.tif20170

[0557] AAtR-126 dA- tRNA(Glu2+Ala2)acc TIFF0007744244000160.tif20170

[0558] AAtR-127 nBuG- tRNA(Glu2)ccc TIFF0007744244000161.tif20170

[0559] AAtR-129 MeG- tRNA(Glu2)agg TIFF0007744244000162.tif20170

[0560] AAtR-130 MeG- tRNA(Glu2)cgg TIFF0007744244000163.tif20170

[0561] AAtR-131 nBuG-tRNA(Asp1+Leu2)aag TIFF0007744244000164.tif19170

[0562] AAtR-132 SPh2Cl-tRNA(Asp1+Leu2)uag TIFF0007744244000165.tif30170

[0563] AAtR-133 SPh2Cl-tRNA(Asp1+Leu2)Lag TIFF0007744244000166.tif30170

[0564] AAtR-134 dA-tRNA(Asp1+Leu2)cag TIFF0007744244000167.tif20170

[0565] AAtR-135 nBuG-tRNA(AsnE2+Leu2)aag TIFF0007744244000168.tif20170

[0566] AAtR-136 SPh2Cl-tRNA(AsnE2+Leu2)uag TIFF0007744244000169.tif32170

[0567] AAtR-137 SPh2Cl-tRNA(AsnE2+Leu2)Lag TIFF0007744244000170.tif32170

[0568] AAtR-138 dA-tRNA(AsnE2+Leu2)cag TIFF0007744244000171.tif20170

[0569] Preparation of initiator aminoacyl-tRNA using aminoacyl-pCpA A reaction mixture containing 25 μM transcribed tRNA(fMet)cau-CA (TR-105), 50 mM HEPES-KOH pH 7.5, 20 mM MgCl2, 1 mM ATP, 0.6 Unit / μL T4 RNA ligase (New England Biolabs), and 0.25 mM aminoacyl pCpA (MT01) was diluted with nuclease-free water and subjected to ligation for 45 minutes at 15°C. Before adding T4 RNA ligase and aminoacyl pCpA, the reaction mixture was heated at 95°C for 2 minutes and then left at room temperature for 5 minutes to allow for tRNA refolding. Sodium acetate was added to the ligation reaction mixture to a concentration of 0.3 M, followed by phenol-chloroform extraction to prepare the initiator aminoacyl-tRNA (AAtR-128). AAtR-128 was recovered by ethanol precipitation and dissolved in 1 mM aqueous sodium acetate before being added to the translation mixture.

[0570] AAtR-128 BdpFL-Phe-tRNA(fMet)cau TIFF0007744244000172.tif22170 Example 5 Preparation of mRNA Template mRNA (MR-1 to MR-8) was synthesized from template DNA (MD-1 to MD-8) by in vitro transcription using the RiboMAX Large Scale RNA production System T7 (Promega, P1300) and purified using the RNeasy mini kit (Qiagen).

[0571] Template DNA SEQ ID NO: 221 (MD-1) DNA sequence: GGCGTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACTTTTATTATTGGTTTTcttATTATTCCGATTGGTTAAGCTTCG

[0572] Template DNA SEQ ID NO: 222 (MD-2) DNA sequence: GGCGTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACTTTTATTATTGGTTTTctgATTATTCCGATTGGTTAAGCTTCG

[0573] Template DNA SEQ ID NO: 223 (MD-3) DNA sequence: GGCGTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACTTTTATTATTGGTTTTcctATTATTGCTATTGGTTAAGCTTCG

[0574] Template DNA SEQ ID NO: 224 (MD-4) DNA sequence: GGCGTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACTTTTATTATTGGTTTTccgATTATTGCTATTGGTTAAGCTTCG

[0575] Template DNA SEQ ID NO: 225 (MD-5) DNA sequence: GGCGTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACTTTTATTATTCTATTTggtATTATTCCGATTCTATAAGCTTCG

[0576] Template DNA SEQ ID NO: 226 (MD-6) DNA sequence: GGCGTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACTTTTATTATTCTATTTgggATTATTCCGATTCTATAAGCTTCG

[0577] Template DNA SEQ ID NO: 227 (MD-7) DNA sequence: GGCGTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACTTTTATTATTCTATTTggaATTATTCCGATTCTATAAGCTTCG

[0578] Template DNA SEQ ID NO: 228 (MD-8) DNA sequence: GGCGTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACTTTTATTATTGGTTTTctaATTATTCCGATTGGTTAAGCTTCG

[0579] Template mRNA SEQ ID NO: 229 (MR-1) RNA sequencing: GGGUUAACUUUAAGAAGGAGAUAUACAUAUGACUUUUUAUUAUUGGUUUUcuuAUUAUUCCGAUUGGUUAAGCUUCG

[0580] Template mRNA SEQ ID NO: 230 (MR-2) RNA sequencing: GGGUUAACUUUAAGAAGGAGAUAUACAUAUGACUUUUAUUAUUGGUUUUcugAUUAUUCCGAUUGGUUAAGCUUCG

[0581] Template mRNA SEQ ID NO: 231 (MR-3) RNA sequencing: GGGUUAACUUUAAGAAGGAGAUAUACAUAUGACUUUUAUUAUUGGUUUUccuAUUAUUGCUAUUGGUUAAGCUUCG

[0582] Template mRNA SEQ ID NO: 232 (MR-4) RNA sequencing: GGGUUAACUUUAAGAAGGAGAUAUACAUAUGACUUUUUAUUAUUGGUUUUccgAUUAUUGCUAUUGGUUAAGCUUCG

[0583] Template mRNA SEQ ID NO: 233 (MR-5) RNA sequencing: GGGUUAACUUUAAGAAGGAGAUAUACAUAUGACUUUUUAUUAUUCUAUUUgguAUUAUUCCGAUUCUAUAAGCUUCG

[0584] Template mRNA SEQ ID NO: 234 (MR-6) RNA sequencing: GGGUUAACUUUAAGAAGGAGAUAUACAUAUGACUUUUUAUUAUUCUAUUUgggAUUAUUCCGAUUCUAUAAGCUUCG

[0585] Template mRNA SEQ ID NO: 235 (MR-7) RNA sequencing GGGUUAACUUUAAGAAGGAGAUAUACAUAUGACUUUUUAUUAUUCUAUUUggaAUUAUUCCGAUUCUAUAAGCUUCG

[0586] Template mRNA SEQ ID NO: 236 (MR-8) RNA sequencing: GGGUUAACUUUAAGAAGGAGAUAUACAUAUGACUUUUAUUAUUGGUUUUcuaAUUAUUCCGAUUGGUUAAGCUUCG

[0587] Example 6 Translational synthesis of peptides Example 6-1 To evaluate the effect of base combinations at positions 32, 33, 37, and 38 of tRNA on translation accuracy, we performed translation experiments in the presence of two types of elongated aminoacyl-tRNAs with anticodons corresponding to different codons within the same codon box. Specifically, template mRNA (MR-2) was prepared using aminoacyl-tRNA (one of AAtR-1 to AAtR-10 and AAtR-105), template mRNA (MR-1) was prepared using aminoacyl-tRNA (one of AAtR-11 to AAtR-20 and AAtR-104), and template mRNA (MR-4) was prepared using aminoacyl-tRNA (one of AAtR-21 to AAtR-30 and AAtR-107). A (MR-3) was translated using aminoacyl-tRNA (AAtR-31 to AAtR-40 and AAtR-106), template mRNA (MR-6) was translated using aminoacyl-tRNA (AAtR-41 to AAtR-50 and AAtR-109), and template mRNA (MR-5) was translated using aminoacyl-tRNA (AAtR-51 to AAtR-60 and AAtR-108), and peptides were translationally synthesized. Note that, in this specification, "AAtR" and "AATR" are used synonymously. That is, "AAtR-105" and "AATR-105" refer to the same tRNA.

[0588] Translation condition 1 We performed a translation experiment to evaluate the amount of misreading of the codon CCG by tRNA carrying the anticodon agg using the PURE system, a reconstituted cell-free protein synthesis system derived from prokaryotes.Specifically, the translation solution (1 mM GTP, 1 mM ATP, 20 mM creatine phosphate, 50 mM HEPES-KOH pH 7.6, 100 mM potassium acetate, 10 mM magnesium acetate, 2 mM spermidine, 1 mM dithiothreitol, 1.5 mg / mL E. coli MRE600 (RNase negative) derived tRNA (Roche), 0.24 μM or 0.26 μM EF-G, 0.24 μM RF2, 0.17 μM RF3, 0.5 μM RRF, 3.7 μM or 4 μg / mL creatine kinase, 2.8 μM or 3 μg / mL myokinase, 1.9 unit / mL or 2 unit / mL inorganic pyrophosphatase, 1.0 μg / mL or 1.1 μg / mL nucleoside diphosphate kinase, 2.5 μM or 2.7 μM IF1, 0.37 μM or 0.4 μM, IF2, 1.4 μM or 1.5 μM, IF3, 37.2 μM or 40 μM, EF-Tu, 49 μM, 54.1 μM, 54.9 μM, or 59 μM, EF-Ts, 0.93 μM or 1 μM, EF-P-Lys, 0.4 unit / μL RNasin® Ribonuclease inhibitor (Promega, N2111), 1.1 μM or 1.2 μM ribosome, 0.5 mM PGA) was added to ARS mix (0.09 μM GlyRS, 0.4 μM or 0.97 μM IleRS, 0.68 μM or 1.64 μM PheRS, 0.16 μM or 0.39 μM ProRS, 0.09 μM or 0.22 μM The translation reaction mixture contained the following: (an ARS (selected from the following amino acid sequences encoded by the mRNA): ThrRS, 2.73 μM AlaRS, 0.04 μM or 0.097 μM LeuRS, 0.04 μM SerRS, and 0.02 μM ValRS); 1 μM template mRNA (MR-4); 0.25 mM each of the natural amino acids encoded by the template mRNA; 10 μM initiator aminoacyl-tRNA (AAtR-128); and 10 μM elongation aminoacyl-tRNAs (AAtR-21 to AAtR-30, and AAtR-107). The mixture was incubated at 37°C for 1 hour to allow translation. Hereinafter, these translation conditions will be referred to as "translation condition 1." The template mRNA was designed so that the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 241 (Pep-3) (amino acid sequences may be represented in this manner with amino acids separated by colons)). If the aminoacyl-tRNA misreads the codon, BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 242 (Pep-4)) is translated.

[0589] A translation experiment was conducted to evaluate the amount of misreading of the codon CCU by tRNAs carrying the anticodon cgg. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-3) and elongated aminoacyl-tRNAs (AAtR-31 to AAtR-40 and AAtR-106) were used. The template mRNA was designed so that the translation product when read correctly was BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 241 (Pep-3)). When misreading by aminoacyl-tRNA occurs, the translation product is BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 242 (Pep-4)).

[0590] A translation experiment was conducted to evaluate the amount of misreading of the codon GGG by tRNAs carrying the anticodon acc. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-6) and elongated aminoacyl-tRNAs (AAtR-41 to AAtR-50 and AAtR-109) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243 (Pep-5)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244 (Pep-6)).

[0591] A translation experiment was conducted to evaluate the amount of misreading of the codon GGU by tRNAs carrying the anticodon ccc. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-5) and elongated aminoacyl-tRNAs (AAtR-51 to AAtR-60 and AAtR-108) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243 (Pep-5)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244 (Pep-6)).

[0592] A translation experiment was conducted to evaluate the amount of misreading of the codon CUG by tRNAs carrying the anticodon aag. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-2) and elongated aminoacyl-tRNAs (AAtR-1 to AAtR-10, and AAtR-105) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239 (Pep-1)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240 (Pep-2)).

[0593] A translation experiment was conducted to evaluate the amount of misreading of the codon CUU by tRNAs carrying the anticodon cag. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-1) and elongated aminoacyl-tRNAs (AAtR-11 to AAtR-20, and AAtR-104) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239 (Pep-1)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240 (Pep-2)).

[0594] Example 6-2 The same experiment as in Example 6-1 was performed using different tRNA bodies. Specifically, template mRNA (MR-2) was translated using aminoacyl-tRNA (AAtR-61 to AAtR-70 and AAtR111), template mRNA (MR-1) was translated using aminoacyl-tRNA (AAtR-71 to AAtR-80 and AAtR-110), template mRNA (MR-2) was translated using aminoacyl-tRNA (AAtR-81 to AAtR-90 and AAtR-113), and template mRNA (MR-1) was translated using aminoacyl-tRNA (AAtR-91 to AAtR-100 and AAtR-112), and peptides were translationally synthesized.

[0595] Translation conditions A translation experiment was conducted to evaluate the amount of misreading of the codon CUG by tRNAs carrying the anticodon aag. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-2) and elongated aminoacyl-tRNAs (AAtR-81 to AAtR-90 and AAtR-113) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239 (Pep-1)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240 (Pep-2)).

[0596] A translation experiment was conducted to evaluate the amount of misreading of the codon CUU by tRNAs carrying the anticodon cag. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-1) and elongated aminoacyl-tRNAs (AAtR-91 to AAtR-100 and AAtR-112) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239 (Pep-1)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240 (Pep-2)).

[0597] A translation experiment was conducted to evaluate the amount of misreading of the codon CUG by tRNAs carrying the anticodon aag. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-2) and elongated aminoacyl-tRNAs (AAtR-61 to AAtR-70 and AAtR-111) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239 (Pep-1)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240 (Pep-2)).

[0598] A translation experiment was conducted to evaluate the amount of misreading of the codon CUU by tRNAs carrying the anticodon cag. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-1) and elongated aminoacyl-tRNAs (AAtR-71 to AAtR-80 and AAtR-110) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeHph:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 239 (Pep-1)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 240 (Pep-2)).

[0599] Example 6-3 Experiments similar to those in Examples 6-1 and 6-2 were carried out using different codons. Specifically, template mRNA (MR-7) was translated using aminoacyl-tRNA (AAtR-44, AAtR-47, or AAtR-48 and AAtR-114), template mRNA (MR-7) was translated using aminoacyl-tRNA (AAtR-101 to AAtR-103 and AAtR-114), and template mRNA (MR-6) was translated using aminoacyl-tRNA (AAtR-101 to AAtR-103 and AAtR-109), to synthesize peptides.

[0600] Translation conditions A translation experiment was conducted to evaluate the amount of misreading of the codon GGA by tRNAs carrying the anticodon acc. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-7) and elongated aminoacyl-tRNAs (AAtR-44, AAtR-47, or AAtR-48, and AAtR-114) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243 (Pep-5)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244 (Pep-6)).

[0601] A translation experiment was conducted to evaluate the amount of misreading of the codon GGA by tRNAs carrying the anticodon gcc. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-7) and elongated aminoacyl-tRNAs (AAtR-101 to AAtR-103 and AAtR-114) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243 (Pep-5)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244 (Pep-6)).

[0602] A translation experiment was conducted to evaluate the amount of misreading of the codon GGG by tRNAs carrying the anticodon gcc. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-6) and elongated aminoacyl-tRNAs (AAtR-101 to AAtR-103 and AAtR-109) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243 (Pep-5)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 244 (Pep-6)).

[0603] Example 6-4 Experiments similar to those in Examples 6-1 to 6-3 were carried out using tRNAs in which the first letter of the anticodon was modified (tRNAs in which the first nucleoside of the anticodon was lysidine). Specifically, template mRNA (MR-1, MR-8, or MR-2) was translated according to the translation condition 1, except that aminoacyl-tRNAs (AAtR-132 or AAtR-133, and AAtR-131 and AAtR-134), or aminoacyl-tRNAs (AAtR-136 or AAtR-137, and AAtR-135 and AAtR-138) were used, and peptides were translationally synthesized. The translation yields of the following translation products were compared. BdpF:Thr:Phe:Ile:Ile:Gly:Phe:nBuG:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 250 (Pep-12)) BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 251 (Pep-13)) BdpF:Thr:Phe:Ile:Ile:Gly:Phe:dA:Ile:Ile:Pro:Ile:Gly(SEQ ID NO: 252(Pep-14))

[0604] Examples 6-5 Experiments similar to those in Examples 6-1 to 6-4 were performed using different amino acids. Specifically, template mRNA (MR-6) was translated using aminoacyl-tRNA (one of AAtR-115 to AAtR-118 and AAtR-109, or one of AAtR-119 to AAtR-122 and AAtR-109, or one of AAtR-123 to AAtR-126 and AAtR-127), template mRNA (MR-4) was translated using aminoacyl-tRNA (one of AAtR-21 to AAtR-30 and AAtR-130), and template mRNA (MR-3) was translated using aminoacyl-tRNA (one of AAtR-31 to AAtR-40 and AAtR-129), and peptides were translationally synthesized.

[0605] Translation conditions A translation experiment was conducted to evaluate the amount of misreading of the codon GGG by tRNAs carrying the anticodon acc. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-6) and elongated aminoacyl-tRNAs (AAtR-115 to AAtR-118 and AAtR-109) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243 (Pep-5)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:Pic2:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 245 (Pep-7)).

[0606] A translation experiment was conducted to evaluate the amount of misreading of the codon GGG by tRNAs carrying the anticodon acc. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-6) and elongated aminoacyl-tRNAs (AAtR-119 to AAtR-122 and AAtR-109) were used. The template mRNA was designed so that the translation product when read correctly was BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeHph:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 243 (Pep-5)). When misreading by aminoacyl-tRNA occurs, the translation product is BdpF:Thr:Phe:Ile:Ile:Leu:Phe:MeG:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 246 (Pep-8)).

[0607] A translation experiment was conducted to evaluate the amount of misreading of the codon GGG by tRNAs carrying the anticodon acc. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-6) and elongated aminoacyl-tRNAs (AAtR-123 to AAtR-126 and AAtR-127) were used. The template mRNA was designed so that the translation product when read correctly would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:nBuG:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 247 (Pep-9)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Leu:Phe:dA:Ile:Ile:Pro:Ile:Leu (SEQ ID NO: 248 (Pep-10)).

[0608] A translation experiment was conducted to evaluate the amount of misreading of the codon CCG by tRNAs carrying the anticodon agg. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-4) and elongated aminoacyl-tRNAs (AAtR-21 to AAtR-30, and AAtR-130) were used. The template mRNA was designed so that the translation product, when read correctly, would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeG:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 249 (Pep-11)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 242 (Pep-4)).

[0609] A translation experiment was conducted to evaluate the amount of misreading of the codon CCU by tRNAs carrying the anticodon cgg. Translation was performed under the same conditions as in the previous section, except that template mRNA (MR-3) and elongated aminoacyl-tRNAs (AAtR-31 to AAtR-40 and AAtR-129) were used. The template mRNA was designed so that the translation product, when read correctly, would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:MeG:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 249 (Pep-11)). When misreading by aminoacyl-tRNA occurs, the translation product would be BdpF:Thr:Phe:Ile:Ile:Gly:Phe:SPh2Cl:Ile:Ile:Ala:Ile:Gly (SEQ ID NO: 242 (Pep-4)).

[0610] Example 7 Analysis of translated peptides The translation product solutions obtained by the translation reactions of Examples 6-1 to 6-5 were diluted 10-fold and analyzed using an LC-FLR-MS system. The retention times of the target translated peptides were identified from the obtained MS data, and the fluorescence peaks at the corresponding retention times were quantified to evaluate the amount of translated peptide. Unless otherwise specified, quantitative evaluation was performed by creating a calibration curve using LCT-67 synthesized in Example 3 as a standard, and calculating the content by relative quantification. Unless otherwise specified, LC-MS analysis was performed according to the conditions of Method 1 in Table 5 below. Furthermore, based on the determined amount of translated peptide, the ratio (%) of misread peptides to the target product was calculated using the following formula. Herein, peptides obtained when correctly read may be referred to as correctly read translation products or target products.

number

[0611] Analysis conditions [Table 5]

[0612] result As a result of the evaluation, the rate of misreading of codons by tRNAs with modified base combinations at positions 32, 33, 37, and 38 tended to increase when the base combinations at positions 32, 33, 37, and 38 were Ser5, Ala1B, and Phe, and tended to decrease when the base combinations at positions 32, 33, 37, and 38 were Pro3, Pro2, Ala2, Leu2, Arg3, and Val2 (Figures 1 to 6, Tables 6 to 11). The tendency for a decrease was particularly large for Pro3, Pro2, Ala2, and Leu2 sequences. Changing the tRNA body did not affect the order of misreading (Figures 7 to 10, Tables 12 to 15). Even when the combination of codons and anticodons to be evaluated was changed, the effect of modifying the base combinations at positions 32, 33, 37, and 38 of the tRNA to specific ones was confirmed to reduce codon misreading (Figures 11 to 13, Tables 16 to 18). Even when the amino acid to be aminoacylated was changed, the effect of modifying the base combinations at positions 32, 33, 37, and 38 of the tRNA to specific ones was confirmed to reduce codon misreading (Figures 14 to 18, Tables 21 to 25). This indicates that modifying the base combinations at positions 32, 33, 37, and 38 of the tRNA can reduce codon misreading. Furthermore, by clarifying the susceptibility of codon misreading for multiple base combinations at positions 32, 33, 37, and 38 of the tRNA, it became possible to customize the tRNA sequence to match the tRNA used, reducing codon misreading while maintaining a certain level of amino acid translation.

[0613] The table below shows the translation results of the CCG codon reading by tRNAs with modified base combinations at positions 32, 33, 37, and 38 and agg or cgg as the anticodon. Compared with tRNA Glu2 (AAtR-24) in which the base combinations at positions 32, 33, 37, and 38 were not modified, tRNAs (AAtR-27 to 30) in which the base combinations were modified to Pro2, Leu2, Ala2, or Pro3 sequences showed a reduction in misreading of the codon (Figure 1).

[0614] [Table 6]

[0615] The table below shows the translation results of the CCU codon read by tRNAs with modified base combinations at positions 32, 33, 37, and 38 and cgg or agg as the anticodon. Compared with tRNA Glu2 (AAtR-34) with unmodified base combinations at positions 32, 33, 37, and 38, tRNAs with modified base combinations at positions 32, 33, 37, and 38 (AAtR33 and AAtR35-40) with modified base combinations to Phe, Arg3, Val2, Pro2, Leu2, Ala2, or Pro3 showed a reduced rate of codon misreading. The tRNAs with modified base combinations to Pro2, Leu2, Ala2, or Pro3 were particularly effective in reducing codon misreading (Figure 2).

[0616] [Table 7]

[0617] The table below shows the translation results of GGG codon reading using tRNAs with modified base combinations at positions 32, 33, 37, and 38 and acc or ccc as the anticodon. Compared with tRNA Glu2 (AAtR-44) without modified base combinations at positions 32, 33, 37, and 38, tRNAs with modified base combinations at positions 32, 33, 37, and 38 (AAtR-43 and AAtR45-50) with modified base combinations to Arg3, Phe, Val2, Pro2, Leu2, Ala2, or Pro3 sequences showed a reduced rate of codon misreading. The effect of reducing codon misreading was particularly high in tRNAs with modified base combinations to Pro2, Leu2, Ala2, or Pro3 sequences (Figure 3).

[0618] [Table 8]

[0619] The table below shows the translation results of GGU codon misreading by tRNAs with altered base combinations at positions 32, 33, 37, and 38 and ccc or acc as the anticodon. Although the amount of peptide translation due to codon misreading was low even in tRNA Glu2 (AAtR-54) with no altered base combinations at positions 32, 33, 37, and 38, the amount of peptide translation due to codon misreading was also kept low in tRNAs (AAtR-52, AAtR-53, and AAtR-55 to 60) with altered base combinations to Phe, Ala1B, Pro2, Leu2, Ala2, Pro3, Arg3, or Val2, demonstrating a similar tendency to that observed in the above examples (Figure 4).

[0620] [Table 9]

[0621] The table below shows the translation results of the CUG codon by tRNAs with modified base combinations at positions 32, 33, 37, and 38 and cag or aag as the anticodon. Compared with tRNA Glu2 (AAtR-4) in which the base combinations at positions 32, 33, 37, and 38 were not modified, tRNAs (AAtR-8 and 9) in which the base combinations were modified to the Pro2 or Pro3 sequence showed reduced codon misreading, and the amount of peptide translation due to codon misreading was kept low even for the Ala2 sequence (Figure 5). [Table 10]

[0622] The table below shows the translation results of the CUU codon read by tRNAs with modified base combinations at positions 32, 33, 37, and 38 and aag or cag as the anticodon. Although the amount of peptide translated due to codon misreading was low even in tRNA Glu2 (AAtR-14) with no modified base combinations at positions 32, 33, 37, and 38, the amount of peptide translated due to codon misreading was also kept low in tRNAs (AAtR-13 and AAtR-15 to -20) in which the base combinations were modified to Phe, Pro, Leu, Ala, Pro, Arg, or Val, showing a similar trend to the above examples (Figure 6).

[0623] [Table 11]

[0624] These results indicate that the rate of misreading of the target peptide is high when the base combination at positions 32, 33, 37, and 38 of the tRNA is Ser5 or Ala1B. The following experiment was conducted to verify whether codon misreading can be reduced by modifying the base combination at positions 32, 33, 37, and 38 of a tRNA having the Ser5 base combination.

[0625] tRNA AsnE2 was selected as a tRNA with a Ser5 sequence at positions 32, 33, 37, and 38. The translation results of the CUG codon read by tRNAs with a tRNA(AsnE2) body, modified nucleotide combinations at positions 32, 33, 37, and 38, and an aag or cag anticodon are shown in the table below. Compared with tRNA(AsnE2) (AAtR-81) with unmodified nucleotide combinations at positions 32, 33, 37, and 38, tRNAs (AAtR-85-90) with modified nucleotide combinations at positions 32, 33, 37, and 38 to Arg3, Val2, Pro2, Leu2, Ala2, or Pro3 sequences showed reduced codon misreading. The tRNAs with modified Pro2, Leu2, Ala2, or Pro3 sequences were particularly effective in reducing codon misreading (Figure 7). [Table 12]

[0626] The table below shows the translation results of the CUU codon read by tRNAs with a tRNA(AsnE2) body, modified base combinations at positions 32, 33, 37, and 38, and cag or aag as the anticodon. Compared with tRNA(AsnE2) (AAtR-91) in which the base combinations at positions 32, 33, 37, and 38 were not modified, tRNAs (AAtR-95 to 100) in which the base combinations were modified to Arg3, Val2, Pro2, Leu2, Ala2, or Pro3 sequences showed a reduced rate of codon misreading. The tRNAs modified to Pro2, Leu2, Ala2, or Pro3 sequences were particularly effective in reducing codon misreading (Figure 8).

[0627] [Table 13]

[0628] These results demonstrate that even in tRNAs prone to codon misreading, codon misreading can be reduced by modifying the base combinations at positions 32, 33, 37, and 38.

[0629] The effect of other tRNA bodies on reducing codon misreading was examined. The translation results of the CUG codon using tRNAs with tRNA(Asp1) bodies, modified base combinations at positions 32, 33, 37, and 38, and anticodons containing either aag or cag, are shown in the table below. Compared with tRNA(Asp1) (AAtR-64) in which the base combinations at positions 32, 33, 37, and 38 were not modified, tRNAs (AAtR-65 to 70) in which the base combinations were modified to Arg3, Val2, Pro2, Leu2, Ala2, or Pro3 sequences showed reduced codon misreading (Figure 9).

[0630] [Table 14]

[0631] The table below shows the translation results of the CUU codon read by tRNAs with a tRNA(Asp1) body, modified base combinations at positions 32, 33, 37, and 38, and cag or aag as the anticodon. Although the amount of peptide translation due to codon misreading was low even with tRNA(Asp1) (AAtR-74) in which the base combinations at positions 32, 33, 37, and 38 were not modified, codon misreading was also reduced in tRNAs (AAtR-76 to 80) in which the base combinations were modified to Phe, Val2, Pro2, Leu2, Ala2, or Pro3, showing a similar trend to the above examples (Figure 10).

[0632] [Table 15]

[0633] The effect of reducing misreading when using a codon with a different third base from the codon used in the above examples was examined. The combinations of bases at positions 32, 33, 37, and 38 were altered, and the translation results for the GGA codon were shown in the table below. When the combinations of bases at positions 32, 33, 37, and 38 of the tRNA with the anticodon acc were altered to the Leu2 sequence or the Pro3 sequence, misreading of the codon GGA was reduced (Figure 11).

[0634] [Table 16]

[0635] The table below shows the translation results of GGA codon reading by tRNAs with ucc or gcc anticodons, in which the base combinations at positions 32, 33, 37, and 38 were altered. When the base combinations at positions 32, 33, 37, and 38 of tRNAs with gcc anticodons were altered to Leu2 or Pro3 sequences, misreading of the GGA codon was reduced (Figure 12).

[0636] [Table 17]

[0637] The table below shows the translation results of GGG codon reading by tRNAs with ccc or gcc anticodons, in which the base combinations at positions 32, 33, 37, and 38 were altered. When the base combinations at positions 32, 33, 37, and 38 of tRNAs with gcc anticodons were altered to Leu2 or Pro3 sequences, misreading of the GGG codon was reduced (Figure 13).

[0638] [Table 18]

[0639] The above results demonstrate that the effect of reducing codon misreading is observed regardless of the type of base at the third letter of the codon. This indicates that the effect of reducing codon misreading according to the present disclosure can be obtained regardless of which codon is used in the codon box. When reprogramming codons to assign multiple different types of amino acids to the same codon box, the effect of the present disclosure can be obtained regardless of the codon used, which is beneficial as it increases the degree of freedom in reprogramming.

[0640] The ability of tRNAs with modified base combinations at positions 32, 33, 37, and 38, and anticodons aag, uag, or cag to read CUU, CUA, or CUG codons was evaluated with and without lysidine modification. tRNA (Asp1+Leu2) and tRNA (AsnE2+Leu2) were used as tRNA bodies. The translation results are shown in the table below. This demonstrated the feasibility of combining this technology with tRNA modification techniques. For quantitative evaluation, a calibration curve was created using LCT-12 as the standard, and the content was calculated by relative quantification. LC-MS analysis was performed according to Method 2 conditions in Table 5.

[0641] [Table 19]

[0642] [Table 20]

[0643] The effect of changing the type of amino acid on reducing codon misreading was examined. The translation results of GGG codon reading using tRNAs with modified base combinations at positions 32, 33, 37, and 38 and acc or ccc as the anticodon are shown in the table below. Codon misreading was reduced in tRNAs with modified base combinations at positions 32, 33, 37, and 38 to Leu2, Ala2, or Pro3 (Figure 14).

[0644] [Table 21]

[0645] The table below shows the translation results of GGG codon reading by tRNAs with modified base combinations at positions 32, 33, 37, and 38 and acc or ccc as the anticodon. A reduction in codon misreading was observed in tRNAs with modified base combinations at positions 32, 33, 37, and 38 to Leu2, Ala2, or Pro3 (Figure 15).

[0646] [Table 22]

[0647] The table below shows the translation results of GGG codon reading by tRNAs with modified base combinations at positions 32, 33, 37, and 38 and acc or ccc as the anticodon. Codon misreading was reduced in tRNAs with modified base combinations at positions 32, 33, 37, and 38 to Leu2, Ala2, or Pro3 (Figure 16).

[0648] [Table 23]

[0649] The table below shows the translation results of CCG codon reading by tRNAs with modified base combinations at positions 32, 33, 37, and 38 and cgg or agg as the anticodon. A reduction in codon misreading was observed in tRNAs with modified base combinations at positions 32, 33, 37, and 38 to Pro2, Leu2, Ala2, or Pro3 (Figure 17).

[0650] [Table 24]

[0651] The table below shows the translation results of CCU codon reading using tRNAs with modified base combinations at positions 32, 33, 37, and 38 and agg or cgg as the anticodon. A reduction in codon misreading was observed in tRNAs with modified base combinations at positions 32, 33, 37, and 38 to Phe, Pro2, Leu2, Ala2, Pro3, Arg3, or Val2, with the Pro2, Leu2, Ala2, and Pro3 sequences showing the most significant effect (Figure 18).

[0652] [Table 25]

[0653] These results indicate that the same effect can be obtained even when the type of amino acid bound to tRNA is changed, and therefore the effect of reducing codon misreading can be obtained regardless of the type of amino acid bound to tRNA or the combination of amino acids.

[0654] While the foregoing invention has been described in detail by way of illustration and illustration for purposes of clarity of understanding, the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties. [Industrial Applicability]

[0655] By using the composition for translation and the method for producing a peptide according to the present disclosure, it is possible to reduce the rate at which an unintended amino acid is erroneously translated due to misreading of a codon by a tRNA when synthesizing a peptide by translating it from a nucleic acid. The composition, method, etc. according to the present disclosure are particularly useful in the field of translational synthesis of peptides.

Claims

1. A composition for translation comprising a first tRNA bound to a first amino acid and a second tRNA bound to a second amino acid, the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, (4) 32U, 33U, 37G, 38U, (5) 32U, 33U, 37A, 38U, or (6) 32C, 33U, 37G, 38A, and the first base of the anticodon of the first tRNA and the second tRNA is different from each other; the second base of the anticodon of the first tRNA and the second tRNA is the same; the third base of the anticodon of the first tRNA and the second tRNA is the same; at least one selected from the first amino acid and the second amino acid is an unnatural amino acid; wherein A is adenine, C is cytosine, G is guanine, and U is uracil (provided that the composition excludes those containing a tRNA in which the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) is 32C, 33U, 37G, 38A and the anticodon is GUC, AUC, AAA, or GAA; excludes those containing a tRNA in which the base sequence at positions 32 to 38 (tRNA numbering rules) is UUGAGGU; and excludes those containing a tRNA in which the base sequence at positions 32 to 38 (tRNA numbering rules) is AUGGGGU).

2. the first base of the anticodon of the first tRNA is A or G, and the first base of the anticodon of the second tRNA is C or U; the first base of the anticodon of the first tRNA is C or U, and the first base of the anticodon of the second tRNA is A or G; The composition of claim 1.

3. The combination of the second and third letter bases of the anticodons of the first and second tRNAs is (i) G as the second character and G as the third character; (ii) A as the second character and G as the third character; (iii) the second character is C and the third character is C; (iv) G as the second character and C as the third character; (v) A as the second character and C as the third character; (vi) G as the second letter and U as the third letter; (vii) the second character is G and the third character is A; or (viii) the second letter is C and the third letter is G; 3. The composition according to claim 1 or 2, wherein

4. The composition of any one of claims 1 to 3, wherein the tRNA body of the first tRNA is a chimeric tRNA body.

5. The composition according to any one of claims 1 to 4, which is a composition for cell-free translation.

6. The composition according to claim 4, wherein the chimeric tRNA body is a chimeric tRNA body in which the bases at positions 32, 33, 37, and 38 (according to the tRNA numbering rules) and the base sequences at other positions are of different origin.

7. The composition according to any one of claims 1 to 6, wherein the first tRNA is an artificial tRNA.

8. The composition according to any one of claims 1 to 7 (however, excluding a composition in which the combination of anticodons of the first tRNA and the second tRNA is GCG and CCG, and a composition in which the combination of anticodons of the first tRNA and the second tRNA is CCG and GCG, when the first tRNA and the second tRNA have the bases specified in (4) of claim 1 at positions 32, 33, 37, and 38 (tRNA numbering rules), and have a chimeric tRNA body having the base sequences of positions 1 to 31 and positions 39 to 74 (tRNA numbering rules) of a tRNA having the base sequence set forth in SEQ ID NO: 255 at positions 1 to 31 and positions 39 to 74 (tRNA numbering rules).

9. The composition of any one of claims 1 to 8, wherein the first tRNA is a tRNA having a chimeric anticodon loop.

10. the combination of bases at positions 32, 33, 37, and 38 (tRNA numbering rules) of the first tRNA is (1) 32U, 33U, 37G, 38A, (2) 32A, 33U, 37G, 38U, (3) 32A, 33U, 37A, 38U, or (4) 32U, 33U, 37G, 38U, The composition according to any one of claims 1 to 9, wherein

11. The composition of any one of claims 1 to 10, wherein the second amino acid is an unnatural amino acid.

12. The method for producing the composition according to any one of claims 1 to 11, comprising the step of preparing the first tRNA by binding the first amino acid to tRNA outside of a translation system, and / or the step of preparing the second tRNA by binding the second amino acid to tRNA outside of a translation system.

13. A method for producing a peptide, comprising translating a nucleic acid using the composition according to any one of claims 1 to 11.

14. A method for reducing misreading of a codon complementary to the anticodon of the second tRNA by the first tRNA, comprising translating a nucleic acid using the composition of any one of claims 1 to 11.

15. A method for reducing misreading of a second codon by a tRNA, the method comprising substituting at least one base selected from the group consisting of positions 32, 33, 37, and 38 (tRNA numbering rules) of a tRNA having an anticodon complementary to a first codon, the first letter of the first codon and the second codon are the same base, the second character of the first codon and the second codon is the same base, The method as described above, wherein the third letters of the first codon and the second codon are different bases.

16. A method for producing a peptide library, comprising translating a nucleic acid library using the composition according to any one of claims 1 to 11.

17. Producing a peptide library using the method of claim 16; and A method for identifying a peptide that binds to a target molecule, comprising the step of contacting the target molecule with the peptide library.

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