High-performance nucleic acid construct and design method for same

Abstract

The present disclosure provides a nucleic acid construct with which protein expression can be controlled. Specifically, the present disclosure provides a nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other. The nucleic acid construct is linked from the 5' side in the order of the 5' UTR, a translated region, and the 3' UTR, and the complementarity between the translated region and the 5' UTR or the 3' UTR is less than 95%. The 3' UTR comprises a portion that is non-complementary to the 5' UTR and a portion that is complementary to the 5' UTR.

Description

High-performance nucleic acid construct and design method thereof

[0001] The present disclosure provides artificial synthetic nucleic acids for enhancing protein expression.

[0002] mRNA medicines are an important modality for COVID-19 vaccines, cancer vaccines, and disease treatments. They exert their therapeutic effects by delivering mRNA into cells to express the active ingredient, a protein. They are considered highly safe because they do not need to be transported into the nucleus and there is a low risk of insertion into the genome.

[0003] Techniques for codon optimization, sequence optimization, and functional sequence addition have been developed to improve translation efficiency. Plasmids encoding the target mRNA are used as starting materials, and after linearization, transcription, capping, and removal of template DNA with DNase are carried out in a test tube. There is also a method in which a poly(A) tail is added enzymatically afterward. Furthermore, a mechanism for promoting translation by circularizing mRNA is known.

[0004] The present inventors provide a nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, wherein the 5' UTR, the translated region, and the 3' UTR are linked in this order from the 5' side, the complementarity between the translated region and the 5' UTR or the 3' UTR is less than 95%, and the 3' UTR comprises a non-complementary portion and a complementary portion to the 5' UTR.

[0005] Thus, the present disclosure provides, for example, the following: <Nucleic Acid Construct A> (Item 1) A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, the 5' UTR, the translated region, and the 3' UTR are linked in this order from the 5' side, the rate of complementarity between the translated region and the 5' UTR or the 3' UTR is less than 95%, and the 3' UTR comprises a non-complementary portion and a complementary portion to the 5' UTR. (Item 2) A nucleic acid construct according to any one of the above items, in which bases in the non-complementary portion have been substituted or deleted. (Item 3) A nucleic acid construct according to any one of the above items, in which all of the non-complementary portions have been deleted. (Item 4) A nucleic acid construct according to any one of the above items, in which the rate of complementarity of the 3' UTR to the 5' UTR is greater than 75% when the length of each non-complementary portion is a single-base substitution. (Item 4-1) The nucleic acid construct according to any one of the above items, wherein the complementarity of the 3'UTR to the 5'UTR is greater than 80% when the length of each of the non-complementary portions is a substitution of only one base. (Item 5) The nucleic acid construct according to any one of the above items, wherein at least one of the non-complementary portions is 2 or more bases in length. (Item 6) The nucleic acid construct according to any one of the above items, wherein at least one of the non-complementary portions is 2 or 3 bases in length, or both. (Item 6-1) The nucleic acid construct according to any one of the above items, wherein the non-complementary portion is 2 or 3 bases in length, or both. (Item 7) The nucleic acid construct according to any one of the above items, wherein at least one of the complementary portions is 5 or more bases in length. (Item 8) The nucleic acid construct according to any one of the above items, wherein at least one of the complementary portions is 5 to 11 bases in length. (Item 9) The nucleic acid construct according to any one of the above items, wherein the non-complementary portions are 2 bases in length. (Item 10) The nucleic acid construct according to any one of the above items, wherein the non-complementary portions are 3 bases in length. (Item 11) The nucleic acid construct according to any one of the above items, wherein the non-complementary portions are all 1 base or more in length and the complementarity rate is higher than 75%. (Item 12) The nucleic acid construct according to any one of the above items, wherein the non-complementary portions are all 1 base or more in length and the complementarity rate is higher than 80%.(Item 13) The nucleic acid construct according to any one of the above items, wherein at least one of the non-complementary portions is 1 base. (Item 14) The nucleic acid construct according to any one of the above items, wherein at least one of the non-complementary portions is 1 base, and the complementary portions are each independently 3 to 11 bases in length. (Item 15) The nucleic acid construct according to any one of the above items, wherein the non-complementary portion is 1 base, and the complementary portions are each independently 3 to 7 bases in length. (Item 16) The nucleic acid construct according to any one of the above items, wherein the non-complementary portion is 2 bases in length, and the complementary portions are each independently 5 to 11 bases in length. (Item 17) The nucleic acid construct according to any one of the above items, wherein the non-complementary portion is 3 bases in length, and the complementary portions are each independently 8 to 11 bases in length. (Item 18) A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, the 5' UTR, the translated region, and the 3' UTR are linked in this order from the 5' side, the 3' UTR comprising a non-complementary portion and a complementary portion to the 5' UTR, the non-complementary portion and the complementary portion alternate, and when the non-complementary portion is two bases, the complementary portion is 5 to 7 bases long in the case of a base substitution and 5 to 11 bases long in the case of a base deletion, and when the non-complementary portion is three bases, the complementary portion is 8 to 9 bases long in the case of a base substitution and 8 to 11 bases long in the case of a base deletion. (Item 18-1) The nucleic acid construct according to any one of the above items, wherein the non-complementary portion is two or more bases. (Item 18-2) The nucleic acid construct according to any one of the above items, wherein the non-complementary portion is two or three bases. (Item 18-3) The nucleic acid construct according to any one of the above items, wherein the non-complementary portion is 2 bases long, and the complementary portion is 5 to 7 bases long in the case of base substitution, or 5 to 11 bases long in the case of base deletion. (Item 18-4) The nucleic acid construct according to any one of the above items, wherein the non-complementary portion is 3 bases long, and the complementary portion is 8 to 9 bases long in the case of base substitution, or 8 to 11 bases long in the case of base deletion. (Item 18-5) The nucleic acid construct according to any one of the above items, wherein the 3'UTR has multiple regions that are partially complementary to the 5'UTR.(Item 19) The nucleic acid construct according to any one of the preceding items, wherein the 5'UTR is selected from at least one nucleic acid sequence selected from the 5'UTR sequences of HBA (α-globin), HBB, HIST1H2BK, ACTB, KRT18, HIST1H1C, HIST1H1E, HIST3H2A, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, PKM, GAPDH, HSD17B4, ETNK1, XAB2, and RRP12, and the nucleic acid sequence set forth in SEQ ID NO: 790, or comprises a nucleic acid sequence of a functional fragment thereof, or is a mutated version of the nucleic acid sequence. (Item 20) The nucleic acid construct according to any one of the above items, wherein the 5'UTR is selected from at least one nucleic acid sequence selected from the 5'UTR sequences of HBA (α-globin), HBB, KLHL11, HIST1H1C, HIST1H2BK, USP11, ACTB, and KRT18, or comprises the nucleic acid sequence of a functional fragment, or is a mutated version of the nucleic acid sequence. <Nucleic Acid Construct B> (Item 21) A nucleic acid construct comprising the nucleic acid construct according to any one of items 1 to 20, further comprising at least one functional untranslated region. (Item 22) The nucleic acid construct according to any one of the above items, wherein the functional untranslated region is contained in the 3'UTR on the 5' side of the 3'UTR portion of a region having partial complementarity with the 5'UTR. (Item 23) The nucleic acid construct according to any one of the preceding items, wherein the functional untranslated region is included in the 3'UTR on the 3' side of the 3'UTR portion of the region having partial complementarity with the 5'UTR. (Item 24) The nucleic acid construct according to any one of the preceding items, wherein the functional untranslated region is included in the 3'UTR on both the 5' and 3' sides of the 3'UTR portion of the region having partial complementarity with the 5'UTR. (Item 25) The nucleic acid construct according to any one of the preceding items, wherein the functional untranslated region is included in the 3'UTR on the 5' side of the 5'UTR portion of the region having partial complementarity with the 5'UTR. (Item 26) The nucleic acid construct according to any one of the preceding items, wherein the functional untranslated region is included in the 3'UTR on the 3' side of the 5'UTR portion of the region having partial complementarity with the 5'UTR.(Item 27) The nucleic acid construct according to any one of the preceding items, wherein the functional untranslated region is included in the 3'UTR on both the 5' and 3' sides of the 5'UTR portion of the region having partial complementarity with the 5'UTR. (Item 27-1) The nucleic acid construct according to any one of the preceding items, wherein the functional untranslated region is included between a region having partial complementarity with the 5'UTR in one 3'UTR and a region having partial complementarity with the 5'UTR in another 3'UTR. (Item 28) The nucleic acid construct according to any one of the above items, wherein the functional non-translated region is at least one selected from a translation control sequence, a nucleic acid construct degradation control sequence, a nucleic acid construct intracellular localization control sequence, a nucleic acid construct extracellular secretion control sequence, a nucleic acid construct extracellular vesicle encapsulation control sequence, a protein / peptide binding sequence, a small molecule compound binding sequence, a small non-coding RNA target sequence such as miRNA, a large molecule non-coding RNA target sequence, a nucleic acid aptamer or its recognition sequence, a ribozyme sequence, a spacer / stuffer sequence, a higher-order structure sequence, a modified nucleic acid-containing sequence, and a labeling compound-containing sequence such as a fluorescent dye. (Item 29) The nucleic acid construct according to any one of the above items, wherein the functional non-translated region is at least one selected from a translation control sequence, a nucleic acid construct degradation control sequence, a nucleic acid construct intracellular localization control sequence, a protein / peptide binding sequence, and a small non-coding RNA target sequence such as miRNA. <Nucleic acid construct C: Composite nucleic acid construct> (Item 30) A nucleic acid construct comprising a repeat sequence of A (adenine) on the 3' side of the nucleic acid construct according to any one of the above items (also referred to as a "composite nucleic acid construct"). (Item 31) The nucleic acid construct according to any one of the above items, wherein the length of the repeat sequence of A is 100 to 300. (Item 32) The nucleic acid construct according to any one of the above items, wherein the length of the repeat sequence of A is 120 to 200.<Design method> (Item 33) A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translation region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are to be substituted or deleted. (Item 34) A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct having a 5'UTR, a translated region encoding the protein, and a 3'UTR linked in this order from the 5' side, the method comprising at least: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and has alternating non-complementary and complementary portions; (2) selecting the base lengths of the non-complementary and complementary portions; and (3) selecting whether bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein and the natural protein is selected from a database related to gene expression. (Item 35) The method for designing a nucleic acid construct according to any one of the above items, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases. (Item 36) A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translation region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, and the natural protein is selected from a database created by a ribosome profiling method.(Item 37) The method for designing a nucleic acid construct according to any one of the above items, further comprising a step of selecting a 5'UTR having a base length of 25 to 100 bases. (Item 37-1) The ribosome profiling is a method for comprehensively analyzing mRNA fragments protected by ribosomes using a next-generation sequencer. This method for designing a nucleic acid construct according to any one of the above items can quantitatively indicate to what extent each mRNA is translated in a cell. (Item 38) A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct having a 5'UTR, a translated region encoding the protein, and a 3'UTR linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and has alternating non-complementary and complementary portions; (2) selecting the base lengths of the non-complementary and complementary portions; and (3) selecting whether bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein and the natural protein is selected from nucleic acid constructs that have been translated in a database related to gene expression. (Item 39) The method for designing a nucleic acid construct according to Item 38, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases. (Item 40) A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translation region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the following steps: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, and the natural protein has one or more ribosome-protected mRNA fragments in ribosome profile analysis or is detected in proteome analysis.(Item 41) The method for designing a nucleic acid construct according to any one of the preceding items, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases. (Item 42) A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translational region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, the natural protein is selected from a database created by ribosome profile analysis, the selected protein contains one or more ribosome-protected mRNA fragments, and the selected protein is within the top 200 of the value obtained by dividing the number of ribosome-protected mRNA fragments by the mRNA expression level. (Item 43) A method for designing a nucleic acid construct according to any one of the above items, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases. (Item 44) A method for designing a nucleic acid construct according to any one of the above items, further comprising the step of selecting the length of a poly-A tail. (Item 45) A method for designing a nucleic acid construct according to any one of the above items, wherein the length of the poly-A tail is selected in the range of 50 to 300. (Item 46) A method for designing a nucleic acid construct according to any one of the above items, comprising the steps of synthesizing the nucleic acid construct, introducing the nucleic acid construct into the desired cell, and evaluating the expression level of the protein. (Item 47) A nucleic acid construct designed by the method according to any one of the above items, in which a 5'UTR, a translated region encoding a desired protein, and a 3'UTR are linked in this order from the 5' side. (Item 48) A nucleic acid construct according to any one of the above items, wherein the complementarity of the 3'UTR to the 5'UTR is higher than 75% when the length of each non-complementary portion is a single-base substitution.(Item 49) The nucleic acid construct according to any one of the preceding items, wherein at least one of the non-complementary portions is two or more bases in length. (Item 50) The nucleic acid construct according to any one of the preceding items, wherein when the non-complementary portion is two bases, the complementary portion is 5 to 7 bases in length in the case of base substitution and 5 to 11 bases in length in the case of base deletion. (Item 51) The nucleic acid construct according to any one of the preceding items, wherein when the non-complementary portion is three bases, the complementary portion is 8 to 9 bases in length in the case of base substitution and 8 to 11 bases in length in the case of base deletion. (Item 52) The nucleic acid construct according to any one of the preceding items, wherein the 5'UTR is selected from at least one nucleic acid sequence selected from the 5'UTR sequences of HBA (α-globin), HBB, HIST1H2BK, ACTB, KRT18, HIST1H1C, HIST1H1E, HIST3H2A, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, PKM, GAPDH, HSD17B4, ETNK1, XAB2, and RRP12, and the nucleic acid sequence set forth in SEQ ID NO: 790, or comprises a nucleic acid sequence of a functional fragment thereof, or is a mutated version of the nucleic acid sequence. (Item 53) The nucleic acid construct according to any one of Items 47 to 52, wherein the 5'UTR is selected from at least one nucleic acid sequence selected from the 5'UTR sequences of HBA (α-globin), HBB, KLHL11, HIST1H1C, HIST1H2BK, USP11, ACTB, and KRT18, or comprises the nucleic acid sequence of a functional fragment, or is a mutated version of the nucleic acid sequence. <Use of Nucleic Acid Construct> (Item 54) A composition comprising the nucleic acid construct according to any one of the above items. (Item 55) A formulation comprising the nucleic acid construct according to any one of the above items. (Item 56) A method for producing the nucleic acid construct, composition, or formulation according to any one of the above items. (Item 57) A method for expressing a protein by introducing the nucleic acid construct, composition, or formulation according to any one of the above items into a cell.(Item 58) A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translation region; 2) a step in which a library of nucleic acid constructs of multiple sequences is constructed, which includes the sequence information of the translation region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap and its related structure and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3. (Item 59) A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR is designed that includes a non-natural sequence that corresponds to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 60) A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translation region; 2) a step in which 5'UTR and 3'UTR are designed, each including complementary and non-complementary portions; 3) a step in which a library of nucleic acid constructs having multiple sequences is constructed, each including the translation region, 5'UTR and 3'UTR designed in steps 1 and 2, and optionally a 5'cap and its analogous structure and a polyA tail; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 4.(Item 61) A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translation region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR is designed that includes a complementary or non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed that includes the translation region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 62) A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR including a naturally occurring sequence is designed; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs of multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 63) A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs of multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.(Item 64) A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs of multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 65) A system comprising: 1) a sequence information providing unit in which a user provides sequence information of a translation region; 2) a library generating unit that generates a nucleic acid construct library containing a plurality of composite sequences each including a nucleic acid sequence encoding the translation region, a 5' cap structural sequence, and an optimized UTR, and performs polyA optimization to optimize the polyA tail as needed; 3) an evaluation unit that evaluates the expression level of a protein by the nucleic acid construct library; and 4) a selection unit that selects, as needed, mRNA that achieves a desired value or level from the nucleic acid construct library. (Item 66) A system for generating an optimized nucleic acid construct, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translation region; 2) a selection unit in which a 5'UTR including a naturally occurring sequence is selected; 3) a design unit in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR of the naturally occurring sequence is designed; 4) a structure selection unit in which a structure of other portions of the nucleic acid construct is selected; 5) an evaluation unit in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated; and 6) a desired selection unit in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.(Item 67) A system for designing one or more nucleic acid constructs that express a protein desired by a user, comprising: 1) a selection unit that selects a 5'UTR structure nucleic acid sequence from a database related to expression of the protein, 2) a structure selection unit that selects a 3'UTR structure nucleic acid sequence including complementary and non-complementary portions thereto, 3) a 5'cap structure sequence, the 5'UTR structure, the translated region of the protein, and the 3'UTR structure, and 4) an optimization unit that optimizes a polyA tail. (Item 68) A system for designing a nucleic acid construct according to any one of the preceding items, wherein the selection unit further comprises an evaluation unit that evaluates the expression level of the protein. (Item 68-1) A system for designing protein expression of a nucleic acid construct having a 5'UTR structure and a 3'UTR structure, comprising: 1) a design unit that designs a nucleic acid sequence including a complementary / non-complementary portion to all or a portion of the 5'UTR, and 2) a sequence adding unit that adds the nucleic acid sequence to the 3'UTR structure. (Item 69) A system for generating an optimized nucleic acid construct, comprising: a sequence information input unit that inputs sequence information of a translated region of a nucleic acid construct having the translated region, a first transmission unit that transmits the sequence information input from the sequence information input unit, a receiving unit that receives the sequence information transmitted from the first transmission unit, a first storage unit that stores the sequence information received by the receiving unit, a second storage unit that stores information on a predetermined untranslated region of the nucleic acid construct, a third storage unit that stores information on a predetermined polyA tail of the nucleic acid construct, and an optimization unit (which may include AI) that optimizes the sequence information. (Item 70) A program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a nucleic acid construct library is generated that includes a plurality of composite sequences each including a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR (including a step of optimizing a polyA tail, if necessary); 3) a step in which the amount of protein expression by the nucleic acid construct library is evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the nucleic acid construct library, if necessary.(Item 71) A program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translation region; 2) a step in which a 5'UTR including a naturally occurring sequence is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR of the naturally occurring sequence is designed; 4) a step in which the structure of other portions of the nucleic acid construct is selected; 5) a step in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the result of step 5. (Item 72) A program encoding a procedure for causing a computer to execute a method for designing one or more nucleic acid constructs that express a protein desired by a user, the method comprising the steps of: 1) selecting a 5'UTR structure nucleic acid sequence from a database related to expression of the protein, 2) selecting a 3'UTR structure nucleic acid sequence including complementary and non-complementary portions thereto, 3) combining a 5'cap structure sequence, the 5'UTR structure, the translated region of the protein, and the 3'UTR structure, and 4) optimizing a polyA tail. (Item 73) The program according to any one of the above items, wherein the method further comprises, in addition to step 1), a step of evaluating the expression level of the protein. (Item 74) A program encoding steps for causing a computer to execute a method for controlling protein expression of a nucleic acid construct having a 5'UTR structure and a 3'UTR structure, the method comprising: 1) designing a nucleic acid sequence including a complementary or non-complementary portion to all or a portion of the 5'UTR; and 2) adding the nucleic acid sequence to the 3'UTR structure.(Item 75) A recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a nucleic acid construct library is generated containing a plurality of composite sequences each containing a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR in combination (including a step of optimizing a polyA tail, if necessary); 3) a step in which the nucleic acid construct library evaluates the amount of protein expression; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the nucleic acid construct library, if necessary. (Item 76) A recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region, 2) a step in which a 5'UTR comprising a naturally occurring sequence, 3) a step in which a 3'UTR comprising portions complementary and non-complementary to the 5'UTR of the naturally occurring sequence, 4) a step in which a structure of other portions of the nucleic acid construct is selected, 5) a step in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated, and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the result of step 5. (Item 77) A method, system, program, or recording medium according to any one of the above or below items, wherein the nucleic acid construct comprises RNA. (Item 78) A method, system, program, or recording medium according to any one of the above or below items, wherein the nucleic acid construct comprises mRNA. (Item 79) A recording medium storing a program encoding the steps for causing a computer to execute a method for designing one or more nucleic acid constructs that express a protein desired by a user, the method comprising the steps of: 1) selecting a 5'UTR structure nucleic acid sequence from a database related to the expression of the protein; 2) selecting a 3'UTR structure nucleic acid sequence including complementary and non-complementary portions thereto; 3) combining a 5'cap structure sequence, the 5'UTR structure, the translation region of the protein, and the 3'UTR structure; and 4) optimizing the poly A tail.(Item 80) The recording medium according to any one of the above items, wherein the method further comprises a step of evaluating the expression level of the protein in step 1). (Item 81) A recording medium storing a program encoding steps for causing a computer to execute a method for controlling protein expression of a nucleic acid construct having a 5'UTR structure and a 3'UTR structure, by: 1) designing a nucleic acid sequence including a complementary / non-complementary portion to all or a portion of the 5'UTR, and 2) adding the nucleic acid sequence to the 3'UTR structure. (Item 82) A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a library construction unit in which a library of nucleic acid constructs of multiple sequences is constructed containing the sequence information of the translated region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap and its analogous structure, and a polyA tail; 3) an evaluation unit in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a desired selection unit in which a nucleic acid construct that achieves a desired value or level is selected from the results of steps 3. (Item 83) A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence design unit that designs a 5'UTR or 3'UTR containing a naturally occurring sequence; 3) a non-natural sequence design unit that designs a 5'UTR or 3'UTR containing a non-natural sequence corresponding to the 5'UTR or 3'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that contain the translated region and 5'UTR and 3'UTR designed in 1 to 3 above, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit that evaluates the functions of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of 5 above.(Item 84) A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing section in which a user provides sequence information of a translated region; 2) a design section that designs 5'UTR and 3'UTR that include complementary and non-complementary portions; 3) a library construction section that constructs a library of nucleic acid constructs of multiple sequences that include the translated region, 5'UTR, and 3'UTR designed in steps 1 and 2, in combination with a 5'cap and its related structure and a polyA tail as needed; 4) an evaluation section that evaluates the functions of the nucleic acid constructs in the library; and 5) a structure selection section that selects nucleic acid constructs that achieve a desired value or level from the results of steps 4 and 5. (Item 85) A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence design unit that designs a 5'UTR or 3'UTR containing a naturally occurring sequence; 3) a complementary / non-complementary design unit that designs a 3'UTR or 5'UTR containing a complementary / non-complementary portion to the 5'UTR or 3'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that contain the translated region, 5'UTR, and 3'UTR designed in accordance with 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit that evaluates the functions of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of 5. (Item 86) A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence design unit that designs a 5'UTR containing a naturally occurring sequence; 3) a complementary / non-complementary design unit that designs a 3'UTR containing a complementary / non-complementary portion to the 5'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that contain the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit that evaluates the functions of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of step 5.(Item 87) A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a sequence selection unit in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a complementary / non-complementary design unit in which a 3'UTR including a complementary / non-complementary portion to the 5'UTR is designed; 4) a library construction unit in which a library of nucleic acid constructs of multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its related structure and a polyA tail as needed; 5) an evaluation unit in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a structure selection unit in which a nucleic acid construct that achieves a desired value or level is selected from the results of steps 5. (Item 88) A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of the translated region; 2) a natural sequence selection unit in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a complementary / non-complementary design unit in which a 3'UTR including a complementary / non-complementary portion to the 5'UTR is designed; 4) a library construction unit in which a library of nucleic acid constructs of multiple sequences including the translated region, 5'UTR and 3'UTR designed in 1 to 3 above, in combination with a 5'cap and its related structure and a polyA tail as needed; 5) an evaluation unit in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a structure selection unit in which a nucleic acid construct that achieves a desired value or level is selected from the results of 5 above. (Item 89) A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the program comprising: 1) a step in which a user provides sequence information of a translation region; 2) a step in which a library of nucleic acid constructs of multiple sequences is constructed, the library including the sequence information of the translation region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap and its related structure and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3.(Item 90) A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR is designed that includes a non-natural sequence that corresponds to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 91) A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which 5'UTR and 3'UTR are designed, each including complementary and non-complementary portions; 3) a step in which a library of nucleic acid constructs having multiple sequences is constructed, each including the translated region, 5'UTR and 3'UTR designed in steps 1 and 2, and optionally a 5'cap and its analogous structure and a polyA tail; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 4. (Item 92) A program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR is designed that includes a complementary or non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.(Item 93) A program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR including a naturally occurring sequence is designed; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed, the library including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 94) A program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs of multiple sequences including the translated region, 5'UTR and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.(Item 95) A program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs of multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 96) A recording medium storing a program encoding the steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the steps including: 1) a step in which a user provides sequence information of a translation region; 2) a step in which a library of nucleic acid constructs of multiple sequences is constructed, which includes the sequence information of the translation region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap and its related structure and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3. (Item 97) A recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the steps including: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR is designed that includes a non-natural sequence that corresponds to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.(Item 98) A recording medium storing a program encoding the steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the steps including: 1) a step in which a user provides sequence information of the translated region; 2) a step in which 5'UTR and 3'UTR are designed, each including complementary and non-complementary portions; 3) a step in which a library of nucleic acid constructs having multiple sequences is constructed, each including the translated region, 5'UTR and 3'UTR designed in steps 1 and 2, and optionally a 5'cap and its analogous structure and a polyA tail; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 4. (Item 99) A recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR is designed that includes a complementary or non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 100) A recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR including a naturally occurring sequence is designed; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed, the library including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.(Item 101) A recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the steps including: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs of multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its related structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5. (Item 102) A recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR is designed, the 3'UTR including a complementary and non-complementary portion to the 5'UTR; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed, the library including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

[0006] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0007] The nucleic acid constructs of the present disclosure are high performance nucleic acid constructs.

[0008] Figure 1 shows the high-performance 5'UTR selection in Example 1. The translation level of each gene was calculated using polysome profiling data from 293 cells in the gene expression information database NCBI Gene Expression Omnibus (GEO). Ribo-seq indicates the number of ribosomes bound, and RNA-seq indicates the RNA quantification value. Ratio indicates the ratio of the Ribo-seq value to the RNA-seq value. The top 200 genes are sorted in descending order of ratio value and shown. Figure 2 shows the high-performance 5'UTR selection in Example 1. The translation level of each gene was calculated using polysome profiling data from mouse skeletal muscle tissue in the gene expression information database NCBI Gene Expression Omnibus (GEO). Ribo-seq indicates the number of ribosomes bound, and RNA-seq indicates the RNA quantification value. Ratio indicates the ratio of Ribo-seq values ​​to RNA-seq values. The top 200 genes are sorted in descending order of ratio value and shown in the figure. Figure 3 shows the selection of high-performance 5'UTRs in Example 1. The translation level of each gene was calculated using polysome profiling data from umbilical cord blood-derived erythroblasts in the gene expression information database NCBI Gene Expression Omnibus (GEO). Ribo-seq indicates the number of ribosomes bound, and RNA-seq indicates the RNA quantification value. Ratio indicates the ratio of Ribo-seq values ​​to RNA-seq values. The top 200 genes are sorted in descending order of ratio value and shown in the figure. Figure 4 shows the selection of high-performance 5'UTRs in Example 1. The table shows the translation levels of the globin gene family in 293 cells, adult peripheral blood-derived erythroblasts (erythroid cells), and umbilical cord blood-derived erythroblasts (erythroid cells). The values ​​in the table indicate ratio values. ND indicates that the level was below the detection limit. Figure 5 shows the verification of the expression level of the high-performance 5'UTR in Example 2. In 293 cells, the expression levels of mRNAs with E2Crimson as the 5'UTR and ORF of highly translated genes were verified. The ratio of the E2Crimson fluorescence measurement value to the calcein fluorescence measurement value was taken as the relative expression level of E2Crimson protein in each mRNA.Measurements were performed on multiple samples for each mRNA, with the average values ​​shown in the bar graph and the individual values ​​for each sample shown in the black plot. Figure 6 shows the verification of the expression level of a high-performance 5'UTR in Example 2. The expression level of mRNAs containing E2Crimson as the 5'UTR and ORF of highly translated genes was verified in C2C12 cells. The ratio of the E2Crimson fluorescence measurement value to the calcein fluorescence measurement value was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, with the average values ​​shown in the bar graph and the individual values ​​for each sample shown in the black plot. Figure 7 shows the improvement in expression efficiency through partial complementarity between the 5'-3' untranslated regions in Example 3. This shows a method for designing non-complementary portions when designing a 3'UTR so that the 5'-3' untranslated regions are partially complementary to each other for mRNAs equipped with a high-performance 5'UTR. Figure 7(1) shows a design method for achieving non-complementarity by substituting specific bases in the 3'UTR. Figure 7(2) shows a design method for achieving non-complementarity by removing specific bases in the 3'UTR. Figure 7(3) shows a design method that combines the above base substitution and base removal. Figure 7(4) shows a design method for achieving non-complementarity by adding specific bases to the 3'UTR. The rectangles in the figures indicate complementary regions. Figure 8 shows the improvement in expression efficiency achieved by partial complementarity between the 5'-3' untranslated regions in Example 3. In 293 cells, the expression levels of mRNAs containing GAPDH as the 5'UTR, E2Crimson as the ORF, and various partially complementary sequences designed by the base substitutions shown in Figure 7(1) as the 3'UTR were examined. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to determine the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, with individual values ​​for each sample plotted in black. Figure 9 shows the improvement in expression efficiency due to partial complementarity between the 5'-3' untranslated regions in Example 3. In 293 cells, the expression levels were examined for mRNAs with various partially complementary sequences designed using the Pfizer sequence as the 5'UTR, E2Crimson as the ORF, and the base substitutions shown in Figure 7(1) as the 3'UTR.The ratio of E2 Crimson fluorescence measurements to calcein fluorescence measurements was taken as the relative expression level of E2 Crimson protein for each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, with individual values ​​for each sample indicated by black plots. Figure 10 shows the improvement in expression efficiency due to partial complementarity between the 5'-3' untranslated regions in Example 3. In 293 cells, the expression levels were examined for mRNAs with the HSD17B4 gene sequence as the 5' UTR, E2 Crimson as the ORF, and various partially complementary sequences designed by the base substitutions shown in Figure 7(1) as the 3' UTR. The ratio of E2 Crimson fluorescence measurements to calcein fluorescence measurements was taken as the relative expression level of E2 Crimson protein for each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, with individual values ​​for each sample indicated by black plots. Figure 11 shows the improvement in expression efficiency due to partial complementarity between the 5'-3' untranslated regions in Example 3. In 293 cells, the expression levels were examined for mRNAs containing the Pfizer sequence as the 5'UTR, various partially complementary sequences designed with the base substitutions shown in Figure 7(2) as the 3'UTR, and SARS CoV2 Spike and E2Crimson fusion protein as ORFs. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, with individual values ​​for each sample plotted in black. Figure 12 shows the improvement in expression efficiency due to partial complementarity between the 5'-3' untranslated regions in Example 3. In 293 cells, the expression levels of mRNAs containing the Pfizer sequence as the 5'UTR, E2Crimson as the ORF, and various partially complementary sequences designed by base deletion as shown in Figure 7(2) as the 3'UTR were examined. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black.Figure 13 shows the improvement in expression efficiency due to partial complementarity between the 5'-3' untranslated regions in Example 3. In 293 cells, the expression levels were examined for mRNAs containing the Pfizer sequence as the 5'UTR, various partially complementary sequences designed by base deletion as shown in Figure 7(2) as the 3'UTR, and SARS CoV2 Spike and E2Crimson fusion protein as ORFs. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black plots. Figure 14 shows the improvement in expression efficiency due to partial complementarity between the 5'-3' untranslated regions in Example 3. A schematic diagram of the nucleic acid construct of the present disclosure is shown. The nucleic acid construct of the present disclosure has a region (1) in its 3' untranslated region that is partially complementary to the 5' untranslated region. Non-complementary portions (2) and complementary portions (3) alternate in region (1), and the fractional portion (4) is at least one base long and is equal to or shorter than the base length of the complementary portion (3). In mRNAs having various partially complementary sequences designed by base substitution as shown in Figure 7 (1), when the non-complementary portion is one base, the complementary portion is preferably 3 to 6 bases long; when the non-complementary portion is two bases, the complementary portion is preferably 5 to 7 bases long; and when the non-complementary portion is three bases, the complementary portion is preferably 8 to 9 bases long. In mRNAs having various partially complementary sequences designed by base removal as shown in Figure 7 (2), when the non-complementary portion is one base, the complementary portion is preferably 3 to 9 bases long; when the non-complementary portion is two bases, the complementary portion is preferably 5 to 11 bases long; and when the non-complementary portion is three bases, the complementary portion is preferably 8 to 11 bases long. 15 shows the verification of the effect of partial complementation in the high-performance 5'UTR of Example 4. In 293 cells, the expression level was verified for mRNA having E2Crimson as the ORF and a sequence in which the complementary-non-complementary ratio becomes 8-2 due to base removal as the 3'UTR.The 5'UTRs examined were HBB, HIST1H1C, HIST1H1E, HIST3H2A, HIST1H2BK, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, ACTB, KRT18, and PKM. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are plotted in black. Figure 16 shows the verification of the effect of partial complementation on the high-performance 5'UTR of Example 4. This is the case of base substitution. In 293 cells, the expression levels of mRNAs with E2Crimson as the ORF and a 3'UTR sequence with a complementary-to-non-complementary ratio of 9-3 due to base substitution were examined. The 5'UTRs examined were HBB, HIST1H1C, HIST1H1E, HIST3H2A, HIST1H2BK, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, ACTB, KRT18, and PKM. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black. Figure 17 shows the verification of the effect of partial complementarity in the high-performance 5'UTR of Example 4. Expression levels were verified for mRNAs with HBB, HIST1H2BK, ACTB, or KRT18 as the 5'UTR, E2Crimson as the ORF, and a 3'UTR with a complementary-noncomplementary sequence of 8-2, 7-2, or 10-3 due to base deletion, or a complementary-noncomplementary sequence of 6-2 or 9-3 due to base substitution. Figure 18 shows the results of Example 5. Expression levels were verified for mRNAs with the Pfizer sequence as the 5'UTR, the E2Crimson ORF, the Pfizer sequence as the functional 3'UTR, and a complementary-noncomplementary sequence of 8-2, 7-2, or 10-3 between the stop codon and this functional 3'UTR, or a complementary-noncomplementary sequence of 6-2 or 9-3 due to base substitution.The ratio of E2 Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2 Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are plotted in black. Figure 19 is a schematic diagram of the system of the present invention. Figure 20 shows the results of Example 5. Expression levels were examined for mRNAs with HBA (α-globin), HIST1H1C, USP11, or HIST1H2BK as the 5'UTR, SARS CoV2 Spike or E2 Crimson fusion protein as the ORF, a stuffer sequence as the functional 3'UTR, and either 8-2, 7-2, or 10-3 complementary-noncomplementary sequences due to base deletion between this functional 3'UTR and the poly(A) tail, or 6-2 or 9-3 complementary-noncomplementary sequences due to base substitution. The ratio of E2 Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2 Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, with the average values ​​shown in the bar graph and the individual values ​​for each sample shown in the black plot. Figure 21 shows the results of Example 5. Expression levels were verified by luciferase luminescence measurement for mRNAs containing HBA (α-globin) as the 5'UTR, the modified luciferase gene luc2 as the ORF, a 5xFlag tag fusion protein, a stuffer sequence as the functional 3'UTR, and either 8-2 or 10-3 complementary-non-complementary sequences due to base deletion, or 6-2 or 9-3 complementary-non-complementary sequences due to base substitution, between this functional 3'UTR and the polyA tail. Measurements were performed on multiple samples for each mRNA, with the average values ​​shown in the bar graph and the individual values ​​for each sample shown in the black plot. Figure 22 shows the results of Example 5. The expression levels of mRNAs were examined, which had HBA (α-globin) as the 5'UTR, E2Crimson as the ORF, HBA (α-globin) 3'UTR as the functional 3'UTR, and either 8-2, 7-2, or 10-3 complementary-non-complementary sequences due to base deletion or 6-2 or 9-3 complementary-non-complementary sequences due to base substitution between this functional 3'UTR and the polyA tail or between the functional 3'UTR and the stop codon.The ratio of E2 Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2 Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, with the average values ​​shown in the bar graph and the individual values ​​for each sample shown in the black plot. Figure 23 shows the results of Example 5. Expression levels were verified by luciferase luminescence measurement for mRNAs containing HBA (α-globin) as the 5'UTR, the modified luciferase gene luc2 as the ORF, a 5xFlag tag fusion protein, CYBA or ARE as the functional 3'UTR, and either 8-2, 7-2, or 10-3 complementary-noncomplementary sequences due to base deletion, or 6-2 or 9-3 complementary-noncomplementary sequences due to base substitution, between this functional 3'UTR and the stop codon. Measurements were performed on multiple samples for each mRNA, with the average values ​​shown in the bar graph and the individual values ​​for each sample shown in the black plot. Figure 24 shows the results of Example 4. The expression levels of mRNAs were verified by luciferase luminescence measurement. The mRNAs had a 5'UTR that was a high-performance 5'UTR but not registered in gene expression databases (non-naturally occurring sequences), an ORF containing the modified luciferase gene luc2, a 5xFlag tag fusion protein, and a 3'UTR containing either a base deletion resulting in a complementary-to-noncomplementary sequence of 8-2 or a base substitution resulting in a complementary-to-noncomplementary sequence of 6-2. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are plotted in black. Figure 25 shows a schematic diagram of mRNAs containing the known sequence KRT18 as the 3'UTR, an ORF containing the modified luciferase gene luc2, a 5xFlag tag fusion protein, and a 5'UTR containing either a base deletion resulting in a complementary-to-noncomplementary sequence of 8-2, 7-2, or 10-3 or a base substitution resulting in a complementary-to-noncomplementary sequence of 6-2 or 9-3.Figure 26 shows schematic diagrams of mRNAs having a known 5'UTR sequence, HBA (α-globin), and a functional 5'UTR sequence, EMCV IRES, an ORF, a modified luciferase gene luc2, a 5xFlag tag fusion protein, and a 3'UTR that results in 8-2, 7-2, or 10-3 complementary-non-complementary sequences due to base deletion, or 6-2 or 9-3 complementary-non-complementary sequences due to base substitution, and mRNAs having a 5'UTR that results in 8-2, 7-2, or 10-3 complementary-non-complementary sequences due to base deletion, or 6-2 or 9-3 complementary-non-complementary sequences due to base substitution, and a functional 5'UTR sequence, EMCV IRES, an ORF, a modified luciferase gene luc2, a 5xFlag tag fusion protein, and KRT18 as the 3'UTR. Figure 27 shows a schematic diagram of an mRNA having the known sequence HBA (α-globin) as the 5'UTR, the modified luciferase gene luc2 and a 5xFlag tag fusion protein as the ORF, a complementary-non-complementary sequence of 8-2 due to base deletion as the 3'UTR, and a functional 3'UTR sequence of CYBA or ARE, and an mRNA having the complementary-non-complementary sequence of 8-2 due to base deletion as the 5'UTR, the modified luciferase gene luc2 and a 5xFlag tag fusion protein as the ORF, a known sequence KRT18 as the 3'UTR, and a functional 3'UTR sequence of CYBA or ARE. Figure 28 shows a schematic diagram of an mRNA having a known 5'UTR sequence, HBA (α-globin), a functional 5'UTR sequence, the EMCV IRES, an ORF, a modified luciferase gene luc2, a 5xFlag tag fusion protein, a 3'UTR sequence, complementary-non-complementary 8-2 by base deletion, and a functional 3'UTR sequence, CYBA or ARE. Figure 29 shows a schematic diagram of an mRNA having a 5'UTR sequence, complementary-non-complementary 8-2 by base deletion, the EMCV IRES, an ORF, a modified luciferase gene luc2, a 5xFlag tag fusion protein, a 3'UTR sequence, KRT18, a known 3'UTR sequence, and a functional 3'UTR sequence, CYBA or ARE.

[0009] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0010] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0011] As used herein, "about" usually refers to significant figures, and if not, typically refers to plus or minus 10% of the indicated value.

[0012] As used herein, the term "untranslated region (UTR)" refers to a region, excluding the cap structure and polyA tail, that is not translated into a substance such as a protein and is located before and after the coding region that is translated into a substance such as a protein in a nucleic acid such as mRNA that contains a portion that encodes information for a substance such as a protein. A UTR located on the 5' side of a nucleic acid is called a 5'UTR, and a UTR located on the 3' side is called a 3'UTR. Note that, when referring to a UTR (e.g., 5'UTR, etc.) or a UTR sequence herein, the gene name alone (e.g., HBA (α-globin) or HBA (α-globin) gene (sequence)) may be used, but in such cases, it is understood that the term refers to the UTR (sequence) (e.g., 5'UTR (sequence)) of the "gene name (sequence)."

[0013] As used herein, the term "open reading frame (ORF)" refers to a region that contains a portion that encodes information about a substance such as a protein.

[0014] As used herein, the term "cap structure" refers to a structure attached to the 5' end of mRNA, such as transcribed precursor mRNA, and is typically formed by the addition of 7-methylguanosine. After the phosphate group at the 5' end of the precursor mRNA is dephosphorylated (removal of two phosphate groups) by a phosphatase (dephosphorylating enzyme), "GTP (three phosphate groups)" reacts with it, resulting in the addition of "guanosine (guanine (base) + ribose (sugar))." Subsequently, the N-7 position of guanine (base) is methylated, resulting in the addition of 7-methylguanosine. This structure plays an important role in inhibiting mRNA degradation and initiating translation.

[0015] As used herein, the term "poly A tail" is interchangeably referred to as a "poly A tail" and refers to a structure in which a sequence of consecutive A's (adenines) (e.g., 20 to 300) is added to the 3' end of a transcribed precursor mRNA. This structure plays an important role in suppressing mRNA degradation.

[0016] As used herein, the term "functional non-translated region" refers to a region having some function. The functions referred to here include, but are not limited to, translation control, nucleic acid construct degradation control, nucleic acid construct intracellular localization control, nucleic acid construct extracellular secretion control, nucleic acid construct extracellular vesicle encapsulation control, protein / peptide binding, low molecular weight compound binding, miRNA and other low molecular weight non-coding RNA binding, high molecular weight non-coding RNA binding, nucleic acid aptamer binding, ribozyme, spacer / stuffer, higher order structure formation, inclusion of modified nucleic acid, inclusion of labeled compound such as fluorescent dye, etc.

[0017] As used herein, a "gene expression database" refers to a database of test and prediction results related to spatiotemporal gene expression, and the targets of the tests and predictions include, but are not limited to, the genome, epigenome, transcriptome, ribosome profile, miRNA profile, non-coding RNA profile, proteome, glycome, lipidome, and metabolome.

[0018] As used herein, the term "database created by the ribosome profiling method" refers to any database generated by the ribosome profiling method. The ribosome profiling method is a method for comprehensively calculating mRNA translation levels using a next-generation sequencer, measuring the amount of RNA bound to ribosomes, and is useful as a more precise method for estimating protein expression levels in a sample. It can be said to be a database related to expression levels.

[0019] As used herein, the terms "mutually" or "complementarity" refer to the specific pairing of adenine with thymine or uracil, or guanine with cytosine or uracil (wobble base pairing) in a nucleic acid base sequence. A "non-complementary portion" refers to a portion of a base sequence that is not complementary, and a "complementary portion" refers to a portion of a base sequence that is complementary. As used herein, complementary portions and non-complementary portions can be specified in single-base increments. The phrase "all non-complementary portions are 1 base in length" refers to the following: when a complementary portion and a non-complementary portion are specified in a portion of a nucleic acid of interest, if there is a single non-complementary portion, the non-complementary portion is 1 base in length; and if there are multiple non-complementary portions, all of the multiple non-complementary portions are 1 base in length. In addition, in a non-complementary portion, either the 5'UTR or the 3'UTR may be 0 bases long. In this case, the term "removal" may be used herein (although it may also be referred to as "addition" when viewed from the other side, the term "removal" is used herein unless otherwise specified, and "removal" is understood in a broad sense to include removal and addition in the narrow sense). Furthermore, when one or more non-complementary bases are present in the 5'UTR and 3'UTR in the non-complementary portion, the term may be used herein as "substitution." Note that, in the non-complementary portion, when the number of bases on one side is different from the number of bases on the other side, the portion with the same number corresponds to "substitution," and in the portion with a different number of bases, the side with fewer bases relative to the side with more bases corresponds to "removal." For example, in the case of 5'-NNNAGNNN 3'-NNNA0NNN (N is the complementary strand, 0 is no base), A for A on the 5' side is a substitution where U would be in the complementary strand, but 0 for G on the 5' side corresponds to removal.

[0020] Furthermore, in the case of 5'-NNNAGNNNU0NNN 3'-NNNA0NNNUGNNN, the AG portion on the 5' side is the same as above, but the portion corresponding to U0, G, can be considered as "addition", but in this specification, it may be described as "removal" in a broad sense.

[0021] Unless otherwise specified, in this specification, deletions, substitutions, and additions are determined by comparing nucleic acids in order from the 5' side.

[0022] As used herein, "partially complementary" refers to complementary sequences over at least a portion (preferably, two or more bases) of the target base sequence. The preferred number of bases in the complementary portion is three or more and 11 or less. An example of a partially complementary sequence is one in which the 3' untranslated region has a region (1) that is partially complementary to the 5' untranslated region. Non-complementary portions (2) and complementary portions (3) alternate in region (1), and the fractional portion (4) is one or more bases long and not longer than the base length of complementary portion (3).

[0023] As used herein, the term "nucleic acid construct" refers to a construct at least partially composed of nucleic acids, and typically refers to a (non-natural) nucleic acid molecule (e.g., recombinant nucleic acid) resulting from the use of recombinant DNA technology. Typically, a nucleic acid construct is a single-stranded or double-stranded nucleic acid molecule that has been modified to contain segments of nucleic acid sequences combined and arranged in a manner not found in nature. A nucleic acid construct may be a "vector" (e.g., a plasmid, an rAAV vector genome, an expression vector, etc.), i.e., a nucleic acid molecule designed to deliver exogenously produced DNA to a host cell. A nucleic acid construct comprising multiple nucleic acid constructs is a single nucleic acid construct, but may be referred to as a "composite nucleic acid construct" based on its composition. Such a composite nucleic acid construct is a single molecule. A collection of multiple nucleic acid constructs is also referred to as a "library (of nucleic acid constructs)."

[0024] As used herein, the term "complementarity (%)" refers to the percentage of identity between the base sequence of a single-stranded nucleic acid and the base sequence of a complementary sequence of a reference sequence in a region of the 3'UTR that is partially complementary to the 5'UTR. The complementarity is calculated based on the length of the reference sequence. When the complementary sequence of the reference sequence and the base sequence of the single-stranded nucleic acid completely match, the complementarity is 100%. In embodiments of the present disclosure in which non-complementary regions are removed, the identity is calculated assuming the presence of non-complementary regions.

[0025] As used herein, the terms "protein," "polypeptide," and "peptide" are used interchangeably to refer to polymers of amino acids of any length. The polymers may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term also encompasses naturally occurring or artificially modified polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling component). Amino acids may be represented in this disclosure by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their commonly accepted single-letter codes. Note that, in this specification, nucleotides may be represented as T even in the case of RNA to comply with the rules of sequence listing; however, those skilled in the art will understand that the designation T means U in the case of RNA.

[0026] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymer of nucleotides of any length. Examples of nucleic acids include DNA, RNA, cDNA, mRNA, rRNA, tRNA, microRNA (miRNA), and large non-coding RNA (lncRNA). This term also includes "polynucleotide derivatives." A "polynucleotide derivative" refers to a polynucleotide that contains a nucleotide derivative or has an unusual internucleotide bond. The term "nucleotide derivative" refers to a nucleotide having a structure different from that of a normal nucleotide used in natural DNA or RNA, and examples thereof include locked nucleic acids (LNA), ethylene nucleic acids such as 2'-O,4'-C-ethylene bridged nucleic acids (ENA), other bridged nucleic acids (BNA), hexitol nucleic acids (HNA), amido-bridged nucleic acids (AmNA), morpholino nucleic acids, tricyclo-DNA (tcDNA), polyether nucleic acids (see, for example, U.S. Pat. No. 5,908,845), cyclohexene nucleic acids (CeNA), and the like. Examples of unusual internucleotide bonds include an interoligonucleotide bond in which a phosphodiester bond is converted to a phosphorothioate bond, an interoligonucleotide bond in which a phosphodiester bond is converted to an N3'-P5' phosphoramidate bond, and an interoligonucleotide bond in which a ribose and phosphodiester bond is converted to a peptide nucleic acid bond.

[0027] As used herein, the term "gene" refers to a nucleic acid moiety that performs a certain biological function, such as encoding a polypeptide or protein, encoding a non-protein-coding functional RNA (e.g., rRNA, tRNA, microRNA (miRNA), lncRNA), regulating the production of a polypeptide, protein, or non-protein-coding functional RNA, specifically binding to a particular protein, or regulating nucleic acid cleavage or replication.

[0028] As used herein, the term "kit" refers to a unit in which the components to be provided (e.g., nucleic acid constructs, instructions, etc.) are provided, usually separated into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit advantageously includes instructions or instructions describing how to use the provided components or how to handle the reagents. When the kit is used herein as a reagent kit, the kit usually includes instructions describing how to use the nucleic acid construct, etc.

[0029] As used herein, "instructions" refers to instructions for users on how to use the present disclosure. These instructions contain text instructing how to use the nucleic acid construct of the present disclosure. These instructions are typically provided in paper form, but are not limited thereto and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, e-mail, etc.).

[0030] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments can be used alone or in combination.

[0031] (Linear RNA structure, nucleic acid construct A) In one aspect, the present disclosure provides a nucleic acid construct (also referred to herein as "nucleic acid construct A") comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, in which, from the 5' side, a cap structure or a structure related thereto (including, but not limited to, a methylated form, etc.) as needed, the 5' UTR, the translated region, and the 3' UTR are linked in this order, wherein the complementarity of the 3' UTR to the 5' UTR is higher than the complementarity of the sequence contained in the translated region to the 5' UTR, and the 3' UTR comprises a non-complementary portion and a complementary portion to the 5' UTR. In another aspect, the present disclosure provides a nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, wherein the 5' UTR, the translated region, and the 3' UTR are linked in this order from the 5' side, the rate of complementarity between the translated region and the 5' UTR or the 3' UTR is less than 95%, and the 3' UTR comprises a non-complementary portion and a complementary portion to the 5' UTR.

[0032] It should be noted that the present disclosure differs from conventional methods in that it describes at least the following steps: (1) selecting a 5'UTR from naturally occurring sequences; and (2) designing a 3'UTR that is partially complementary to the 5'UTR. Reference information is provided in Figures 5, 6, 7, and 14. One important part of nucleic acid construct A is the combination with a natural 5'UTR, but this is not a limitation. Furthermore, the present disclosure preferably describes all the components of the nucleic acid construct designed using the mRNA design service scheme, and also indicates the necessary design requirement of low affinity between the 5'UTR and ORF, but this is not a limitation.

[0033] The nucleic acid constructs of the present disclosure are capable of regulating protein expression. Preferably, the nucleic acid constructs of the present disclosure are capable of increasing protein expression.

[0034] In one embodiment of nucleic acid construct A, the nucleic acid construct of the present disclosure may include a 3' poly-A tail.

[0035] In some embodiments of nucleic acid construct A, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 non-complementary portions may be 2 or 3 bases in length, respectively. In a preferred embodiment, all non-complementary portions are 2 or 3 bases in length.

[0036] In another embodiment of nucleic acid construct A, the length of each of the non-complementary portions may be 1 base, in which case the complementarity of the 3'UTR to the 5'UTR may be greater than 75%, for example, greater than 75% and less than 90%, greater than 75% and less than 89%, greater than 80%, 80% to 90%, or 81% to 89%.

[0037] In another embodiment of nucleic acid construct A, non-complementary portions with different numbers of bases may be present, for example, portions with a length of 1 and portions with a length of 2 may be present. Also, portions with a length of 1 and portions with a length of 3 may be present. Also, portions with a length of 1, 2, and 3 may be present. Also, non-complementary portions with a length of 4 or more may be present.

[0038] In some embodiments of nucleic acid construct A, the length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 complementary portions can be 5 bases or more, for example, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 15 bases, or 20 bases. In preferred embodiments, the length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 complementary portions can be 5 to 11 bases. The lengths of the complementary portions may all be the same or different, but are preferably all the same.

[0039] In some embodiments, when the length of each of the non-complementary portions is 2 or 3 bases, the complementarity rate may be at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more, or at least 90% or more. In certain embodiments, when the length of each of the non-complementary portions is 2 or 3 bases, the complementarity rate may be higher than 75%, for example, higher than 75% but not higher than 90%, higher than 75% but not higher than 89%, 80% or higher but not higher than 90%, or higher than 80%, or 81% or higher but not higher than 89%. The lengths of the complementary and non-complementary portions can be determined appropriately depending on the desired complementarity rate, but typically, the length of each of the non-complementary portions is independently 1 to 3 bases, and the length of each of the complementary portions is independently 3 to 11 bases.

[0040] In one embodiment of nucleic acid construct A, the bases in the non-complementary portions are substituted or removed.

[0041] In one embodiment of nucleic acid construct A, all of the non-complementary portions are removed.

[0042] In one embodiment of nucleic acid construct A, when the length of each non-complementary portion is a substitution of only one base, the rate of complementarity of the 3'UTR to the 5'UTR is greater than 75%. When the length of each non-complementary portion is only one base, the rate of complementarity of the 3'UTR to the 5'UTR may be other than this, for example, greater than 75% and less than 90%, greater than 75% and less than 89%, greater than 80%, and less than 80% and less than 90%, or less than 81% and less than 89%.

[0043] In one embodiment of nucleic acid construct A, at least one of the non-complementary portions is 2 or more bases in length. In some embodiments, the non-complementary portions are 2 bases in length, and the complementary portions are each independently 5 to 7 bases in length.

[0044] In one embodiment of nucleic acid construct A, the length of at least one of the non-complementary portions is 2 or 3 bases. In some embodiments, the length of the non-complementary portion may be 3 bases, and the length of each of the complementary portions may independently be 8 to 11 bases.

[0045] In one embodiment of the nucleic acid construct A, at least one of the complementary portions has a length of 5 bases or more.

[0046] In one embodiment of nucleic acid construct A, at least one of the complementary portions has a length of 5 to 11 bases.

[0047] In one embodiment of the nucleic acid construct A, the non-complementary portions have a length of 2 bases, and the complementary portions have a length of 5 to 7 bases, respectively.

[0048] In one embodiment of the nucleic acid construct A, the non-complementary portions have a length of 3 bases, and the complementary portions have a length of 8 to 11 bases, respectively.

[0049] In one embodiment of the nucleic acid construct A, the non-complementary portions are all at least one base in length, and the rate of complementarity is higher than 75%.

[0050] In one embodiment of the nucleic acid construct A, the non-complementary portions are all one base or more in length, and the complementarity rate is greater than 80%, for example, 81% to 89%.

[0051] In one embodiment of nucleic acid construct A, at least one of the non-complementary portions is a single base.

[0052] In one embodiment of the nucleic acid construct A, at least one of the non-complementary portions has a length of 1 base, and the complementary portions each have a length of 3 to 11 bases.

[0053] In one embodiment of the nucleic acid construct A, the non-complementary portion has a length of 1 base, and the complementary portions each have a length of 3 to 7 bases.

[0054] In one embodiment of the nucleic acid construct A, the non-complementary portions have a length of 2 bases, and the complementary portions have a length of 5 to 7 bases, respectively.

[0055] In one embodiment of the nucleic acid construct A, the non-complementary portions have a length of 3 bases, and the complementary portions have a length of 8 to 11 bases, respectively.

[0056] In another aspect of nucleic acid construct A, the present disclosure provides a nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, the 5'-UTR being linked in the following order from the 5' end: cap structure, 5' UTR, translated region, 3' UTR, the 3' UTR comprising a non-complementary portion and a complementary portion to the 5' UTR, the non-complementary portion and the complementary portion alternately existing. Alternatively, the present disclosure provides a nucleic acid construct comprising a region (1) in the 3' UTR that is partially complementary to the 5' UTR, the 3' UTR comprising a non-complementary portion (2) and a complementary portion (3) to the other UTR, the non-complementary portion and the complementary portion alternately existing. In one embodiment, the portion other than the alternating portions (also referred to as the fractional portion (4)) is at least one base long but not longer than the length of the complementary portion.

[0057] In one embodiment of the nucleic acid construct A, when the non-complementary portion (2) is 1 base, the complementary portion (3) is 3 to 6 bases long in the case of a base substitution, or 3 to 9 bases long in the case of a base deletion.

[0058] In one embodiment, when the non-complementary portion (2) is 2 bases long, the complementary portion (3) is 5 to 7 bases long in the case of base substitution, or 5 to 11 bases long in the case of base deletion.

[0059] In one embodiment of the nucleic acid construct A, when the complementary portion (2) is 3 bases long, the complementary portion (3) is 8 to 9 bases long in the case of base substitution, and 8 to 11 bases long in the case of base deletion.

[0060] In one embodiment of the nucleic acid construct A, the non-complementary portion (2) is usually 0 to 3 bases long, preferably 2 or 3 bases long.

[0061] In this way, the appropriate base length may vary depending on the substitution or removal, but those skilled in the art can design it appropriately based on the description in this specification.

[0062] In one embodiment of nucleic acid construct A, non-complementary portion (2) may have two or more different lengths in the untranslated region. In this case, it is advantageous for at least one of the above-mentioned preferred base lengths to be satisfied, and it is more advantageous for all of them to be satisfied. When the untranslated region has a mixture of lengths, the above-mentioned preferred base length number can be interpreted as the average value obtained by dividing the total number of bases in non-complementary portion (2) by the number of non-complementary portions (2) contained in the nucleic acid construct. For example, non-complementary portion 2 includes an average length of 1.5 to 2.5, and non-complementary portion 3 includes an average length of 2.5 to 3.5. When the lengths of non-complementary portions are mixed, the average length of the non-complementary portions is preferably greater than 1 and equal to or less than 3.

[0063] In one embodiment of the nucleic acid construct A, the non-complementary portion (2) may have a mixture of substitutions and deletions in the untranslated region. In this case, it is advantageous for at least one of the above-mentioned preferred base lengths to be satisfied, and it is also advantageous for all of them to be satisfied.

[0064] In one embodiment of nucleic acid construct A, the UTRs are selected from the group consisting of GAPDH, HSD17B4, PSMB3, RPL31, RPL32, RPL35, RPL21, Albumin7, LDHB, ACAT2, ATP5A1, Ndufa4, Mp68, NOSIP, SLC7A3, TUBB4B, UBQLN2, mRPL35A, mRPL21, AIG1, COX6C, α-globin, β-globin, RPS8, TOP, MCP-1, RPL12s.c., Ang-2, HSP70, H3.3. , Galectin-9, GADD34, EDN1, HSP70m5, E-selectin, ICAM-1, IL-6, or vWF UTRs. Other examples of UTRs are disclosed in JP-A-2021-501572, JP-A-2015-517803, and JP-A-2022-164843.

[0065] In one embodiment of nucleic acid construct A, the 5'UTR is selected from the nucleic acid sequences of at least one of HBA (α-globin), HBB, HIST1H2BK, ACTB, KRT18, HIST1H1C, HIST1H1E, HIST3H2A, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, PKM, GAPDH, HSD17B4, ETNK1, XAB2, and RRP12, or comprises the nucleic acid sequence of a functional fragment thereof, or is a mutated version of the nucleic acid sequence.

[0066] In one embodiment of nucleic acid construct A, the 5'UTR is selected from the nucleic acid sequences of at least one of HBA (α-globin), HBB, HIST1H2BK, ACTB, and KRT18, or comprises a nucleic acid sequence of a functional fragment thereof, or is a mutated version of the nucleic acid sequence. (Nucleic Acid Construct B)

[0067] In one aspect, the present disclosure provides a nucleic acid construct (also referred to as "nucleic acid construct B") in which at least one functional untranslated region is further added to the nucleic acid construct of the present disclosure (nucleic acid construct A). It should be noted that in the present disclosure, the final nucleic acid construct does not differ regardless of the order in which the "partially complementary 3' UTR" or the "functional 3' UTR" is added later. For example, in Figure 18, "our design" corresponds to the "partially complementary 3' UTR," and the "Pfizer sequence" present in the 3' UTR corresponds to the "functional 3' UTR."

[0068] In one embodiment of nucleic acid construct B, the functional untranslated region is contained in the 3'UTR 5' to the 3'UTR portion within region (1) having partial complementarity with the 5'UTR.

[0069] In one embodiment of nucleic acid construct B, the functional untranslated region is contained in the 3'UTR 3' to the 3'UTR portion within region (1) having partial complementarity with the 5'UTR.

[0070] In one embodiment of nucleic acid construct B, the functional untranslated region is contained in the 3'UTR both 5' and 3' to the 3'UTR portion within region (1) that is partially complementary to the 5'UTR.

[0071] In one embodiment of nucleic acid construct B, the functional untranslated region is contained in the 3'UTR 5' to the 5'UTR portion within region (1) having partial complementarity with the 5'UTR.

[0072] In one embodiment of nucleic acid construct B, the functional untranslated region is contained in the 3'UTR 3' to the 5'UTR portion within region (1) having partial complementarity with the 5'UTR.

[0073] In one embodiment of nucleic acid construct B, the functional untranslated region is contained in the 3'UTR both 5' and 3' to the 5'UTR portion within region (1) that is partially complementary to the 5'UTR.

[0074] In one embodiment of nucleic acid construct B, the functional untranslated region is selected from at least one of a translation control sequence, a nucleic acid construct degradation control sequence, a nucleic acid construct intracellular localization control sequence, a nucleic acid construct extracellular secretion control sequence, a nucleic acid construct extracellular vesicle encapsulation control sequence, a protein / peptide binding sequence, a low molecular weight compound binding sequence, a low molecular weight non-coding RNA target sequence such as miRNA, a high molecular weight non-coding RNA target sequence, a nucleic acid aptamer or its recognition sequence, a ribozyme sequence, a spacer / stuffer sequence, a higher order structure sequence, a modified nucleic acid-containing sequence, and a labeling compound-containing sequence such as a fluorescent dye.

[0075] In one embodiment of nucleic acid construct B, the functional untranslated region is selected from at least one of a translation control sequence, a nucleic acid construct degradation control sequence, a nucleic acid construct intracellular localization control sequence, a protein / peptide binding sequence, and a small non-coding RNA target sequence such as miRNA.

[0076] (Nucleic Acid Construct C) In one aspect, the present disclosure provides a nucleic acid construct of the present disclosure (nucleic acid construct A or nucleic acid construct B), or a nucleic acid construct comprising a 5'UTR and a 3'UTR that are at least partially complementary to each other, wherein the 5'UTR, translated region, and 3'UTR are linked in this order from the 5' side, with the rate of complementarity between the translated region and the 5'UTR or the 3'UTR being less than 95%, and the 3'UTR comprising a non-complementary portion and a complementary portion to the 5'UTR, and a nucleic acid construct (also referred to herein as "nucleic acid construct C" or "composite nucleic acid construct") comprising a repeat sequence of A (adenine) on the 3' side of the nucleic acid construct.

[0077] In one embodiment, the length of the repeat sequence of A is 100 to 300.

[0078] In one embodiment, the length of the repeat sequence of A is 120 to 200. (Design method, etc.)

[0079] The present disclosure provides a method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct that has a 5'UTR, a translated region encoding the protein, and a 3'UTR linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and complementary portions; and (3) selecting whether bases in the non-complementary portions are substituted or deleted. Each embodiment of the method of the present disclosure can optionally apply one or more features of various embodiments of the nucleic acid construct described elsewhere in this specification.

[0080] In one embodiment of the design, etc., there is provided a method for optimizing the expression level of a desired protein in a desired cell using a nucleic acid construct in which a 5'UTR, a translated region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and complementary portions; and (3) selecting whether bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, and the natural protein is selected from a database related to gene expression. Each embodiment of the method of the present disclosure can optionally apply one or more features of the various embodiments of the nucleic acid construct described elsewhere in this specification.

[0081] In one embodiment of the design, a method for optimizing the expression level of a desired protein in a desired cell is provided for a nucleic acid construct that is linked in the following order from the 5' end: a 5'UTR, a translated region encoding the desired protein, and a 3'UTR. The method includes at least the following steps: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary and complementary portions alternate; (2) selecting the base lengths of the non-complementary and complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted. The 5'UTR is derived from a natural protein, and the natural protein is selected from a database created by a ribosome profiling method. Ribosome profiling is a method that comprehensively analyzes ribosome-protected mRNA fragments using a next-generation sequencer. This method can quantitatively indicate which mRNAs are translated to what extent in cells. Each embodiment of the method disclosed herein can optionally apply one or more features of various embodiments of the nucleic acid constructs described elsewhere in this specification.

[0082] In one embodiment of the design, etc., there is provided a method for optimizing the expression level of a desired protein in a desired cell using a nucleic acid construct in which a 5'UTR, a translated region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary and complementary portions alternate; (2) selecting the base lengths of the non-complementary and complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, and the natural protein is selected from mRNA translated in a database related to gene expression or a database created by a ribosome profiling method. Each embodiment of the method of the present disclosure may optionally apply one or more features of the various embodiments of the nucleic acid construct described elsewhere in this specification.

[0083] In one embodiment of the design, etc., there is provided a method for optimizing the expression level of a desired protein in a desired cell using a nucleic acid construct in which a 5'UTR, a translated region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, and the natural protein is one in which one or more ribosome-protected mRNA fragments are present in a ribosome profile analysis or which has been detected in a proteome analysis. Each embodiment of the method of the present disclosure may optionally apply one or more features of various embodiments of the nucleic acid construct described elsewhere in this specification.

[0084] In one embodiment of the design, the method is for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translational region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the steps of: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, the natural protein is selected from a database created by a ribosome profiling method, the selected protein contains one or more ribosome-protected mRNA fragments, and the selected protein is ranked high (for example, but not limited to, within the top 200) when the number of ribosome-protected mRNA fragments in the selected protein is divided by the mRNA expression level. More preferably, the 5'UTR has a base length of 25 to 100 bases. Each embodiment of the disclosed method can optionally apply one or more features of various embodiments of the nucleic acid constructs described elsewhere herein.

[0085] In one aspect, the present disclosure provides a system including: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a library generating unit that generates an mRNA library containing a plurality of composite sequences each containing a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR (optimized by the techniques described herein or other methods; the same applies herein), and optionally optimizes the polyA tail; 3) an evaluation unit that evaluates the protein expression level of the mRNA library; and 4) a selection unit that, if necessary, selects from the mRNA library an mRNA that achieves a desired value or level. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0086] In another aspect, the present disclosure provides a system for generating an optimized nucleic acid construct, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a selection unit in which a 5'UTR comprising a naturally occurring sequence; 3) a design unit in which a 3'UTR comprising a complementary and non-complementary portion to the 5'UTR of the naturally occurring sequence; 4) a structure selection unit in which a structure of other portions of a nucleic acid construct is selected; 5) an evaluation unit in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated; and 6) a desired selection unit in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0087] In one aspect, the present disclosure provides a system for designing one or more mRNAs that express a protein desired by a user, the system comprising: 1) a selection section for selecting a 5'UTR structure nucleic acid sequence from a database related to the expression of the protein, 2) a structure selection section for selecting a 3'UTR structure nucleic acid sequence including complementary and non-complementary portions thereto, 3) a step of combining a 5'cap structure sequence, the 5'UTR structure, the translated region of the protein, and the 3'UTR structure, and 4) an optimization strain for optimizing a polyA tail. Each embodiment of the system of the present disclosure may optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0088] In one embodiment of the present disclosure, the selection unit further includes an evaluation unit that evaluates the expression level of the protein.

[0089] In another aspect, there is provided a system for controlling protein expression of an mRNA having a 5'UTR structure and a 3'UTR structure, the system comprising: 1) a design unit that designs a nucleic acid sequence including a portion complementary or non-complementary to all or a portion of the 5'UTR, and 2) a sequence addition unit that adds the nucleic acid sequence to the 3'UTR structure. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0090] In another aspect, the present disclosure provides a system for generating optimized mRNA, comprising: a sequence information input unit that inputs sequence information of a translation region of an mRNA having the translation region, a first transmission unit that transmits the sequence information input from the customer sequence information input unit, a receiving unit that receives the sequence information transmitted from the first transmission unit, a first storage unit that stores the sequence information received by the receiving unit, a second storage unit that stores information on a predetermined untranslated region of the mRNA, a third storage unit that stores information on a predetermined PolyA strand of the mRNA, and an optimization unit (which may include AI) that optimizes the sequence information. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0091] In another aspect, the present disclosure provides a program encoding procedures for causing a computer to execute a method for generating optimized mRNA, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which an mRNA library is generated containing a plurality of composite sequences each containing a combination of a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR (including a step of optimizing a polyA tail, as necessary); 3) a step in which the amount of protein expression by the mRNA library is evaluated; and 4) a step in which an mRNA that achieves a desired value or level is selected from the mRNA library, as necessary. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0092] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region, 2) a step in which a 5'UTR comprising a naturally occurring sequence, 3) a step in which a 3'UTR comprising a complementary and non-complementary portion to the 5'UTR of the naturally occurring sequence, 4) a step in which a structure of other portions of the nucleic acid construct is selected, 5) a step in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated, and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the result of step 5. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0093] In another aspect, the present disclosure provides a program encoding procedures for causing a computer to execute a method for designing one or more mRNAs that express a protein desired by a user, the method comprising the steps of: 1) selecting, for a protein desired by a user, a nucleic acid sequence for a 5'UTR structure from a database related to expression of the protein, 2) selecting a nucleic acid sequence for a 3'UTR structure including complementary and non-complementary portions thereto, 3) combining a 5'cap structural sequence, the 5'UTR structure, the translated region of the protein, and the 3'UTR structure, and 4) optimizing a polyA tail. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0094] In another aspect, the present invention provides a program encoding instructions for causing a computer to execute a method, wherein the method of the program further includes a step of evaluating the expression level of the protein in step 1). Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0095] In another aspect, the present disclosure provides a program encoding procedures for causing a computer to execute a method for controlling protein expression of an mRNA having a 5'UTR structure and a 3'UTR structure, by: 1) designing a nucleic acid sequence including a complementary or non-complementary portion to all or a portion of the 5'UTR, and 2) adding the nucleic acid sequence to the 3'UTR structure. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0096] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized mRNA, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which an mRNA library is generated containing a plurality of composite sequences each including a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR (optionally including a step of optimizing a polyA tail); 3) a step in which the mRNA library is evaluated for protein expression; and 4) a step in which, option ally, an mRNA that achieves a desired value or level is selected from the mRNA library. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0097] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region, 2) a step in which a 5'UTR comprising a naturally occurring sequence, 3) a step in which a 3'UTR comprising portions complementary and non-complementary to the 5'UTR of the naturally occurring sequence, 4) a step in which a structure of other portions of the nucleic acid construct is selected, 5) a step in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated, and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the result of step 5. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0098] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for designing one or more mRNAs that express a protein desired by a user, the method comprising the steps of: 1) selecting, for a protein desired by a user, a nucleic acid sequence for a 5'UTR structure from a database related to expression of the protein, 2) selecting a nucleic acid sequence for a 3'UTR structure including complementary and non-complementary portions thereto, 3) combining a 5'cap structural sequence, the 5'UTR structure, the translated region of the protein, and the 3'UTR structure, and 4) optimizing a polyA tail. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0099] In another aspect, the method of the present disclosure further comprises, in addition to step 1), a step of evaluating the expression level of the protein.

[0100] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for controlling protein expression of an mRNA having a 5'UTR structure and a 3'UTR structure, by: 1) designing a nucleic acid sequence including a complementary or non-complementary portion to all or a portion of the 5'UTR, and 2) adding the nucleic acid sequence to the 3'UTR structure. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0101] In one embodiment of the design, the method further comprises the step of selecting the length of the poly-A tail.

[0102] In one embodiment of the design, the length of the polyA tail is selected in the range of 50 to 300.

[0103] In one embodiment of the design, the steps include synthesizing the nucleic acid construct, introducing the nucleic acid construct into the desired cells, and evaluating the expression level of the protein.

[0104] The present disclosure provides a nucleic acid construct (also referred to as nucleic acid construct D) designed by any of the methods described herein, in which a 5'UTR, a translation region encoding a desired protein, and a 3'UTR are linked in this order from the 5' side.

[0105] In one embodiment of such a design, when the length of each non-complementary portion is all single base substitution, the complementarity of the 3'UTR to the 5'UTR is greater than 75%.

[0106] In one embodiment of nucleic acid construct D, at least one of the non-complementary portions has a length of 2 or more bases.

[0107] In one embodiment of nucleic acid construct D, when the non-complementary portion is 2 bases, the complementary portion is 5 to 7 bases long in the case of base substitution, or 5 to 11 bases long in the case of base deletion.

[0108] In one embodiment of nucleic acid construct D, when the non-complementary portion is 3 bases, the complementary portion is 8 to 9 bases long in the case of base substitution, and 8 to 11 bases long in the case of base deletion.

[0109] In one embodiment of nucleic acid construct D, the 5'UTR is selected from the nucleic acid sequences of at least one of HBA (α-globin), HBB, HIST1H2BK, ACTB, KRT18, HIST1H1C, HIST1H1E, HIST3H2A, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, PKM, GAPDH, HSD17B4, ETNK1, XAB2, and RRP12, or comprises the nucleic acid sequence of a functional fragment thereof, or is a mutated version of the nucleic acid sequence.

[0110] In one embodiment of nucleic acid construct D, the 5'UTR is selected from the nucleic acid sequences of at least one of HBA (α-globin), HBB, HIST1H2BK, ACTB, and KRT18, or comprises a nucleic acid sequence of a functional fragment thereof, or is a mutated version of the nucleic acid sequence. (Use of Nucleic Acid Construct)

[0111] In another aspect, the present disclosure provides a pharmaceutical, vaccine, diagnostic agent, veterinary drug, agricultural chemical, or reagent comprising a nucleic acid construct (A, B, C, D, etc.) of the present disclosure or a complex of the present disclosure, wherein the nucleic acid construct or complex may be used alone or in combination, or a specific individual combination or selection of each embodiment may be used.

[0112] The technology of the present disclosure can include transcription as part of the process.These methods include transcription-mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA), DNA and RNA sequencing and other nucleic acid extension reactions known in the art.Those skilled in the art will understand that other methods, including the variants of transcription reactions that will be developed in the future, can be used instead of or together with transcription methods.

[0113] In another aspect, the present disclosure provides a nucleic acid construct (A, B, D, etc.) of the present disclosure or a composite nucleic acid construct of the present disclosure, which is used for various purposes such as medicine, vaccine, diagnostic agent, veterinary drug, agricultural chemical, reagent, etc. Here, the nucleic acid construct or composite nucleic acid construct may be used alone or in combination, and specific individual combinations or selections of each embodiment may be used.

[0114] The present invention also provides methods and medicaments for introducing the nucleic acid construct, or a source or complex thereof, into a cell for use as a therapeutic agent to treat a cellular medical condition. The nucleic acid construct of the present disclosure can be introduced into a cell that utilizes it to generate mRNA containing the nucleic acid construct to produce a protein that can have a therapeutic effect on the host cell.

[0115] One method for using a nucleic acid construct to treat or prevent a disease, disorder or symptom or a particular condition may comprise administering a nucleic acid construct or complex of the present disclosure or a composition comprising such a complex to a subject having or suspected of having a condition whose symptoms / symptoms may be reduced in severity or eliminated.

[0116] The nucleic acid constructs of the present disclosure, when formulated in a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable salt at a concentration of 4 mg / ml or less, are effective in providing a reduction in symptoms and / or symptomology of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to an untreated individual using a pharmaceutically acceptable carrier alone.

[0117] To treat a specific condition, pharmaceutical compositions can be formulated for administration by injection or other suitable route known to those skilled in the art.Injectable compositions for parenteral administration usually contain active compounds in suitable solutions such as sterile saline and / or pharmaceutical carriers.Compositions can also be formulated as suspensions in lipids or phospholipids, in liposomal suspensions, or in aqueous emulsions.

[0118] Methods for preparing various compositions and / or formulations are known to those skilled in the art; see Remington's Pharmaceutical Sciences (19th ed., Williams & Wilkins, 1995). The composition to be administered contains a pharmaceutically safe, effective amount of the selected compound to increase expression of the desired protein in the target cell or tissue.

[0119] In some embodiments, the pharmaceutical composition contains at least 0.1% (w / v) of the compound, as noted above, in some embodiments, the pharmaceutical composition contains more than 0.1%, in some embodiments, the pharmaceutical composition contains up to about 10%, in some embodiments, the pharmaceutical composition contains up to about 5%, and in some embodiments, the pharmaceutical composition contains up to about 1% (w / v) of the compound. Selection of an appropriate concentration will depend on factors such as the desired dose, frequency, and method of delivery of the active agent.

[0120] For treatment of a subject, such as a mammal or human, dosage is determined based on factors such as the subject's weight and overall health, the condition being treated, the severity of the symptoms, etc. Dosage amounts and concentrations are determined to provide the desired benefit while avoiding any undesirable side effects. Typical dosages of the subject compounds range from about 0.0005 to 500 mg / day for human patients, and in some embodiments, range between about 1 to 100 mg / day. For example, higher dosages can include, for example, 50 to 100, 75 to 100, or 50 to 75 mg / day, while lower dosages include, for example, 1 to 50, 25 to 50, or 1 to 25 mg / day.

[0121] The present disclosure provides raw materials for pharmaceuticals, vaccines, diagnostic agents, veterinary drugs, agricultural chemicals, reagents, etc., which contain the nucleic acid constructs (A, B, C, D, etc.) of the present disclosure or the composite nucleic acid constructs of the present disclosure. Here, the nucleic acid constructs or composite nucleic acid constructs may be used alone or in combination, and specific individual combinations or selections of each embodiment may be used.

[0122] In another aspect, the present disclosure provides a formulation comprising a nucleic acid construct (A, B, C, D, etc.) of the present disclosure or a composite nucleic acid construct of the present disclosure, wherein the nucleic acid construct or composite nucleic acid construct may be used alone or in combination, or a specific individual combination or selection of each embodiment may be used.

[0123] In another aspect, the present disclosure provides methods for producing the nucleic acid constructs (A, B, C, D, etc.) of the present disclosure or the composite nucleic acid constructs, compositions, pharmaceuticals, diagnostic agents, veterinary drugs, agricultural chemicals, reagents, etc. of the present disclosure, or raw materials or formulations thereof.

[0124] In another aspect, the present disclosure provides a method for introducing a nucleic acid construct (A, B, C, D, etc.) of the present disclosure or a composite nucleic acid construct, composition, pharmaceutical, diagnostic agent, veterinary drug, agricultural chemical, reagent, etc., or a raw material or formulation thereof into a cell to express a protein.

[0125] In an aspect of the present disclosure, the nucleic acid construct of the present disclosure may be provided as a kit.

[0126] The present disclosure can be applied in the application field of nucleic acid technology (e.g., pharmaceuticals). For example, a nucleic acid construct (e.g., mRNA) of the present disclosure can be used as a drug substance for a nucleic acid (e.g., mRNA) drug. Furthermore, a plasmid DNA of the present disclosure can be used to create a cell bank for use in manufacturing a nucleic acid construct (e.g., mRNA) drug substance. Furthermore, a linear template DNA of the present disclosure can be used as a raw material in the manufacturing process of a nucleic acid construct (e.g., mRNA) drug substance.

[0127] (Application) In another aspect, the present disclosure provides a method for generating an optimized nucleic acid construct (e.g., mRNA), comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a nucleic acid construct (e.g., mRNA) library is generated, the nucleic acid construct (e.g., mRNA) library containing a plurality of composite sequences each including a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR (including a step of optimizing a polyA tail, if necessary); 3) a step in which the amount of protein expression by the nucleic acid construct (e.g., mRNA) library is evaluated; and 4) a step in which a nucleic acid construct (e.g., mRNA) that achieves a desired value or level is selected from the nucleic acid construct (e.g., mRNA) library, if necessary.

[0128] In another aspect, the present disclosure provides a system for generating an optimized nucleic acid construct (e.g., mRNA), comprising: a sequence information input unit that inputs sequence information of a translation region of a nucleic acid construct (e.g., mRNA) having the translation region; a first transmission unit that transmits the sequence information input from the customer sequence information input unit; a receiving unit that receives the sequence information transmitted from the first transmission unit; a first storage unit that stores the sequence information received by the receiving unit; a second storage unit that stores information on a predetermined untranslated region of the nucleic acid construct (e.g., mRNA); a third storage unit that stores information on a predetermined PolyA strand of the nucleic acid construct (e.g., mRNA); and an optimization unit (which may include AI) that optimizes the sequence information.

[0129] In one aspect, the present disclosure provides a method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a library of nucleic acid constructs having multiple sequences is constructed, the library including the sequence information of the translated region, in combination with multiple combinations of 5'UTR and 3'UTR, and optionally, a 5'cap or its analogous structure, and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3. Each embodiment of the method of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0130] In one embodiment, the step 1) in which a user provides sequence information of a translation region can be performed by any method, including, but not limited to, obtaining a target DNA or RNA sequence, identifying the translation initiation point (start codon such as ATG), determining the range to the termination codon (TAA, TAG, TGA, etc.), extracting the nucleic acid sequence in this range, and providing the sequence from the start codon to the termination codon as is.

[0131] In one embodiment, the step of 2) constructing a library of nucleic acid constructs of multiple sequences containing the sequence information of the translated region in combination with multiple types of 5'UTR and 3'UTR, and optionally in combination with a 5'cap or its analogous structure and a polyA tail, can be carried out by appropriately referring to known techniques and the techniques described herein. For example, the library can be provided by selecting a target gene or sequence, amplifying and synthesizing each sequence using PCR or a synthesis technique, inserting each obtained sequence into a vector, synthesizing the sequence using the vector as a template by an in vitro transcription reaction, enzymatically or chemically modifying the transcript as needed, and further verifying it by sequencing or the like as needed, but is not limited to these methods.

[0132] In one embodiment, 3) the step of evaluating the function of the nucleic acid constructs in the library can be carried out using any technique known in the art. The step of evaluating the function of the nucleic acid construct can be achieved, for example, by introducing the nucleic acid construct into a target cell or a model organism, measuring the expression levels of RNA or protein (using techniques such as RT-PCR or Western blotting) to confirm expression and function after introduction, evaluating biological effects such as changes in cell morphology, growth rate, and metabolic activity (fluorescence microscopy or flow cytometry can be used for functional evaluation), and analyzing the obtained data to comprehensively evaluate the function of the nucleic acid construct, but is not limited to these.

[0133] In one embodiment, the step of 4) selecting a nucleic acid construct that achieves a desired value or level from the results of step 3 can be carried out using any technique known in the art. The value or level can be selected appropriately depending on the purpose. For example, the step of selecting a nucleic acid construct that achieves a desired value or level can be carried out by, but is not limited to, obtaining multiple candidate nucleic acid constructs from a library, introducing each construct into appropriate cells or model organisms, measuring the expression and function of the target gene, identifying constructs that meet the desired criteria for expression level or biological effect based on the obtained data, conducting additional experiments to confirm reproducibility of the selected candidates as necessary, selecting nucleic acid constructs that exhibit the desired performance, and performing detailed characterization as necessary.

[0134] In one aspect, the present disclosure provides a method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR comprising a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR comprising a non-naturally occurring sequence corresponding to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

[0135] In one embodiment, 2) the step of designing a 5'UTR or 3'UTR comprising a naturally occurring sequence can be carried out using any method known in the art for designing a sequence, taking naturally occurring sequences into appropriate consideration. For example, the step of designing a 5'UTR or 3'UTR comprising a naturally occurring sequence can be carried out by obtaining the naturally occurring 5'UTR or 3'UTR sequence of a gene of interest from a database or literature, analyzing these sequences, identifying important elements associated with the function, optimizing the sequence while maintaining these elements as necessary, and inserting the designed UTR sequence upstream or downstream of the translated region of the gene of interest.

[0136] In one embodiment, 3) the step of designing a 5'UTR or 3'UTR comprising a non-naturally occurring sequence corresponding to the 5'UTR or 3'UTR can be carried out using any design technique known in the art. For example, the step of designing a 5'UTR or 3'UTR comprising a non-naturally occurring sequence can be carried out by adding a mutation to the naturally occurring 5'UTR or 3'UTR sequence of a gene of interest, substituting an artificially designed sequence, or inserting the artificially designed sequence, and, if necessary, optimizing the sequence while maintaining the basic structure, and then inserting the designed UTR sequence upstream or downstream of the translated region of the gene of interest.

[0137] In one embodiment, the step of 4) constructing a library of nucleic acid constructs of multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail, as needed, can be carried out using any technique known in the art for constructing a library. For example, the library can be provided by, but is not limited to, selecting a gene or sequence of interest, amplifying and synthesizing each sequence using PCR or a synthesis technique, inserting each obtained sequence into a vector, synthesizing the sequence using the vector as a template in an in vitro transcription reaction, enzymatically or chemically modifying the transcription product as needed, storing the resulting nucleic acid construct library, confirming the quality of the library, and verifying it by sequencing or other methods as needed.

[0138] In one embodiment, the other steps can be performed in any manner described herein.

[0139] In one aspect, the present disclosure provides a method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which 5'UTR and 3'UTR containing complementary and non-complementary portions to each other; 3) a step in which a library of nucleic acid constructs having multiple sequences containing the coding region, 5'UTR, and 3'UTR designed in steps 1 and 2, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 4. Each embodiment of the method of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0140] In one embodiment, the step 2) of designing 5'UTR and 3'UTR containing complementary and non-complementary portions can be carried out using any method known in the art for designing sequences to contain complementary and non-complementary portions. For example, the step can be carried out by obtaining the natural sequences of the 5'UTR and 3'UTR of a gene of interest from a database or literature, analyzing these sequences, designing sequences that are completely complementary to the analyzed sequences, substituting or deleting appropriate nucleotide sequences in the designed complementary sequences to change them to non-complementary portions, and then inserting the designed sequences as 5'UTR or 3'UTR upstream or downstream of the translated region of the gene of interest.

[0141] In one embodiment, the step of 3) constructing a library of nucleic acid constructs of multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 and 2, in combination with a 5'cap or its analogous structure and a polyA tail, can be carried out using any technique known in the art for constructing a library. For example, the library can be provided by selecting a gene or sequence of interest, amplifying and synthesizing each sequence using PCR or a synthesis technique, inserting each obtained sequence into a vector, synthesizing the sequence using the vector as a template by an in vitro transcription reaction, enzymatically or chemically modifying the transcription product as needed, storing the resulting nucleic acid construct library, confirming the quality of the library, and verifying it by sequencing or other methods as needed, but is not limited to this.

[0142] In one embodiment, the other steps can be performed in any manner described herein.

[0143] In one aspect, the present disclosure provides a method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR comprising a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR comprising a complementary and non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the method of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0144] In one embodiment, the steps can be performed in any manner described herein.

[0145] In one aspect, the present disclosure provides a method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR comprising a naturally occurring sequence; 3) a step in which a 3'UTR comprising a complementary and non-complementary portion to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the method of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0146] In one embodiment, 2) the step of designing a 5'UTR comprising a naturally occurring sequence can be carried out using any technique known in the art for designing a sequence, taking the naturally occurring sequence into appropriate consideration.

[0147] In one embodiment, the other steps can be performed in any manner described herein.

[0148] In one aspect, the present disclosure provides a method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs having multiple sequences including the coding region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the method of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0149] In one embodiment, the step 2) of selecting a 5′ UTR sequence derived from a natural protein selected from a database related to gene expression can be performed using any method known in the art for designing a sequence, taking natural sequences into appropriate consideration, or any method described herein.

[0150] In one embodiment, the step 3) of designing a 3'UTR containing complementary and non-complementary portions to the 5'UTR can be performed using any method known in the art for designing sequences containing complementary and non-complementary portions, or any method described herein.

[0151] In one embodiment, the other steps can be performed in any manner described herein.

[0152] In one aspect, the present disclosure provides a method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs having multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the method of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0153] In one embodiment, 2) the step of selecting a 5'UTR sequence derived from a natural protein selected by the ribosome profiling method can be carried out using any method known in the art for designing a sequence, taking natural sequences into appropriate consideration.

[0154] In one embodiment, the other steps can be performed in any manner described herein.

[0155] In another aspect, the present disclosure provides a system including: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a library generating unit that generates an mRNA library containing a plurality of composite sequences each including a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR, and optionally optimizes the polyA tail; 3) an evaluation unit that evaluates the amount of protein expression by the mRNA library; and 4) a selection unit that, as necessary, selects mRNAs from the mRNA library that achieve a desired value or level. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0156] In one embodiment, 1) the sequence information providing section, in which the user provides sequence information of the translated region, can be realized by any method known in the art. This section receives, analyzes, and processes nucleic acid sequences. First, a DNA or RNA sequence provided by the user may be received as input. In this case, the start codon (e.g., ATG) and stop codon (e.g., TAA, TAG, TGA) may be detected and the translated region may be extracted. The extracted sequence may be saved and formatted for provision to the user. The obtained sequence information of the translated region may also be returned to the user for further analysis or confirmation as needed.

[0157] In one embodiment, 2) the library generation section for generating an mRNA library containing a plurality of composite sequences each including a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR, and optimizing the polyA tail as needed, can be realized by any method known in the art. This section is used to combine designed nucleic acid sequences and create a library. First, an mRNA library may be generated by randomly combining nucleic acid sequences encoding separately designed translated regions, 5' cap structural sequences, and optimized UTR sequences, and then polyA tails of random lengths may be combined.

[0158] In one embodiment, 3) the evaluation step of evaluating the expression level of proteins from the mRNA library can be performed using any technique known in the art. Evaluation of the function of a nucleic acid construct can be achieved, for example, by introducing the nucleic acid construct into a target cell or a model organism, measuring the expression levels of RNA or protein (using techniques such as RT-PCR or Western blotting) to confirm the expression and function after introduction, evaluating biological effects such as changes in cell morphology, growth rate, and metabolic activity (fluorescence microscopy or flow cytometry can be used for functional evaluation), and analyzing the obtained data to comprehensively evaluate the function of the nucleic acid construct, but is not limited to this. These evaluations can also be performed in a virtual space (see, for example, Filippo MD, Damiani C, Vanoni M, Maspero D, Mauri G, Alberghina L, Pescini D. Single-cell Digital Twins for Cancer Preclinical Investigation. Methods Mol Biol. 2020; 2088: 331-343. doi: 10.1007 / 978-1-0716-0159-4_15. PMID: 31893381, Honardoost MA, Naghavian R, Ahmadinejad F, Hosseini A, Ghaedi K. Integrative computational mRNA-miRNA interaction analyzes of the autoimmune-deregulated miRNAs and well-known Th17 differentiation regulators: An attempt to discover new potential miRNAs involved in Th17 differentiation. Gene. 2015 Nov 10;572(2):153-62. doi:10.1016 / j.gene.2015.08.043. Epub 2015 Aug 22. PMID:26307197. etc.

[0159] 4) The selection step of selecting mRNAs that achieve a desired value or level from the mRNA library can be carried out using any technique known in the art. The value or level can be selected appropriately depending on the purpose. For example, the step of selecting nucleic acid constructs that achieve a desired value or level can be carried out by, but is not limited to, obtaining multiple candidate nucleic acid constructs from the library, simulating gene expression and function when each construct is introduced into appropriate cells or model organisms, and identifying constructs whose expression level or biological effect meets the desired criteria based on the obtained prediction results. If necessary, additional simulations are performed on the selected candidates to confirm reproducibility, selecting nucleic acid constructs that exhibit the desired performance, and, if necessary, performing detailed characterization.

[0160] 1) to 4) may be configured so that one part carries out multiple functions, or may be configured as separate sections.

[0161] In another aspect, the present disclosure provides a system for generating an optimized nucleic acid construct, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a selection unit in which a 5'UTR comprising a naturally occurring sequence; 3) a design unit in which a 3'UTR comprising a complementary and non-complementary portion to the 5'UTR of the naturally occurring sequence; 4) a structure selection unit in which a structure of other portions of a nucleic acid construct is selected; 5) an evaluation unit in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated; and 6) a desired selection unit in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0162] In another aspect, the present disclosure provides a system for designing one or more mRNAs that express a protein desired by a user, the system comprising: 1) a selection unit that selects a 5'UTR structure nucleic acid sequence from a database related to the expression of the protein, 2) a structure selection unit that selects a 3'UTR structure nucleic acid sequence including complementary and non-complementary portions thereto, 3) a step of combining a 5'cap structure sequence, the 5'UTR structure, the translated region of the protein, and the 3'UTR structure, and 4) an optimization unit that optimizes a polyA tail. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0163] In one embodiment, the selection unit further includes an evaluation unit that evaluates the expression level of a protein.

[0164] In one aspect, the present disclosure provides a system for controlling protein expression of an mRNA having a 5'UTR structure and a 3'UTR structure, the system comprising: 1) a design unit that designs a nucleic acid sequence including a portion complementary or non-complementary to all or a portion of the 5'UTR, and 2) a sequence addition unit that adds the nucleic acid sequence to the 3'UTR structure. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0165] In another aspect, the present disclosure provides a system for generating optimized mRNA, comprising: a sequence information input unit that inputs sequence information of a translation region of an mRNA having the translation region, a first transmission unit that transmits the sequence information input from the customer sequence information input unit, a receiving unit that receives the sequence information transmitted from the first transmission unit, a first storage unit that stores the sequence information received by the receiving unit, a second storage unit that stores information on a predetermined untranslated region of the mRNA, a third storage unit that stores information on a predetermined PolyA strand of the mRNA, and an optimization unit (which may include AI) that optimizes the sequence information. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0166] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating optimized mRNA, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which an mRNA library is generated containing a plurality of composite sequences each containing a combination of a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR (including a step of optimizing a polyA tail, as necessary); 3) a step in which the amount of protein expression by the mRNA library is evaluated; and 4) a step in which an mRNA that achieves a desired value or level is selected from the mRNA library, as necessary. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0167] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region, 2) a step in which a 5'UTR comprising a naturally occurring sequence, 3) a step in which a 3'UTR comprising portions complementary and non-complementary to the 5'UTR of the naturally occurring sequence, 4) a step in which a structure of other portions of the nucleic acid construct is selected, 5) a step in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated, and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0168] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for designing one or more mRNAs that express a protein desired by a user, the method comprising the steps of: selecting, for a protein desired by a user, a nucleic acid sequence for a 5'UTR structure from a database related to the expression of the protein, selecting a nucleic acid sequence for a 3'UTR structure including complementary and non-complementary portions thereto, combining a 5'cap structure sequence, the 5'UTR structure, the translated region of the protein, and the 3'UTR structure, and optimizing a polyA tail. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0169] In one embodiment, the method of the present disclosure further comprises, in addition to step 1), a step of evaluating the expression level of the protein.

[0170] In another aspect, the present disclosure provides a program encoding procedures for causing a computer to execute a method for controlling protein expression of an mRNA having a 5'UTR structure and a 3'UTR structure, by: 1) designing a nucleic acid sequence including a complementary or non-complementary portion to all or a portion of the 5'UTR, and 2) adding the nucleic acid sequence to the 3'UTR structure. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0171] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized mRNA, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which an mRNA library is generated containing a plurality of composite sequences each including a nucleic acid sequence encoding the translated region, a 5' cap structural sequence, and an optimized UTR (optionally including a step of optimizing a polyA tail); 3) a step in which the mRNA library is evaluated for protein expression; and 4) optional steps in which an mRNA that achieves a desired value or level is selected from the mRNA library. Each embodiment of the recording medium of the present disclosure can optionally be applied with one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0172] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region, 2) a step in which a 5'UTR comprising a naturally occurring sequence, 3) a step in which a 3'UTR comprising portions complementary and non-complementary to the 5'UTR of the naturally occurring sequence, 4) a step in which a structure of other portions of the nucleic acid construct is selected, 5) a step in which the function of the nucleic acid construct generated in steps 1 to 4 is evaluated, and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the recording medium of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0173] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for designing one or more mRNAs that express a protein desired by a user, the method comprising the steps of: 1) selecting a 5'UTR structure nucleic acid sequence from a database related to the expression of the protein, 2) selecting a 3'UTR structure nucleic acid sequence including complementary and non-complementary portions thereto, 3) combining a 5'cap structure sequence, the 5'UTR structure, the translated region of the protein, and the 3'UTR structure, and 4) optimizing a polyA tail. Each embodiment of the recording medium of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0174] In one embodiment, the present disclosure provides the method, wherein step 1) further comprises a step of evaluating the expression level of the protein.

[0175] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for controlling protein expression of an mRNA having a 5'UTR structure and a 3'UTR structure, by: 1) designing a nucleic acid sequence including a complementary or non-complementary portion to all or a portion of the 5'UTR, and 2) adding the nucleic acid sequence to the 3'UTR structure. Each embodiment of the recording medium of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0176] In another aspect, the present disclosure provides a system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences containing the sequence information of the translated region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap and its analogous structure and a polyA tail; 3) an evaluation unit that evaluates the function of the nucleic acid constructs in the library; and 4) a desired selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of step 3. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0177] In another aspect, the present disclosure provides a system for generating an optimized nucleic acid construct, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence design unit that designs a 5'UTR or 3'UTR comprising a naturally occurring sequence; 3) a non-natural sequence design unit that designs a 5'UTR or 3'UTR comprising a non-natural sequence corresponding to the 5'UTR or 3'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that include the translated region and 5'UTR and 3'UTR designed in steps 1 to 3 above, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit that evaluates the function of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the result of step 5. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0178] In another aspect, the present disclosure provides a system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which the user provides sequence information of the translated region; 2) a design unit in which 5'UTR and 3'UTR containing complementary and non-complementary portions; 3) a library construction unit in which a library of nucleic acid constructs having multiple sequences containing the translated region, 5'UTR, and 3'UTR designed in steps 1 and 2, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 4) an evaluation unit in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a structure selection unit in which nucleic acid constructs achieving a desired value or level are selected from the results of steps 4. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0179] In another aspect, the present disclosure provides a system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a native sequence design unit that designs a 5'UTR or 3'UTR containing a naturally occurring sequence; 3) a complementary / non-complementary design unit that designs a 3'UTR or 5'UTR containing a complementary / non-complementary portion to the 5'UTR or 3'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that include the translated region, 5'UTR, and 3'UTR designed in accordance with steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit that evaluates the function of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of step 5. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0180] In another aspect, the present disclosure provides a system for generating an optimized nucleic acid construct, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence design unit that designs a 5'UTR comprising a naturally occurring sequence; 3) a complementary / non-complementary design unit that designs a 3'UTR comprising a complementary / non-complementary portion to the 5'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit that evaluates the function of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the result of step 5. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0181] In another aspect, the present disclosure provides a system for generating an optimized nucleic acid construct, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a sequence selection unit in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression; 3) a complementary / non-complementary design unit in which a 3'UTR including a complementary / non-complementary portion to the 5'UTR is designed; 4) a library construction unit in which a library of nucleic acid constructs having multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3 above, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a structure selection unit in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5 above. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0182] In another aspect, the present disclosure provides a system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence selection unit in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a complementary / non-complementary design unit in which a 3'UTR including a complementary / non-complementary portion to the 5'UTR is designed; 4) a library construction unit in which a library of nucleic acid constructs having multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3 above, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a structure selection unit in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5 above. Each embodiment of the system of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere herein.

[0183] In another aspect, the present disclosure provides a program encoding procedures for causing a computer to execute a method for generating optimized nucleic acid constructs, the steps including: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a library of nucleic acid constructs of multiple sequences is constructed, comprising the sequence information of the translated region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap or its analogous structure, and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0184] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR comprising a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR comprising a non-naturally occurring sequence corresponding to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0185] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which 5'UTR and 3'UTR containing complementary and non-complementary portions; 3) a step in which a library of nucleic acid constructs having multiple sequences containing the translated region, 5'UTR, and 3'UTR designed in steps 1 and 2, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 4. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0186] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR comprising a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR comprising a complementary or non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0187] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR comprising a naturally occurring sequence; 3) a step in which a 3'UTR comprising a complementary and non-complementary portion to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0188] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs having multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the function of the nucleic acid constructs in the library is evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0189] In another aspect, the present disclosure provides a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs having multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the program of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0190] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a library of nucleic acid constructs having multiple sequences is constructed, the library including the sequence information of the translated region, in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap or its analogous structure, and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3. Each embodiment of the recording medium of the present disclosure can optionally be applied with one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0191] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR comprising a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR comprising a non-naturally occurring sequence corresponding to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the recording medium of the present disclosure can optionally be applied with one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0192] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which 5'UTR and 3'UTR containing complementary and non-complementary portions; 3) a step in which a library of nucleic acid constructs having multiple sequences containing the translated region, 5'UTR, and 3'UTR designed in steps 1 and 2, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 4. Each embodiment of the recording medium of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0193] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR includes a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR includes a complementary and non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the recording medium of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0194] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR comprising a naturally occurring sequence; 3) a step in which a 3'UTR comprising a complementary and non-complementary portion to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the recording medium of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0195] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs having multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the function of the nucleic acid constructs in the library is evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the recording medium of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0196] In another aspect, the present disclosure provides a recording medium storing a program encoding steps for causing a computer to execute a method for generating an optimized nucleic acid construct, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR including a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs having multiple sequences including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the function of the nucleic acid constructs in the library is evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5. Each embodiment of the recording medium of the present disclosure can optionally apply one or more features of various embodiments of the methods, systems, and nucleic acid constructs described elsewhere in this specification.

[0197] The system according to the present embodiment described above can be implemented in various ways using a hardware configuration capable of realizing each of the above-described functions. For example, the information analysis system may be realized by a single device, or may be a system in which multiple devices each realize some of the functions and each device appropriately exchanges data via a network or recording medium to realize all of the functions as a whole.

[0198] The system configuration can be realized by a hardware configuration similar to that of a general computer system. The computer system 10 shown in Figure 19, for example, includes a processor 11, RAM 12, ROM 13, an internal hard disk drive 14, a removable memory 15 such as an external hard disk drive, CD, DVD, USB memory, memory stick, or SD card, an input / output user interface 16 (touch panel, keyboard, mouse, speaker, microphone, lamp, etc.) through which a user exchanges data with the computer system 10, a wired / wireless communication interface 17 capable of communicating with other computer devices, and a display 18. Each function of the system described above can be realized, for example, by the processor 11 reading a program stored in advance in the hard disk drive 14, ROM 13, removable memory 15, etc. into a memory such as RAM 12, and executing the program while appropriately reading the data required for processing from the hard disk drive 14, ROM 13, removable memory 15, etc.

[0199] As described above, the system according to this embodiment may be realized by one computer device or by multiple computer devices. When the information analysis system 1 according to this embodiment is configured by multiple computer devices, each computer device may have the computer system configuration shown in Fig. 19. Note that the hardware configuration shown in Fig. 19 is merely an example and is not limited to this.

[0200] In the sequence calculation, gene sequence information is input into a sequence design tool to calculate candidate sequences for modification, which are candidates for gene sequences of a host cell for modifying the expression level of a key protein. The "sequence design tool" may be an existing tool such as GeneOptimizer (registered trademark) from Thermofisher Scientific.

[0201] In one aspect, the present disclosure provides any nucleic acid construct produced by the disclosed technique. The nucleic acid construct of the present disclosure can regulate protein expression. Preferably, the nucleic acid construct of the present disclosure can increase protein expression.

[0202] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range" of "two values," the range also includes the two values ​​themselves.

[0203] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0204] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0205] The reagents used were specifically the products described in the Examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

[0206] (Molecular Biology Experimental Procedures) General DNA, RNA, and gene recombination experimental procedures were performed according to standard protocols (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)).

[0207] (Production Example) (Purification of DNA Fragments Using Low-Melting-Point Agarose Gel) DNA fragments were separated by electrophoresis for 1 hour at 100 V using a 0.7% low-melting-point agarose gel prepared using 2-hydroxyethyl agarose (Sigma-Aldrich) and 1x TAE buffer (Nacalai). After electrophoresis, the gel was stained for 30 minutes with 1x TAE buffer containing GelRed nucleic acid gel stain (Fujifilm Wako Pure Chemical Industries, Ltd.), and the target DNA fragments were recovered from the agarose gel by visualization under long-wavelength ultraviolet light (366 nm). The agarose gel containing the DNA fragments was dissolved by incubating it in the presence of Thermostable β-Agarase (Nippon Gene) at 63°C for 5 minutes and then at 60°C for 10 minutes. TE-saturated phenol (Nacalai) was then added and mixed thoroughly. The phenol and aqueous phases were separated by centrifugation (20,000 × g, 10 minutes), and the aqueous phase was collected in a new tube. To the collected aqueous phase, 1-butanol was added, thoroughly mixed, and then centrifuged (20,000 × g, 10 minutes) to separate the 1-butanol and aqueous phases. The separated 1-butanol phase was removed. This series of steps was repeated three times to remove the phenol and reduce the volume of the aqueous phase. DNA fragments were precipitated by adding 3M potassium acetate-acetic acid buffer (pH 5.2) and ethanol to the aqueous phase, thoroughly mixing, and then centrifuging (20,000 × g, 10 minutes). The precipitated DNA fragments were washed with 70% ethanol and then dissolved in TE buffer (Nacalai).

[0208] (Annealing of synthetic oligonucleotides) Two types of 100 μM synthetic oligonucleotides, each 5 μL, were mixed and incubated at 99° C. for 10 seconds using a TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio), followed by gradual cooling to 40° C. over 90 minutes to form 50 μM double-stranded oligonucleotides. These double-stranded oligonucleotides were diluted to 0.5 μM with distilled water.

[0209] (Ligation, E. coli transformation, plasmid preparation) 1 μl of a 1-20 ng / μl restriction enzyme-treated plasmid solution, 1 μl of a 0.1-20 ng / μl DNA fragment solution or 1 μl of a 0.5 μM double-stranded oligonucleotide solution, and 2 μl of DNA Ligation Kit <Mighty Mix> (Takara Bio) were mixed and incubated at 16°C for 1-4 hours to carry out a ligation reaction. 1 μl of this reaction solution was mixed with 10 μl of E. coli JM109 competent cells (Takara Bio), left on ice for 1 hour, and then incubated in a 42°C water bath for 1 minute. After incubation, the E. coli was left on ice for 2 minutes, after which 50 μl of SOC medium was added, and the mixture was cultured at 37°C for 1 hour at 30 rpm using a small rotary incubator RT-50 (Taitec). After cultivation, the E. coli was smeared onto an LB agar plate containing 10 μg / ml carbenicillin and cultured overnight at 37°C. The E. coli colonies formed on the agar plate were inoculated into 2 ml of LB liquid medium containing 10 μg / ml carbenicillin and cultured overnight at 37°C at 220 rpm using a medium-sized thermostatic shaker BR-53FP (Taitec). After cultivation, the E. coli was recovered by centrifugation (20,000 × g, 1 minute), and plasmids were prepared using a QIAprep Spin Miniprep Kit and a QIAcube nucleic acid extraction and purification system (Qiagen) according to the manufacturer's instructions.

[0210] (Construction of a Plasmid to Serve as a Template for RNA Synthesis) A DNA fragment consisting of a T7 promoter sequence, a Kozak sequence, a stop codon, and a 101-base-long poly(A) continuous sequence was amplified by PCR using primers Common-F and Common-R (SEQ ID NOs: 3 and 4) and KOD-Plus-Ver. 2 (Toyobo) as a template. The DNA fragment was made by annealing synthetic oligonucleotides shown in SEQ ID NOs: 1 and 2, which have partially complementary 3' ends, and converting the single-stranded portions of the oligonucleotides into double-stranded fragments by polymerase extension reaction from 5' to 3' using KOD-Plus-Ver. 2, which is a Toyobo product. This DNA fragment contains a BsaI site for inserting a 5'UTR between the T7 promoter sequence and the Kozak sequence, a BspQI site for inserting an ORF between the Kozak sequence and the stop codon, a PaqCI site for inserting a 3'UTR between the stop codon and the poly A sequence, and a BsmBI site for linearizing the template DNA downstream of the poly A sequence. The amplified DNA fragment was purified using the MinElute PCR Purification Kit (Qiagen) and then inserted into the XcmI site of the pBR322_ΔtypeIIS plasmid to construct the pT7_TL_pA100 plasmid (SEQ ID NO: 135).

[0211] The DNA fragment encoding the fluorescent protein E2Crimson was prepared by annealing synthetic oligonucleotides of SEQ ID NOS: 5, 6, 7, 8, and 9, 10, each of which has a partially complementary 3' end, and then converting the single-stranded portions of these oligonucleotides to double strands by polymerase extension in the 5' to 3' direction using KOD-Plus-Ver. 2 (Toyobo). Using this double-stranded product as a template, the DNA fragment was amplified by PCR using primers Common-F and Common-R (SEQ ID NOS: 3, 4) and KOD-Plus-Ver. 2. The amplified DNA fragment was inserted into the XcmI site of the pBR322_ΔtypeIIS plasmid to construct the pBR_E2Crimson plasmid. The pT7_TL_E2Crimson_pA100 plasmid was constructed by inserting the E2Crimson DNA fragment obtained by treating pBR_E2Crimson with BspQI into the PaqCI site of pT7_TL_pA100. The DNA fragment encoding the SARS CoV-2 spike protein is a messenger RNA encoding the full-length SARS-CoV-2 spike glycoprotein Sept. 2020 document 11889; the sequence described in the 19th Health Science Council Immunization and Vaccine Subcommittee Materials (https: / / www.mhlw.go.jp / stf / shingi2 / 0000192554_00004.html). This DNA fragment was inserted upstream of the E2Crimson coding region in the pT7_TL_E2Crimson_pA100 plasmid to construct the pT7_TL_Spk_E2Crimson_pA100 plasmid. The DNA fragment encoding the modified luciferase gene luc2, 5x Flag tag fusion protein (luc2Flag) was obtained by PCR amplification in the same manner as E2Crimson. Luc2Flag was further provided with a BsmBI site for inserting a 5'UTR, a PaqCI site for inserting a 3'UTR, and a BsaI site downstream of the poly(A) continuous sequence for linearizing the template DNA.This DNA fragment was inserted into the XcmI site of the pBR322_ΔtypeIIS plasmid to construct p23-054_pT7_TL2_luc2Flag plasmid (SEQ ID NO: 711).

[0212] The Pfizer 5'UTR sequence was obtained by annealing the synthetic oligonucleotides shown in SEQ ID NOs: 13 and 14. The pT7_TL_5Pf_E2Crimson_pA100 or pT7_TL_5Pf_Spk_E2Crimson_pA100 plasmid was constructed by inserting this DNA fragment into the BsaI site of pT7_TL_E2Crimson_pA100 or pT7_TL_5Pf_Spk_E2Crimson_pA100. The pT7_TL_5Pf_luc2Flag_pA100 plasmid was constructed by inserting this DNA fragment into the BsmBI site of p23-054_pT7_TL2_luc2Flag. The Pfizer 3'UTR sequence was amplified by PCR using primers Common-F and Common-R (SEQ ID NOs: 3 and 4) and KOD-Plus-Ver. 2 (Toyobo) as a template. Synthetic oligonucleotides shown in SEQ ID NOs: 11 and 12, which are partially complementary at the 3' end, were annealed, and the resulting single-stranded portions were polymerase-extended from 5' to 3' using KOD-Plus-Ver. 2. This PCR fragment was cloned into the XcmI site of the pBR322_ΔtypeIIS plasmid to construct the pBR-3Pf plasmid. The Pfizer 3'UTR sequence obtained by treating this pBR-3Pf plasmid with BsaI was inserted into the PaqCI site of pT7_TL_5Pf_E2Crimson_pA100 or pT7_TL_5Pf_Spk_E2Crimson_pA100 and pT7_TL_5Pf_luc2Flag_pA100 to construct pT7_TLpA_5Pf_E2Crimson_3Pf_pA100 (SEQ ID NO: 136) or pT7_TL_5Pf_Spk_E2Crimson_3Pf_pA100 (SEQ ID NO: 300) and pT7_TL_5Pf_luc2Flag_3Pf_pA100 (SEQ ID NO: 711).

[0213] The 5'UTR sequence of the GAPDH gene was obtained by annealing the synthetic oligonucleotides shown in SEQ ID NOs: 15 and 16. The pT7_TL_5GAP_E2Crim_pA100 plasmid was constructed by inserting this DNA fragment into the BsaI site of pT7_TL_E2Crimson_pA100. The 3'UTR sequence, which is partially complementary to the 5'UTR sequence of the GAPDH gene, was obtained by annealing the synthetic oligonucleotides described below. The pT7_TL_5GAP_E2Crim_3UTR_pA100 plasmid was constructed by introducing this DNA fragment into the PaqCI site of pT7_TL_5GAP_E2Crim_pA100. The sequence numbers of the synthetic oligonucleotides and the combinations of the constructed plasmids are listed in Table 1 below.

[0214]

[0215] A 3'UTR sequence partially complementary to the Pfizer 5'UTR sequence was obtained by annealing the synthetic oligonucleotides described below. This DNA fragment was introduced into the PaqCI site of pT7_TL_5Pf_E2Crimson_pA100 to construct the pT7_TL_5Pf_E2Crim_3UTR-100_pA100 plasmid. The sequence numbers of the synthetic oligonucleotides and the combination of the constructed plasmid are described below.

[0216]

[0217]

[0218]

[0219]

[0220] The 5'UTR sequence of the HSD17B4 gene was obtained by annealing the synthetic oligonucleotides shown in SEQ ID NOs: 99 and 100. The pT7_TL_5HSD_E2Crim_pA100 plasmid was constructed by inserting this DNA fragment into the BsaI site of pT7_TL_E2Crimson_pA100. The 3'UTR sequence, which is partially complementary to the 5'UTR sequence of the HSD17B4 gene, was obtained by annealing the synthetic oligonucleotides described below. The pT7_TL_5HSD_E2Crim_3UTR_pA100 plasmid was constructed by introducing this DNA fragment into the PaqCI site of pT7_TL_5HSD_E2Crim_pA100. The sequence numbers of the synthetic oligonucleotides and the combination of the constructed plasmids are as follows:

[0221]

[0222] The 5'UTR sequences of the genes selected from the database analysis were obtained by annealing the synthetic oligonucleotides described below. Plasmids described in SEQ ID NOs: 371 to 391 were constructed by inserting this DNA fragment into the BsaI site of pT7_TL_E2Crimson_pA100. 3'UTR sequences partially complementary to the 5'UTR sequences of these genes were obtained by annealing the synthetic oligonucleotides described below. Plasmids described in SEQ ID NOs: 331 to 450 were constructed by introducing this DNA fragment into the PaqCI site of pT7_TL_5HSD_E2Crim_pA100. The sequence numbers of the synthetic oligonucleotides and the combinations of the constructed plasmids are as follows:

[0223]

[0224]

[0225] For plasmids with functional 3'UTR sequences, the 5'UTR and 3'UTR sequences were obtained by annealing the synthetic oligonucleotides listed in Table 5. These DNA fragments were constructed by inserting the 5'UTR sequence into the BsaI site and the 3'UTR sequence into the PaqCI site of pT7_TL_E2Crimson_pA100 or pT7_TL_Spk_E2Crimson_pA100. The sequence numbers of the synthetic oligonucleotides and the combinations of the constructed plasmids are listed in Table 5.

[0226] A 3'UTR sequence partially complementary to the Pfizer 5'UTR sequence or a non-naturally occurring 5'UTR sequence was obtained by annealing the synthetic oligonucleotides listed in Table 6. This DNA fragment was introduced into the PaqCI site of pT7_TL_5Pf_luc2Flag_pA100 to construct the pT7_TL_5Pf_luc2Flag_3UTR_pA100 plasmid. The sequence numbers of the synthetic oligonucleotides and the combination of the constructed plasmids are listed in Table 6.

[0227] (In vitro transcription by T7 RNA polymerase) To terminate RNA synthesis by dissociating RNA polymerase from the template DNA and to generate a continuous A nucleotide sequence at the 3' end of the synthesized RNA, the template DNA plasmid was linearized with the restriction enzyme BsmBI (New England Biolab). After BsmBI treatment, TE-saturated phenol was added to the plasmid and mixed thoroughly, followed by centrifugation (20,000 × g, 10 minutes) to separate it into an organic phase and an aqueous phase, and the aqueous phase was recovered in a new tube. 1-butanol was added to the recovered aqueous phase, mixed thoroughly, and then centrifuged (20,000 × g, 10 minutes) to separate it into a 1-butanol phase and an aqueous phase, and the separated 1-butanol phase was removed. This procedure was repeated three times to completely remove the phenol and reduce the volume of the aqueous phase. To this aqueous phase, 3M sodium acetate solution (pH 5.2) (Nacalai) and ethanol were added, mixed thoroughly, and then centrifuged (20,000 × g, 10 minutes) to precipitate the DNA fragments. The precipitated DNA fragments were washed with 70% ethanol and then dissolved in distilled water to obtain linear template DNA. In vitro transcription reactions were performed by incubating at 42°C for 3 hours under the conditions of 25 ng / μl linear template DNA, 1x T7 RNA polymerase buffer (Takara Bio), 5 mM DTT, 1.6 mM CleanCap (TriLink), 0.4 mM GTP, 2.0 mM ATP, 2.0 mM CTP, and 2.0 mM N1-methylpseudo-UTP (TriLink or Yamasa Shoyu), 1 U / μl Recombinant RNase Inhibitor (Takara Bio), 2 U / ml inorganic pyrophosphatase (New England Biolab), and 2.5 U / μl T7 RNA polymerase (Takara Bio). After this reaction, to remove the remaining linear template DNA, Recombinant DNase I (Takara Bio) was added to a final concentration of 0.125 U / μl, and the mixture was further incubated at 37° C. for 15 minutes.After the reaction, the RNA-containing solution was mixed thoroughly with a 1:1 mixture of citrate-saturated phenol (pH 4.3) (Nacalai) and chloroform (Nacalai), and then centrifuged (20,000 × g, 10 minutes) to separate the organic and aqueous phases. Chloroform was added to the recovered aqueous phase, mixed thoroughly, and then centrifuged (20,000 × g, 10 minutes) to separate the chloroform and aqueous phases. This procedure was repeated twice to completely remove the phenol contained in the aqueous phase. 3M sodium acetate solution (pH 5.2) and ethanol were added to the aqueous phase, mixed thoroughly, and then centrifuged (20,000 × g, 10 minutes) to precipitate the RNA. The precipitated RNA was washed with 70% ethanol and then dissolved in 50 μl of distilled water. This RNA solution was applied to a MicroSpin S-200 Column (Cytiva) that had previously been centrifuged at 700 × g for 1 minute. After centrifugation at 700 × g for 2 minutes, 50 μl of distilled water was added and centrifuged at 700 × g for 2 minutes to remove unreacted nucleotides. To the eluate, 3 M sodium acetate solution (pH 5.2) and ethanol were added, mixed thoroughly, and centrifuged (20,000 × g, 10 minutes) to precipitate the RNA. The precipitated RNA was washed with 70% ethanol and then dissolved in 20 μl of distilled water. RNA concentration was quantified using a Nano Drop One microspectrophotometer (Thermofisher) and a Qubit RNA Broad Range Assay Kit (Thermofisher) according to the manufacturer's instructions.

[0228] (Quantitative Example) (RNA Transfection into Cultured Cells and Quantification of Fluorescent Protein and Luciferase Protein Expression Amounts) 293 cells were cultured in DMEM (Thermofisher or Nakarai) supplemented with 10% FBS (Thermofisher) and a penicillin-streptomycin mixed solution (Nacalai) under 5% CO ２ The cells were cultured at 37°C in the presence of 1.5 ml of medium per well of a 6-well culture plate. ５Cells were seeded onto the plate and cultured at 37°C for 24 hours before RNA transfection. A mixture of 48.5 μl of Opti-MEM (Thermofisher) and 1.5 μl of Lipofectamine messengerMAX (Thermofisher) and 50 μl of Opti-MEM containing 50 ng of RNA were prepared per well, and then mixed and incubated at room temperature for 10 minutes. This solution was added to the culture wells and cultured for an additional 24 hours at 37°C. After RNA transfection, 293 cells were washed with 1 ml of DPBS (Thermofisher) per well and then dissociated from the bottom of the culture plate with 0.5 ml of trypsin-EDTA (Nacalai). After dissociation, the cells were transferred to a 1.5 ml tube with the addition of 1 ml of DPBS and then collected by centrifugation at 3,000 x g for 3 minutes. The collected cells were resuspended in 100 μl of DPBS, and 30 μl of the suspension was dispensed into a 384-well, flat-bottom, black microplate (Greiner). The relative amount of E2Crimson protein contained in each well was calculated by fluorescence measurement at an excitation wavelength of 606 nm and an emission wavelength of 651 nm using an Infinite 200 PRO (TECAN) plate reader. After the fluorescence measurement, 3 μl of DPBS containing 5 μg / ml Calcein-AM (Dojindo Laboratories) was added to the cell suspension dispensed into each well, mixed, and incubated at 37°C for 30 minutes. The relative number of viable cells in each well was calculated by measuring the fluorescence of calcein, a hydrolysis product of calcein-AM by intracellular esterase, at an excitation wavelength of 480 nm and a fluorescence wavelength of 533 nm. It was separately confirmed that the fluorescence of the E2 Crimson protein did not interfere with the fluorescence of calcein. The ratio of the E2 Crimson fluorescence measurement value to the calcein fluorescence measurement value was used to represent the relative expression level of the E2 Crimson protein in each mRNA. For quantification of luciferase protein expression, 1 x 10 cells were added to 0.1 ml of medium per well of a 96-well culture plate. ４Cells were seeded and cultured at 37°C for 24 hours before RNA transfection. A mixture of 24.25 μl of Opti-MEM (Thermofisher) and 0.25 μl of Lipofectamine messengerMAX (Thermofisher) and 25 μl of Opti-MEM containing 25 ng of RNA were prepared per well, and then mixed and incubated at room temperature for 10 minutes. 6.7 μl of this mixture was added to the culture wells and cultured for an additional 24 hours at 37°C. After RNA transfection, 80 μl of culture medium was removed from the 293 cells per well, and 20 μl of Steady-Glo® Reagent (Promega) was added to the remaining 20 μl of culture medium and shaken for 10 minutes. 30 μl of this cell lysate was dispensed into a 384-well flat-bottom white microplate (Greiner). The luminescence intensity of the luciferase protein contained in each well was calculated by luminescence measurement using a plate reader Infinite 200 PRO (TECAN).

[0229] (Example 1: Selection of high-performance 5'UTR) In this example, the translation level was calculated by ribosome profiling. The number of ribosomes bound and RNA quantitative values ​​for each gene were extracted from data registered in a gene expression information database, and the translation level was calculated. Based on these results, high-performance 5'UTRs were selected.

[0230] (Results) Polysome profiling data for 293 cells (accession number GSE94460, Zhang et al., Nat Commun. 2017, 23, 8(1):1749.), mouse skeletal muscle tissue (accession number GSE160917), and human adult erythroblasts and umbilical cord blood-derived erythroblasts (accession number GSE131809, Basak et al., Nat Genet. 2020, 52(2):138-145) were downloaded from the gene expression information database NCBI Gene Expression Omnibus (GEO). The number of ribosomes bound per kb of coding region and RNA quantification values ​​for each gene were extracted from these datasets. The translation level of each gene was calculated as the ratio of the number of ribosomes bound to the RNA quantification value.

[0231] The results for 293 cells, mouse skeletal muscle tissue, and cord blood-derived erythroblasts are shown in Figures 1, 2, and 3, respectively. Ribo-seq indicates the number of ribosomes bound, and RNA-seq indicates RNA quantification. Ratio indicates the ratio of the Ribo-seq value to the RNA-seq value. The top 200 genes are sorted by ratio and shown. Figure 4 shows the translation levels of the globin gene family in 293 cells, adult erythroblasts, and cord blood-derived erythroblasts. The numbers in the table indicate ratio values. ND indicates that the expression was below the detection limit. While expression of the HBA1, HBA2, HBB, HBD, HBE1, HBG1, HBG2, HBM, HBQ1, and HBZ genes was below the detection limit in 293 cells, expression was confirmed in adult erythroblasts and cord blood-derived erythroblasts.

[0232] (Example 2: Verification of expression levels of mRNAs carrying high-performance 5'UTRs) Based on the results of translation levels in 293 cells shown in Figure 1, 21 highly translated genes were selected. In this example, the expression levels of mRNAs carrying the 5'UTRs of these highly translated genes were verified. Construct production and expression level quantification were performed based on the above production example and quantification example.

[0233] (Results) The results for 293 cells and C2C12 cells are shown in Figures 5 and 6, respectively. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein for each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are plotted in black. Of the 21 types of 5'UTR examined, the expression levels were high in the RRP12, XBA2, KRT18, ACTB, and HIST1H2BK genes.

[0234] (Example 3: Improving expression efficiency by partial complementarity between 5'-3' untranslated regions) In this example, the effect of partial complementarity between 5'-3' untranslated regions on mRNA expression levels in 293 cells was examined. Construct production and expression level quantification were performed based on the above production example and quantification example.

[0235] (Results) Figure 7 shows the design method for non-complementary portions when designing a 3'UTR so that the 5'-3' untranslated region is partially complementary to an mRNA equipped with a high-performance 5'UTR. Figure 7(1) shows a design method in which a specific base in the 3'UTR is substituted to make it non-complementary, Figure 7(2) shows a design method in which a specific base in the 3'UTR is removed to make it non-complementary, Figure 7(3) shows a design method in which the above-mentioned base substitution and base removal are combined, and Figure 7(4) shows a design method in which a specific base is added to the 3'UTR to make it non-complementary. The rectangles in the figures indicate complementary portions.

[0236] Figure 8 shows the expression levels of mRNAs with GAPDH as the 5'UTR and various partially complementary sequences designed with the base substitutions shown in Figure 7(1) as the 3'UTR. For comparison, mRNAs with Pfizer sequences for both the 5'UTR and 3'UTR were used. The ratio of E2Crimson fluorescence measurements to Calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, with individual values ​​for each sample plotted in black. When the non-complementary portion was one base, high expression levels were observed at a complementarity rate of 75-89% and a complementary portion of 3-7 bases. Expression levels decreased when the complementary portion exceeded seven bases. Furthermore, high expression levels were observed when the non-complementary portion was two bases and the complementary portion was six bases, and when the non-complementary portion was three bases and the complementary portion was 11 bases.

[0237] Figure 9 shows the expression levels of mRNAs with the Pfizer sequence as the 5'UTR and various partially complementary sequences designed with the base substitutions shown in Figure 7(1) as the 3'UTR. For comparison, mRNAs with the Pfizer sequence in both the 5'UTR and 3'UTR were used. The ratio of the E2Crimson fluorescence measurement value to the calcein fluorescence measurement value was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, with individual values ​​for each sample plotted in black. Complementary / non-complementary: Expression levels were enhanced compared to the Pfizer sequence in mRNAs 4-1, 3-1, 6-2, 5-2, and 9-3, increasing by approximately 50% at most. Expression levels tended to be higher when the number of complementary bases was shorter.

[0238] Figure 10 shows the expression levels of mRNAs with the HSD17B4 gene sequence as the 5'UTR and various partially complementary sequences designed with the base substitutions shown in Figure 7(1) as the 3'UTR. For comparison, mRNAs with Pfizer sequences for both the 5'UTR and 3'UTR were used. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, with individual values ​​for each sample plotted in black. Complementary / non-complementary: Expression levels were enhanced compared to the Pfizer sequence in mRNAs 4-1, 3-1, 6-2, 5-2, 9-3, and 8-3.

[0239] Figure 11 shows the expression levels of mRNAs with the Pfizer sequence as the 5'UTR, various partially complementary sequences designed with the base substitutions shown in Figure 7(1) as the 3'UTR, and SARS CoV2 Spike and E2Crimson fusion proteins as ORFs. For comparison, mRNAs with the Pfizer sequence in both the 5'UTR and 3'UTR were used. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are plotted in black. Complementary-non-complementary: In 6-1, 3-1, 11-2, 8-2, 7-2, 6-2, 5-2, 12-3, 11-3, 10-3, 9-3, and 8-3, expression levels were enhanced compared to the Pfizer sequence, increasing by approximately twofold at most. This was equivalent to the results for mRNA with the E2Crimson protein as the ORF shown in Figure 9. The ORF for the SARS CoV2 Spike and E2Crimson fusion protein is 4.5 kbp, while the ORF for the E2Crimson protein is 0.7 kbp, demonstrating that the effect of the partially complementary 5' untranslated region (UTR) and 3'UTR is not limited by the type of ORF or its length.

[0240] In summary, in mRNAs having various partially complementary sequences designed by base substitution as shown in Figure 7 (1), when the non-complementary portion (2) is one base, the complementary portion (3) is preferably 3 to 6 bases long; when the non-complementary portion (2) is two bases long, the complementary portion (3) is preferably 5 to 7 bases long; and when the non-complementary portion (2) is three bases long, the complementary portion (3) is preferably 8 to 9 bases long (Figure 14). The nucleic acid construct of the present disclosure has a region (1) in its 3' untranslated region that is partially complementary to the 5' untranslated region. In region (1), non-complementary portion (2) and complementary portion (3) alternate, and the fractional portion (4) is one or more bases long but not longer than the base length of complementary portion (3).

[0241] Figure 12 shows the expression levels of mRNAs with the Pfizer sequence as the 5'UTR and various partially complementary sequences designed by base deletion as shown in Figure 7(2) as the 3'UTR. For comparison, mRNAs with both the Pfizer sequence in the 5'UTR and 3'UTR were used. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, with individual values ​​for each sample plotted in black. Complementary / non-complementary: Expression levels were enhanced compared to the Pfizer sequence in 9-1, 5-1, 4-1, 3-1, 9-2, 8-2, 7-2, 6-2, 5-2, 11-3, 10-3, 9-3, and 8-3, with a maximum increase of approximately 60%. When the non-complementary portions shown in Figure 6 were substituted with bases, the expression levels were enhanced in mRNAs with complementary-non-complementary sequences: 4-1, 3-1, 6-2, 5-2, 9-3, and 8-3, compared to the Pfizer sequence. This indicates that when bases are removed, the expression level tends to be higher in mRNAs with longer complementary strands and higher complementarity rates.

[0242] Figure 13 shows the expression levels of mRNAs with the Pfizer sequence as the 5'UTR, various partially complementary sequences designed by base deletion as shown in Figure 7(2) as the 3'UTR, and SARS CoV2 Spike and E2Crimson fusion proteins as ORFs. For comparison, mRNAs with Pfizer sequences in both the 5'UTR and 3'UTR were used. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are plotted in black. In complementary-noncomplementary sequences: 9-1, 6-1, 5-1, 11-2, 10-2, 9-2, 8-2, 7-2, 11-3, 10-3, and 8-3, the expression level was enhanced compared to the Pfizer sequence, increasing by up to approximately 40%. When the noncomplementary portion shown in Figure 16 was substituted with a base, the expression level was enhanced compared to the Pfizer sequence in complementary-noncomplementary sequences: 6-1, 11-2, 8-2, 7-2, 6-2, 5-2, 12-3, 11-3, 10-3, 9-3, and 8-3. This indicates that base removal in long mRNAs also tends to result in higher expression levels in mRNAs with long complementary strands and high complementarity.

[0243] In summary, in mRNAs having various partially complementary sequences designed by base deletion as shown in Figure 7 (2), when the non-complementary portion (2) is 1 base, the complementary portion (3) is preferably 3 to 9 bases; when the non-complementary portion (2) is 2 bases, the complementary portion (3) is preferably 5 to 11 bases; and when the non-complementary portion (2) is 3 bases, the complementary portion (3) is preferably 8 to 11 bases (Figure 14). The nucleic acid construct of the present disclosure has a region (1) in its 3' untranslated region that is partially complementary to the 5' untranslated region. In region (1), non-complementary portions (2) and complementary portions (3) alternate, and the fractional portion (4) is at least 1 base and is equal to or shorter than the base length of the complementary portion (3).

[0244] (Example 4: Verification of the effect of partial complementarity on high-performance 5'UTRs) In this example, the effect of partial complementarity on mRNA expression levels was verified using 293 cells for the high-performance 5'UTRs found in Examples 1 and 2, or for high-performance 5'UTRs not included in gene expression data (derived from non-natural sequences). Construct production and expression level quantification were performed based on the above production example and quantification example.

[0245] (Results) Figures 15 and 16 show the expression levels of mRNAs containing the following 5'UTRs among the high-performance 5'UTRs found in Examples 1 and 2: HBB, HIST1H1C, HIST1H1E, HIST3H2A, HIST1H2BK, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, ACTB, KRT18, and PKM. In Figure 15, the 3'UTR had a sequence in which the complementary-non-complementary ratio was 8-2 due to base deletion. In Figure 16, the 3'UTR had a sequence in which the complementary-non-complementary ratio was 9-3 due to base substitution. The ratio of the E2Crimson fluorescence measurement value to the calcein fluorescence measurement value was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples of each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black plots. The effect of partial complementation was observed in HBB, HIST1H1C, HIST3H2A, HIST1H2BK, SFT2D2, KLHL11, PHGDH, USP11, TKT, ACTN4, MDH2, BROX, ACTB, KRT18, and PKM, using sequences in which the complementary-noncomplementary ratio is 9-3 due to base substitution. The effect of partial complementation was observed in HBB, HIST1H1C, SFT2D2, KLHL11, USP11, TKT, ACTN4, ACTB, KRT18, and PKM, using sequences in which the complementary-noncomplementary ratio is 8-2 due to base removal.

[0246] Figure 17 examines the expression levels of mRNAs with either HBB, HIST1H2BK, ACTB, or KRT18 as the 5'UTR, and either 8-2, 7-2, or 10-3 complementary-to-non-complementary sequences as the 3'UTR due to base deletion, or 6-2 or 9-3 complementary-to-non-complementary sequences due to base substitution. For comparison, mRNAs with both the 5'UTR and 3'UTR Pfizer sequences were used. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are plotted in black. Four mRNAs with a 5'UTR of HBB, three with HIST1H2BK, two with ACTB, and two with KRT18 were identified for which expression levels were enhanced compared to the Pfizer sequence.

[0247] Figure 24 shows the mRNA expression levels of a 5'UTR that is a high-performance 5'UTR that is not registered in a gene expression database (a non-naturally occurring sequence), and a 3'UTR that has either a complementary-non-complementary sequence of 8-2 due to base deletion or a complementary-non-complementary sequence of 6-2 due to base substitution. The expression level was enhanced by the partially complementary sequence.

[0248] (Example 5: Addition of a partially complementary sequence to an mRNA having a functional UTR enhances the expression level of the translated region) This example demonstrates that partial complementarity to a functional 3'UTR, such as an miRNA regulatory sequence or a translation regulatory sequence, additively enhances the expression level.

[0249] Figure 18 shows the expression levels of mRNAs with the Pfizer sequence as the 5'UTR, the Pfizer sequence as the functional 3'UTR, and a partial complementary sequence between the stop codon and this functional 3'UTR, either 8-2, 7-2, or 10-3 complementary-to-non-complementary sequences due to base deletion, or 6-2 or 9-3 complementary-to-non-complementary sequences due to base substitution. For comparison, mRNAs with both the Pfizer sequence in the 5'UTR and 3'UTR were used. The ratio of E2Crimson fluorescence measurements to calcein fluorescence measurements was used to represent the relative expression level of E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black. Expression levels were enhanced compared to the Pfizer sequence for all mRNAs.

[0250] The expression level can also be verified for mRNAs having a Pfizer sequence as the 5'UTR, a Pfizer sequence as the functional 3'UTR, and a partial complementary sequence between this functional 3'UTR and the polyA tail, where either a base deletion results in a complementary-non-complementary sequence of 8-2, 7-2, or 10-3, or a base substitution results in a complementary-non-complementary sequence of 6-2 or 9-3. mRNAs prepared using SEQ ID NOs: 547, 548, 549, 550, or 551 as templates are used. It is expected that the expression level of any of these mRNAs will be enhanced compared to the Pfizer sequence. The expression level can also be verified for mRNAs having a Pfizer sequence as the 5'UTR, two Pfizer sequences as the functional 3'UTR, and a partial complementary sequence between these two functional 3'UTRs, where either a base deletion results in a complementary-non-complementary sequence of 8-2, 7-2, or 10-3, or a base substitution results in a complementary-non-complementary sequence of 6-2 or 9-3. It is expected that the expression level of both mRNAs will be higher than that of the Pfizer sequence.

[0251] Figure 20 shows the expression levels of mRNAs containing HBA (α-globin), HIST1H1C, USP11, or HIST1H2BK as the 5'UTR, SARS CoV2 Spike or E2Crimson fusion protein as the ORF, a stuffer sequence as the functional 3'UTR, and either a base deletion resulting in a complementary-noncomplementary sequence of 8-2, 7-2, or 10-3 between this functional 3'UTR and the poly(A) tail, or a base substitution resulting in a complementary-noncomplementary sequence of 6-2 or 9-3. Even when the 3'UTR contained a stuffer sequence, a functional 3'UTR, the expression level was enhanced in certain designs using partial complementarity between the 5'UTR and 3'UTR.

[0252] Figure 21 shows the expression levels of mRNAs containing HBA (α-globin) as the 5'UTR, the modified luciferase gene luc2 as the ORF, a 5xFlag tag fusion protein, a stuffer sequence as the functional 3'UTR, and either a base deletion resulting in a complementary-noncomplementary sequence of 8-2 or 10-3 between this functional 3'UTR and the polyA tail, or a base substitution resulting in a complementary-noncomplementary sequence of 6-2 or 9-3 between this functional 3'UTR and the polyA tail. mRNAs with Pfizer sequences for both the 5'UTR and 3'UTR were used for comparison. Even when the 3'UTR contained a stuffer sequence, which is a functional 3'UTR, the expression level was enhanced in a specific design using partial complementarity between the 5'UTR and 3'UTR.

[0253] Figure 22 shows the expression levels of mRNAs with HBA (α-globin) as the 5'UTR, E2Crimson as the ORF, HBA (α-globin) 3'UTR as the functional 3'UTR, and either a base deletion resulting in a complementary-non-complementary sequence of 8-2, 7-2, or 10-3, or a base substitution resulting in a complementary-non-complementary sequence of 6-2 or 9-3, between this functional 3'UTR and the polyA tail or between the functional 3'UTR and the stop codon. For comparison, mRNAs with both the 5'UTR and 3'UTR sequences from Pfizer were used. Expression levels were enhanced whether the 3'UTR sequence that was partially complementary to the 5'UTR was located between the functional 3'UTR and the polyA tail or between the functional 3'UTR and the stop codon.

[0254] Figure 23 shows the expression levels of mRNAs containing HBA (α-globin) as the 5'UTR, the modified luciferase gene luc2 as the ORF, a 5xFlag tag fusion protein, CYBA or ARE as the functional 3'UTR, and either 8-2 complementary-non-complementary sequences due to base deletion or 6-2 complementary-non-complementary sequences due to base substitution between this functional 3'UTR and the stop codon, as verified by luciferase luminescence measurement. For comparison, mRNA without a 3'UTR that is partially complementary to the 5'UTR was used. The expression level was enhanced by the partially complementary 3'UTR.

[0255] Figure 26 shows a schematic diagram of mRNAs with a known 5'UTR sequence, HBA (α-globin), and a functional 5'UTR sequence, EMCV IRES; an ORF, a modified luciferase gene luc2, or a 5xFlag tag fusion protein; and a 3'UTR with either 8-2, 7-2, or 10-3 complementary-to-non-complementary configurations due to base deletion, or 6-2 or 9-3 complementary-to-non-complementary configurations due to base substitution. mRNAs prepared using SEQ ID NOS: 728-739 as templates may be used for investigation. mRNAs with partially complementary sequences (SEQ ID NOS: 729-733, 735-739) are expected to have higher expression levels than the mRNAs without these partially complementary sequences (SEQ ID NOS: 728, 734). The diagram shows a schematic diagram of an mRNA having a 5'UTR with a complementary-non-complementary sequence of 8-2, 7-2, or 10-3 due to base deletion, or a complementary-non-complementary sequence of 6-2 or 9-3 due to base substitution, a functional 5'UTR sequence, the EMCV IRES, a modified luciferase gene luc2, or a 5xFlag tag fusion protein as an ORF, and KRT18 as a 3'UTR. mRNAs prepared using SEQ ID NOS: 740-745 as templates may be used for testing. mRNAs with partially complementary sequences (SEQ ID NOS: 741-750) are expected to have higher expression levels than the mRNA (SEQ ID NOS: 740) that does not have the partially complementary sequence.

[0256] Figure 27 shows a schematic diagram of an mRNA having a known 5'UTR sequence, HBA (α-globin), a modified luciferase gene luc2, a 5xFlag tag fusion protein, and a 3'UTR sequence with a complementary-non-complementary sequence of 8-2 due to base deletion, and a functional 3'UTR sequence, CYBA or ARE. mRNAs prepared using SEQ ID NOS: 751-754, 787, and 788 as templates may be used for testing. mRNAs with partially complementary sequences (SEQ ID NOS: 753, 754, 787, and 788) are expected to have higher expression levels than mRNAs without these partially complementary sequences (SEQ ID NOS: 751 and 752). The figure shows a schematic diagram of an mRNA having a 5'UTR with a complementary-non-complementary sequence of 8-2 due to base deletion, a modified luciferase gene luc2 as the ORF, a 5xFlag tag fusion protein, a known sequence of KRT18 as the 3'UTR, and a CYBA or ARE as the functional 3'UTR. mRNAs prepared using SEQ ID NOS: 755-762 as templates may be used for testing. mRNAs with partially complementary sequences (SEQ ID NOS: 757, 758, 761, 762) are expected to have higher expression levels than mRNAs without these partially complementary sequences (SEQ ID NOS: 755, 756, 759, 760).

[0257] Figure 28 shows a schematic diagram of an mRNA having the known sequence HBA (α-globin) and the functional 5'UTR sequence EMCV IRES as the 5'UTR, the modified luciferase gene luc2 and a 5xFlag tag fusion protein as the ORF, and the functional 3'UTR sequence CYBA or ARE and the known sequence KRT18 as the 3'UTR. mRNAs prepared using SEQ ID NOS: 763-774 as templates may be used for testing. mRNAs with partially complementary sequences (SEQ ID NOS: 765-768, 771-774) are expected to have higher expression levels than mRNAs without these partially complementary sequences (SEQ ID NOS: 763, 764, 769, 770).

[0258] Figure 29 shows a schematic diagram of mRNAs with a 5'UTR in which the complementary-non-complementary sequence is 8-2 due to base deletion, a functional 5'UTR sequence, the EMCV IRES, a modified luciferase gene luc2, and a 5xFlag tag fusion protein as ORFs, a known sequence, KRT18, as a 3'UTR, and a functional 3'UTR sequence, CYBA or ARE. mRNAs prepared using SEQ ID NOS: 775-786 as templates may be used for testing. mRNAs with partially complementary sequences (SEQ ID NOS: 777-780, 783-786) are expected to have higher expression levels than mRNAs without these partially complementary sequences (SEQ ID NOS: 775, 776, 781, 782).

[0259] Example 6: Partial complementarity synergistically increases the efficiency of expression with poly(A) tails. In this example, the effect of partial complementarity between the 5'-3' untranslated regions on the expression level of mRNA in 293 cells is examined using mRNAs with poly(A) tails of 100 bases, 150 bases, and 200 bases.

[0260] The expression level can be verified for mRNAs having the Pfizer sequence as the 5' UTR and a 3' UTR in which the complementary-noncomplementary sequence is 8-2, 7-2, or 10-3 due to base deletion, or the complementary-noncomplementary sequence is 6-2 or 9-3 due to base substitution. For a 100-base poly-A tail, mRNAs prepared using SEQ ID NOs: 268, 269, 274, 172, or 176 are used; for a 150-base poly-A tail, mRNAs prepared using SEQ ID NOs: 557, 558, 559, 560, or 561 are used; and for a 200-base poly-A tail, mRNAs prepared using SEQ ID NOs: 561, 562, 563, 564, or 565 are used. It is expected that the mRNA expression level increases with increasing poly-A tail length, whether the complementary-noncomplementary sequence is 8-2, 7-2, or 10-3 due to base deletion, or the complementary-noncomplementary sequence is 6-2 or 9-3 due to base substitution.

[0261] (Example 7: Expression levels are enhanced by inserting a sequence partially complementary to a known 3'UTR sequence into the 5'UTR.) Figure 25 shows a schematic diagram of an mRNA having the known 3'UTR sequence, KRT18, and a 5'UTR with either 8-2, 7-2, or 10-3 complementary-non-complementary sequence due to base deletion, or 6-2 or 9-3 complementary-non-complementary sequence due to base substitution. mRNAs prepared using SEQ ID NOS: 722 to 727 as templates may be used for investigation. mRNAs with partially complementary sequences (SEQ ID NOS: 722, 723, 724, 725, and 726) are expected to have enhanced expression levels compared to the mRNA (SEQ ID NOS: 727) that does not have a partially complementary sequence.

[0262] (Note) As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted solely by the claims. It is understood that the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2024-113709, filed on July 16, 2024, with the Japan Patent Office, the entire contents of which are incorporated by reference herein.

[0263] The present disclosure can be applied in the application fields of nucleic acid technology (e.g., medicine).

[0264] SEQ ID NOs: 1 to 798: See the tables in the specification and the tables below.

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

Claims

1. A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, linked in the order of 5' UTR, translated region, and 3' UTR from the 5' side, wherein the complementarity between the translated region and the 5' UTR or 3' UTR is less than 95%, and the 3' UTR comprises a non-complementary portion and a complementary portion to the 5' UTR.

2. The nucleic acid construct according to claim 1, wherein the bases in the non-complementary portions are substituted or deleted.

3. The nucleic acid construct of claim 1 or 2, wherein all of the non-complementary portions are removed.

4. A nucleic acid construct according to any one of claims 1 to 3, wherein the complementarity of the 3'UTR to the 5'UTR is greater than 75% when the length of each non-complementary portion is a single-base substitution.

5. A nucleic acid construct according to any one of claims 1 to 4, wherein at least one of the non-complementary portions is two or more bases in length.

6. A nucleic acid construct according to any one of claims 1 to 5, wherein the non-complementary portion has a length of 2 or 3 bases.

7. A nucleic acid construct according to any one of claims 1 to 6, wherein at least one of the complementary portions has a length of 5 bases or more.

8. A nucleic acid construct according to any one of claims 1 to 7, wherein at least one of the complementary portions has a length of 5 to 11 bases.

9. A nucleic acid construct according to any one of claims 1 to 8, wherein the non-complementary portion has a length of 2 bases.

10. A nucleic acid construct according to any one of claims 1 to 8, wherein the non-complementary portion has a length of 3 bases.

11. A nucleic acid construct according to any one of claims 1 to 10, wherein the length of each of the non-complementary portions is at least one base, and the rate of complementarity is higher than 75%.

12. A nucleic acid construct according to any one of claims 1 to 11, wherein the length of each of the non-complementary portions is at least one base, and the rate of complementarity is higher than 80%.

13. A nucleic acid construct according to any one of claims 1 to 12, wherein at least one of the non-complementary portions is a single base.

14. A nucleic acid construct according to any one of claims 1 to 13, wherein at least one of the non-complementary portions is 1 base long, and the complementary portions are each independently 3 to 11 bases long.

15. A nucleic acid construct according to any one of claims 1 to 14, wherein the non-complementary portion is 1 base, and the complementary portions are each independently 3 to 7 bases in length.

16. A nucleic acid construct according to any one of claims 1 to 14, wherein the non-complementary portions are 2 bases in length and the complementary portions are each independently 5 to 11 bases in length.

17. A nucleic acid construct according to any one of claims 1 to 14, wherein the non-complementary portions are 3 bases in length and the complementary portions are each independently 8 to 11 bases in length.

18. A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, the 5' UTR, the translated region, and the 3' UTR are linked in this order from the 5' side, the 3' UTR comprising a non-complementary portion and a complementary portion to the 5' UTR, the non-complementary portion and the complementary portion alternate, and when the non-complementary portion is 2 bases, the complementary portion is 5 to 7 bases long in the case of a base substitution and 5 to 11 bases long in the case of a base deletion, and when the non-complementary portion is 3 bases, the complementary portion is 8 to 9 bases long in the case of a base substitution and 8 to 11 bases long in the case of a base deletion.

19. The nucleic acid construct of any one of claims 1 to 18, wherein the 5'UTR is selected from at least one of the 5'UTR sequences of HBA (α-globin), HBB, HIST1H2BK, ACTB, KRT18, HIST1H1C, HIST1H1E, HIST3H2A, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, PKM, GAPDH, HSD17B4, ETNK1, XAB2, and RRP12, and the nucleic acid sequence set forth in SEQ ID NO: 790, or comprises the nucleic acid sequence of a functional fragment thereof, or is a mutated version of the nucleic acid sequence.

20. A nucleic acid construct according to any one of claims 1 to 19, wherein the 5'UTR is selected from at least one nucleic acid sequence selected from the 5'UTR sequences of HBA (α-globin), HBB, KLHL11, HIST1H1C, HIST1H2BK, USP11, ACTB, and KRT18, or comprises the nucleic acid sequence of a functional fragment thereof, or is a mutated version of said nucleic acid sequence.

21. A nucleic acid construct comprising the nucleic acid construct according to any one of claims 1 to 20, further comprising at least one functional untranslated region.

22. The nucleic acid construct of claim 21, wherein the functional untranslated region is contained in the 3'UTR on the 5' side of the 3'UTR portion of a region having partial complementarity with the 5'UTR.

23. A nucleic acid construct according to claim 21 or 22, wherein the functional untranslated region is contained in the 3'UTR on the 3' side of the 3'UTR portion of the region having partial complementarity with the 5'UTR.

24. A nucleic acid construct according to any one of claims 21 to 23, wherein the functional untranslated region is contained in the 3'UTR on both the 5' and 3' sides of the 3'UTR portion of the region that is partially complementary to the 5'UTR.

25. A nucleic acid construct according to any one of claims 21 to 24, wherein the functional untranslated region is contained in the 3'UTR on the 5' side of the 5'UTR portion of a region that is partially complementary to the 5'UTR.

26. A nucleic acid construct according to any one of claims 21 to 25, wherein the functional untranslated region is contained in the 3'UTR on the 3' side of the 5'UTR portion of a region that is partially complementary to the 5'UTR.

27. A nucleic acid construct according to any one of claims 21 to 26, wherein the functional untranslated region is contained in the 3'UTR on both the 5' and 3' sides of the 5'UTR portion of the region that is partially complementary to the 5'UTR.

28. The nucleic acid construct according to any one of claims 21 to 27, wherein the functional non-translated region is at least one selected from a translation control sequence, a nucleic acid construct degradation control sequence, a nucleic acid construct intracellular localization control sequence, a nucleic acid construct extracellular secretion control sequence, a nucleic acid construct extracellular vesicle encapsulation control sequence, a protein / peptide binding sequence, a low molecular weight compound binding sequence, a low molecular weight non-coding RNA target sequence such as miRNA, a high molecular weight non-coding RNA target sequence, a nucleic acid aptamer or its recognition sequence, a ribozyme sequence, a spacer / stuffer sequence, a higher-order structure sequence, a modified nucleic acid-containing sequence, and a labeling compound-containing sequence such as a fluorescent dye.

29. A nucleic acid construct according to any one of claims 21 to 28, wherein the functional untranslated region is at least one selected from a translation control sequence, a nucleic acid construct degradation control sequence, a nucleic acid construct intracellular localization control sequence, a protein / peptide binding sequence, and a small non-coding RNA target sequence such as miRNA.

30. A nucleic acid construct comprising a repeat sequence of A (adenine) on the 3' side of the nucleic acid construct according to any one of claims 1 to 29.

31. The nucleic acid construct according to claim 30, wherein the length of the repeat sequence of A is 100 to 300.

32. The nucleic acid construct according to claim 30 or 31, wherein the length of the repeat sequence of A is 120 to 200.

33. A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translation region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the following steps: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are to be substituted or deleted.

34. A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translation region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the following steps: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, and the natural protein is selected from a database related to gene expression.

35. A method for designing a nucleic acid construct according to claim 33 or 34, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases.

36. A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translational region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the following steps: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, and the natural protein is selected from a database created by a ribosome profiling method.

37. The method for designing a nucleic acid construct according to claim 36, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases.

38. A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translated region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the following steps: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and the complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein and the natural protein is selected from nucleic acid constructs that have been translated in a database related to gene expression.

39. The method for designing a nucleic acid construct according to claim 38, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases.

40. A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translation region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the following steps: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, and the natural protein is one in which one or more ribosome-protected mRNA fragments are present in a ribosome profile analysis or which has been detected in a proteome analysis.

41. The method for designing a nucleic acid construct according to claim 40, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases.

42. A method for optimizing the expression level of a desired protein in a desired cell for a nucleic acid construct in which a 5'UTR, a translational region encoding the desired protein, and a 3'UTR are linked in this order from the 5' side, the method comprising at least the following steps: (1) selecting a 5'UTR and a 3'UTR that is at least partially complementary to the 5'UTR and in which non-complementary portions and the complementary portions alternate; (2) selecting the base lengths of the non-complementary portions and complementary portions; and (3) selecting whether the bases in the non-complementary portions are substituted or deleted; wherein the 5'UTR is derived from a natural protein, the natural protein is selected from a database created by ribosome profile analysis, the selected protein contains one or more ribosome-protected mRNA fragments, and the selected protein is within the top 200 of the value obtained by dividing the number of ribosome-protected mRNA fragments by the mRNA expression level.

43. The method for designing a nucleic acid construct according to claim 42, further comprising the step of selecting a 5'UTR having a base length of 25 to 100 bases.

44. A method for designing a nucleic acid construct according to any one of claims 33 to 43, further comprising the step of selecting the length of a polyA tail.

45. The method for designing a nucleic acid construct according to claim 44, wherein the length of the polyA tail is selected in the range of 50 to 300.

46. ​​A method for designing a nucleic acid construct according to any one of claims 33 to 45, comprising the steps of: synthesizing the nucleic acid construct; introducing the nucleic acid construct into the desired cell; and evaluating the expression level of the protein.

47. A nucleic acid construct designed by the method of any one of claims 33 to 46, in which a 5'UTR, a translation region encoding a desired protein, and a 3'UTR are linked in this order from the 5' side.

48. The nucleic acid construct of claim 47, wherein the complementarity of the 3'UTR to the 5'UTR is greater than 75% when the length of each non-complementary portion is a single-base substitution.

49. The nucleic acid construct of claim 47, wherein at least one of the non-complementary portions is two or more bases in length.

50. A nucleic acid construct according to claim 47 or 49, wherein when the non-complementary portion is two bases, the complementary portion is 5 to 7 bases in length in the case of a base substitution, or 5 to 11 bases in length in the case of a base deletion.

51. A nucleic acid construct according to claim 47 or 49, wherein when the non-complementary portion is 3 bases, the complementary portion is 8 to 9 bases long in the case of a base substitution, or 8 to 11 bases long in the case of a base deletion.

52. The nucleic acid construct of any one of claims 47 to 51, wherein the 5'UTR is selected from at least one nucleic acid sequence selected from the 5'UTR sequences of HBA (α-globin), HBB, HIST1H2BK, ACTB, KRT18, HIST1H1C, HIST1H1E, HIST3H2A, SFT2D2, GPI, KLHL11, PHGDH, USP11, TKT, ACLY, ACTN4, MDH2, BROX, PKM, GAPDH, HSD17B4, ETNK1, XAB2, and RRP12, and the nucleic acid sequence set forth in SEQ ID NO: 790, or comprises the nucleic acid sequence of a functional fragment thereof, or is a mutated version of the nucleic acid sequence.

53. A nucleic acid construct according to any one of claims 47 to 52, wherein the 5'UTR is selected from at least one nucleic acid sequence selected from the 5'UTR sequences of HBA (α-globin), HBB, KLHL11, HIST1H1C, HIST1H2BK, USP11, ACTB, and KRT18, or comprises the nucleic acid sequence of a functional fragment thereof, or is a mutated version of said nucleic acid sequence.

54. A composition comprising a nucleic acid construct according to any one of claims 1 to 32 and 47 to 53.

55. A formulation comprising a nucleic acid construct according to any one of claims 1 to 32 and 47 to 53.

56. A method for producing a nucleic acid construct, composition or formulation according to any one of claims 1 to 32 and 47 to 53.

57. A method for expressing a protein by introducing the nucleic acid construct, composition or preparation according to any one of claims 1 to 32 and 47 to 53 into a cell.

58. A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a library of nucleic acid constructs with multiple sequences is constructed, which includes the sequence information of the translated region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap and its related structure and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3.

59. A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which a 5'UTR or 3'UTR comprising a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR comprising a non-naturally occurring sequence corresponding to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the coding region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

60. A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which 5'UTR and 3'UTR are designed, each containing complementary and non-complementary portions; 3) a step in which a library of nucleic acid constructs having multiple sequences is constructed, each containing the coding region, 5'UTR, and 3'UTR designed in steps 1 and 2, and optionally a 5'cap or its analogous structure and a polyA tail; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 4.

61. A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which a 5'UTR or 3'UTR containing a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR containing a complementary or non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences containing the coding region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

62. A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which a 5'UTR comprising a naturally occurring sequence; 3) a step in which a 3'UTR comprising a complementary and non-complementary portion to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the coding region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

63. A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR is designed, comprising complementary and non-complementary portions to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed, comprising the coding region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

64. A method for generating an optimized nucleic acid construct, comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR is designed, comprising complementary and non-complementary portions to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed, comprising the coding region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

65. A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that contain the sequence information of the translated region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap and its related structure and a polyA tail; 3) an evaluation unit that evaluates the functions of the nucleic acid constructs in the library; and 4) a desired selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of 3).

66. A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence design unit that designs a 5'UTR or 3'UTR containing a naturally occurring sequence; 3) a non-natural sequence design unit that designs a 5'UTR or 3'UTR containing a non-natural sequence corresponding to the 5'UTR or 3'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that contain the translated region, 5'UTR, and 3'UTR designed in accordance with 1 to 3 above, in combination with, if necessary, a 5'cap and its analogous structure and a polyA tail; 5) an evaluation unit that evaluates the functions of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of 5 above.

67. A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a design unit that designs 5'UTR and 3'UTR containing complementary and non-complementary portions; 3) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences containing the translated region, 5'UTR and 3'UTR designed in 1 and 2 above, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 4) an evaluation unit that evaluates the functions of the nucleic acid constructs in the library; and 5) a structure selection unit that selects nucleic acid constructs that achieve desired values ​​or levels from the results of 4 above.

68. A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence design unit that designs a 5'UTR or 3'UTR containing a naturally occurring sequence; 3) a complementary / non-complementary design unit that designs a 3'UTR or 5'UTR containing a complementary / non-complementary portion to the 5'UTR or 3'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that contain the translated region, 5'UTR, and 3'UTR designed in accordance with steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit that evaluates the functions of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of steps 5.

69. A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence design unit that designs a 5'UTR containing a naturally occurring sequence; 3) a complementary / non-complementary design unit that designs a 3'UTR containing a complementary / non-complementary portion to the 5'UTR; 4) a library construction unit that constructs a library of nucleic acid constructs of multiple sequences that contain the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit that evaluates the functions of the nucleic acid constructs in the library; and 6) a structure selection unit that selects nucleic acid constructs that achieve a desired value or level from the results of step 5.

70. A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a sequence selection unit in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a complementary / non-complementary design unit in which a 3'UTR containing a complementary / non-complementary portion to the 5'UTR is designed; 4) a library construction unit in which a library of nucleic acid constructs of multiple sequences containing the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3 above in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a structure selection unit in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5 above.

71. A system for generating optimized nucleic acid constructs, comprising: 1) a sequence information providing unit in which a user provides sequence information of a translated region; 2) a natural sequence selection unit in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a complementary / non-complementary design unit in which a 3'UTR containing a complementary / non-complementary portion to the 5'UTR is designed; 4) a library construction unit in which a library of nucleic acid constructs of multiple sequences containing the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3 above in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) an evaluation unit in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a structure selection unit in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5 above.

72. A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the steps including: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a library of nucleic acid constructs with multiple sequences is constructed, which includes the sequence information of the translated region in combination with multiple combinations of 5'UTR and 3'UTR, and optionally a 5'cap or its analogous structure and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3.

73. A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR is designed that includes a non-naturally occurring sequence that corresponds to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

74. A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which 5'UTR and 3'UTR are designed, each containing complementary and non-complementary portions; 3) a step in which a library of nucleic acid constructs having multiple sequences is constructed, each containing the translated region, 5'UTR, and 3'UTR designed in steps 1 and 2, and optionally a 5'cap, its analogous structure, and a polyA tail; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 4.

75. A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR is designed that includes a complementary or non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

76. A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of a coding region; 2) a step in which a 5'UTR containing a naturally occurring sequence is designed; 3) a step in which a 3'UTR containing a complementary and non-complementary portion to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed, the library comprising the coding region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

77. A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR is designed, the 3'UTR comprising complementary and non-complementary portions to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5.

78. A program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR is designed, the 3'UTR comprising complementary and non-complementary portions to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5.

79. A recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the steps including: 1) a step in which a user provides sequence information of a translated region; 2) a step in which a library of nucleic acid constructs with multiple sequences is constructed, which includes the sequence information of the translated region in combination with multiple combinations of 5'UTR and 3'UTR, and, if necessary, a 5'cap and its related structure and a polyA tail; 3) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 4) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 3.

80. A recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the steps including: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 5'UTR or 3'UTR is designed that includes a non-naturally occurring sequence that corresponds to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with, if necessary, a 5'cap or its analogous structure and a polyA tail; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

81. A recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the steps including: 1) a step in which a user provides sequence information of the translated region; 2) a step in which 5'UTR and 3'UTR containing complementary and non-complementary portions; 3) a step in which a library of nucleic acid constructs having multiple sequences containing the translated region, 5'UTR, and 3'UTR designed in steps 1 and 2, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 4) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 5) a step in which nucleic acid constructs that achieve a desired value or level are selected from the results of step 4.

82. A recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR or 3'UTR is designed that includes a naturally occurring sequence; 3) a step in which a 3'UTR or 5'UTR is designed that includes a complementary or non-complementary portion to the 5'UTR or 3'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed that includes the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap or its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

83. A recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the method comprising: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR containing a naturally occurring sequence is designed; 3) a step in which a 3'UTR containing complementary and non-complementary portions to the 5'UTR is designed; 4) a step in which a library of nucleic acid constructs having multiple sequences is constructed, the library comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

84. A recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the steps including: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected from a database related to gene expression is selected; 3) a step in which a 3'UTR is designed, the 3'UTR including complementary and non-complementary portions to the 5'UTR; 4) a step in which a library of nucleic acid constructs having multiple sequences, the library including the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct that achieves a desired value or level is selected from the results of step 5.

85. A recording medium storing a program encoding steps for causing a computer to execute a method for generating optimized nucleic acid constructs, the steps including: 1) a step in which a user provides sequence information of the translated region; 2) a step in which a 5'UTR sequence derived from a natural protein selected by a ribosome profiling method is selected; 3) a step in which a 3'UTR is designed, the 3'UTR comprising complementary and non-complementary portions to the 5'UTR; 4) a step in which a library of nucleic acid constructs of multiple sequences is constructed, the library comprising the translated region, 5'UTR, and 3'UTR designed in steps 1 to 3, in combination with a 5'cap and its analogous structure and a polyA tail as needed; 5) a step in which the functions of the nucleic acid constructs in the library are evaluated; and 6) a step in which a nucleic acid construct achieving a desired value or level is selected from the results of step 5.

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