Artificial synthetic nucleic acids for enhancing protein expression

Abstract

The present disclosure provides a nucleic acid construct capable of controlling protein expression. In particular, 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 at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, and when each of the non-complementary portions is all 1 base in length, the 3' UTR has a complementarity rate of more than 75% to the 5' UTR.

Description

[Technical field]

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

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

[0003] Technologies for optimizing codons and sequences have been developed to increase translation efficiency. The plasmid encoding the target mRNA is used as the starting material, and after linearization, transcription, capping, and removal of template DNA with DNase are performed in a test tube. There is also a method in which a polyA tail is added enzymatically afterwards. A mechanism for promoting translation by circularizing mRNA is also known. Summary of the Invention [Means for solving the problem]

[0004] The present inventors have newly discovered that in a nucleic acid construct, protein expression can be controlled by the complementarity ratio between the 3' untranslated region and the 5' untranslated region and a specific pattern of complementary and non-complementary portions. Thus, 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, and having a specific complementary pattern.

[0005] Thus, the present disclosure provides: [Item X1] A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR which are at least partially complementary to each other, wherein at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, and the length of each of the non-complementary portions is a single base substitution, and the complementarity of the 3' UTR to the 5' UTR is greater than 75%. [Item X2] The nucleic acid construct according to the preceding item, wherein bases in the non-complementary portions are substituted or removed. [Item X2A] The nucleic acid construct of any one of the preceding claims, wherein all of the non-complementary portions are substitutions. [Item X2B] The nucleic acid construct according to any one of the preceding items, wherein all of the non-complementary portions are substitutions and each is one base in length. [Item X2C] The nucleic acid construct according to any one of the preceding claims, wherein all of the non-complementary portions are removed. [Item X3] 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 X4] The nucleic acid construct according to any one of the preceding items, wherein at least one of the non-complementary portions is 2 or 3 bases in length. [Item X5] The nucleic acid construct according to any one of the preceding items, wherein at least one of the complementary portions is 5 bases or longer in length. [Item X6] The nucleic acid construct according to any one of the preceding items, wherein at least one of the complementary portions has a length of 5 to 11 bases. [Item X7] The nucleic acid construct according to any one of the preceding items, wherein the non-complementary portions are 2 bases long, and the complementary portions are each independently 5 to 7 bases long. [Item X8] The nucleic acid construct according to any one of the preceding items, wherein the non-complementary portions are 3 bases long, and the complementary portions are each independently 8 to 11 bases long. [Item X9] The nucleic acid construct according to any one of the preceding items, wherein the length of each of the non-complementary portions is at least one base, and the complementarity rate is higher than 75%. [Item X10] The nucleic acid construct according to any one of the preceding items, wherein the complementarity is 80% to 90%. [Item X11] The nucleic acid construct according to any one of the preceding items, wherein the complementarity is 81% to 89%. [Item X12] The nucleic acid construct according to any one of the preceding claims, wherein at least one of the non-complementary portions is a single base. [Item X13] The nucleic acid construct according to any one of the preceding items, wherein the complementary portions each independently have a length of 3 to 11 bases. [Item X14] The nucleic acid construct according to any one of the preceding items, wherein the non-complementary portion is 1 base, and the complementary portions are each independently 3 to 7 bases in length. [Item X15] The nucleic acid construct according to any one of the preceding items, wherein the non-complementary portions are 2 bases long, and the complementary portions are each independently 5 to 7 bases long. [Item X16] The nucleic acid construct according to any one of the preceding items, wherein the non-complementary portions are 3 bases long, and the complementary portions are each independently 8 to 11 bases long. [Item X17] A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, characterized in that at least one UTR contains non-complementary and complementary portions to the other UTR, and the non-complementary and complementary portions alternate. [Item X17A] The nucleic acid construct according to any one of the preceding items, wherein the portion other than the alternating portions (also referred to as the fractional portion (4)) is one or more bases in length not exceeding the base length of the complementary portion. [Item X17B] The nucleic acid construct according to any one of the preceding items, wherein when the non-complementary portion (2) is one base, the complementary portion (3) is 3 to 6 bases long in the case of base substitution, or 3 to 9 bases long in the case of base deletion. [Item X18] The nucleic acid construct according to any one of the preceding items, wherein when the non-complementary portion is 2 bases, 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. [Item X19] The nucleic acid construct according to any one of the preceding items, wherein when the non-complementary portion is 3 bases, 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 X19A] The nucleic acid construct according to any one of the preceding items, wherein the non-complementary portion (2) is 2 or 3 bases long. [Item X19B] The nucleic acid construct according to any one of the preceding items, wherein the non-complementary portions are all at least one base in length and have a complementarity rate of more than 75%. [Item X19C] The nucleic acid construct according to any one of the preceding items, wherein the complementarity is 80% to 90%. [Item X19D] The nucleic acid construct according to any one of the preceding items, wherein the complementarity is 81% to 89%. [Item X20] The nucleic acid construct according to any one of the preceding items, wherein either the 5'UTR or the 3'UTR does not contain a non-complementary portion. [Item X21] The nucleic acid construct according to any one of the preceding items, wherein the free energy change (ΔG) is a predetermined value or range thereof. [Item X22] The nucleic acid construct according to any one of the preceding items, wherein the predetermined value or range is a value or range of free energy change that has an increase rate of 0% to 60% relative to the average value of the maximum and minimum values ​​of free energy change (ΔG). [Item X23] The nucleic acid construct according to any one of the preceding items, wherein the predetermined value or range is a value or range of free energy change that has an increase rate of 10% to 50% relative to the average value of the maximum and minimum values ​​of free energy change (ΔG). [Item X24] A method for producing a nucleic acid construct, the nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, wherein at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, the method comprising the steps of designing a plurality of candidate nucleic acid constructs, calculating a free energy change (ΔG) for the plurality of designed candidate nucleic acid constructs, selecting a candidate nucleic acid construct whose free energy change (ΔG) is a predetermined value or within a predetermined range, and, if necessary, measuring the expression level of the selected candidate nucleic acid construct in a cell. [Item X25] A method for producing a nucleic acid construct, the nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR which are at least partially complementary to each other, and at least one UTR comprising a non-complementary portion and a complementary portion to the other UTR, the method comprising the steps of: designing a plurality of candidate nucleic acid constructs; calculating the free energy change (ΔG) for the plurality of designed candidate nucleic acid constructs; selecting a candidate nucleic acid construct having a free energy change value with an increase rate of 0% to 60% relative to the average value of the maximum and minimum values ​​of the free energy change (ΔG); and, if necessary, measuring the expression level of the selected candidate nucleic acid construct in a cell. [Item X26] The method according to any one of the preceding items, wherein the increase rate relative to the average value is 10% to 50%. [Item X27] A nucleic acid construct according to any one of the preceding items, produced according to the method according to any one of the preceding items.

[0006] The present disclosure also provides the following: (Item 1) A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR which are at least partially complementary to each other, wherein at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, and when each of the non-complementary portions is all 1 base in length, the 3' UTR has a complementarity to the 5' UTR of greater than 75%. (Item 2) 2. The nucleic acid construct according to item 1, wherein at least one of the non-complementary portions is two or more bases in length. (Item 3) 3. The nucleic acid construct according to item 1 or 2, wherein at least one of the non-complementary portions is 2 or 3 bases in length. (Item 4) 4. The nucleic acid construct according to any one of items 1 to 3, wherein at least one of the complementary portions has a length of 5 bases or more. (Item 5) 4. The nucleic acid construct according to any one of items 1 to 3, wherein at least one of the complementary portions has a length of 5 to 11 bases. (Item 6) 2. The nucleic acid construct according to item 1, wherein the non-complementary portions are 2 bases long, and the complementary portions are each independently 5 to 7 bases long. (Item 7) 2. The nucleic acid construct according to item 1, wherein the non-complementary portions are 3 bases long and the complementary portions are each independently 8 to 11 bases long. (Item 8) 2. The nucleic acid construct according to item 1, wherein the length of each of the non-complementary portions is at least one base, and the complementarity rate is higher than 75%. (Item 9) 9. The nucleic acid construct according to item 8, wherein the complementarity is 80% to 90%. (Item 10) 9. The nucleic acid construct according to item 8, wherein the complementarity is 81% to 89%. (Item 11) 8. The nucleic acid construct according to item 7, wherein at least one of the non-complementary portions is a single base. (Item 12) 12. The nucleic acid construct according to any one of items 8 to 11, wherein the length of each of the complementary portions is independently 3 to 11 bases. (Item 13) 9. The nucleic acid construct according to item 8, wherein the non-complementary portion is 1 base, and the complementary portions are each independently 3 to 7 bases in length. (Item 14) 9. The nucleic acid construct according to item 8, wherein the non-complementary portions are 2 bases long and the complementary portions are each independently 5 to 7 bases long. (Item 15) 9. The nucleic acid construct according to item 8, wherein the non-complementary portions are 3 bases long and the complementary portions are each independently 8 to 11 bases long. (Item 16) 2. The nucleic acid construct according to item 1, wherein either the 5'UTR or the 3'UTR does not contain a non-complementary portion. (Item 17) 17. The nucleic acid construct according to any one of items 1 to 16, wherein the free energy change (ΔG) is a predetermined value or within a predetermined range. (Item 18) 18. The nucleic acid construct according to item 17, wherein the predetermined value or the range thereof is a value or range of a free energy change having an increase rate of 0% to 60% relative to the average value of the maximum and minimum values ​​of the free energy change (ΔG). (Item 19) 18. The nucleic acid construct according to item 17, wherein the predetermined value or the range thereof is a value or range of a free energy change having an increase rate of 10% to 50% relative to the average value of the maximum and minimum values ​​of the free energy change (ΔG). (Item 20) A method for producing a nucleic acid construct, the nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, wherein at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, the method comprising the steps of designing a plurality of candidate nucleic acid constructs, calculating a free energy change (ΔG) for the plurality of designed candidate nucleic acid constructs, selecting a candidate nucleic acid construct whose free energy change (ΔG) is a predetermined value or within a predetermined range, and, if necessary, measuring the expression level of the selected candidate nucleic acid construct in a cell. (Item 21) A method for producing a nucleic acid construct, the nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR which are at least partially complementary to each other, and at least one UTR comprising a non-complementary portion and a complementary portion to the other UTR, the method comprising the steps of: designing a plurality of candidate nucleic acid constructs; calculating the free energy change (ΔG) for the plurality of designed candidate nucleic acid constructs; selecting a candidate nucleic acid construct having a free energy change value with an increase rate of 0% to 60% relative to the average value of the maximum and minimum values ​​of the free energy change (ΔG); and, if necessary, measuring the expression level of the selected candidate nucleic acid construct in a cell. (Item 22) 22. The method according to item 21, wherein the increase rate relative to the average value is 10% to 50%. (Item 23) A nucleic acid construct according to any one of Items 1 to 19, produced according to the method according to any one of Items 20 to 22. Effect of the Invention

[0007] The nucleic acid construct of the present disclosure is an improved nucleic acid construct that exhibits higher protein expression levels compared to conventional nucleic acid constructs. [Brief description of the drawings]

[0008] [Figure 1]Figure 1 shows the results of verifying the expression levels of mRNAs whose 5'UTR is the GAPDH gene sequence (left), the Pfizer sequence (Messenger RNA encoding the full-length SARS-CoV-2 spike glycoprotein Sept. 2020 document 11889; see the 19th Health Science Council Vaccination and Vaccine Subcommittee Document (https: / / www.mhlw.go.jp / stf / shingi2 / 0000192554_00004.html)) (center), and the HSD17B4 gene sequence (right). For comparison, mRNAs whose 5'UTR and 3'UTR are both Pfizer sequences were used. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was used as 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 bars and the individual values ​​for each sample are shown in black plots. [Diagram 2] Figure 2 shows the results of examining the expression level of mRNA in which the 5'UTR is a GAPDH gene sequence and the 3'UTR is a sequence that has 100%, 94%, or 75% complementarity with the 5'UTR. The upper left of the figure shows a schematic of the mRNA structure. The lower left of the figure shows the base sequences of the 5'UTR and 3'UTR, and in each sequence, the 5'-end of the 5'UTR sequence is written to the left in the upper row and the 5'-end of the 3'UTR sequence is written to the right in the lower row. The ORF is omitted. The bases that are complementary between the 5'UTR and 3'UTR are circled. The right of the figure is a graph showing the results of examining the expression level of each mRNA in 293 cells. For comparison, mRNA with Pfizer sequences for both the 5'UTR and 3'UTR was used. 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, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black plots. [Diagram 3]In Figure 3, the 5'UTR is the GAPDH gene sequence, and the 3'UTR shows the base sequences of the 5'UTR and 3'UTR of various mRNAs that are partially complementary to the 5'UTR. They are classified according to the number of bases in the non-complementary portion. In each sequence, the upper row shows the 5'UTR sequence with the 5' end on the left, and the lower row shows the 3'UTR sequence with the 5' end on the right. The ORF is omitted. The bases that are complementary between the 5'UTR and 3'UTR are circled. [Figure 4] Figure 4 is a graph showing the results of verifying the expression levels in 293 cells for mRNAs that have the GAPDH gene sequence as the 5'UTR and various sequences that are partially complementary to this 5'UTR as the 3'UTR (left). The right graph shows the distribution of the complementarity rate versus the relative expression level (ratio of E2Crimson fluorescence measurement value to Calcein fluorescence measurement value) for the left graph. For comparison, mRNAs whose 5'UTR and 3'UTR both have Pfizer sequences were used. The ratio of E2Crimson fluorescence measurement value to 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, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black plots. [Diagram 5] In Figure 5, the 5'UTR is the Pfizer sequence, and the 3'UTR shows the base sequences of the 5'UTR and 3'UTR of various mRNAs that are partially complementary to the 5'UTR. They are classified according to the number of bases in the non-complementary portion. In each sequence, the 5'UTR sequence is written with the 5' end on the left in the upper row, and the 3'UTR sequence is written with the 5' end on the right in the lower row. The ORF is omitted. The bases that are complementary between the 5'UTR and 3'UTR are enclosed. [Figure 6]Figure 6 is a graph showing the results of examining the expression levels in 293 cells of mRNAs with the Pfizer sequence as the 5'UTR and various sequences partially complementary to the 5'UTR 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 taken as 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 7] In Figure 7, the 5'UTR is the HSD17B4 gene sequence, and the 3'UTR shows the base sequences of the 5'UTR and 3'UTR of various mRNAs that are partially complementary to the 5'UTR. They are classified according to the number of bases in the non-complementary portion. In each sequence, the upper row shows the 5'UTR sequence with the 5' end on the left, and the lower row shows the 3'UTR sequence with the 5' end on the right. The ORF is omitted. The bases that are complementary between the 5'UTR and 3'UTR are enclosed. [Figure 8] Figure 8 is a graph showing the results of examining the expression levels in 293 cells of mRNAs with the HSD17B4 gene sequence as the 5'UTR and various sequences partially complementary to this 5'UTR as the 3'UTR. For comparison, mRNAs with Pfizer sequences 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 taken as 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 9] In Figure 9, the 5'UTR is a GAPDH gene sequence, and the 3'UTR shows the base sequences of the 5'UTR and 3'UTR of various mRNAs that are partially complementary to the 5'UTR. They are listed in order of increasing complementarity. In each sequence, the 5'UTR sequence is written with the 5' end on the left in the upper row, and the 3'UTR sequence is written with the 5' end on the right in the lower row. The ORF is omitted. The bases that are complementary between the 5'UTR and 3'UTR are shown in boxes. [Figure 10] FIG. 10 shows the results of verifying the expression level in 293 cells for mRNAs with the GAPDH gene sequence as the 5'UTR and various sequences partially complementary to the 5'UTR as the 3'UTR. For comparison, mRNAs with Pfizer sequences for both the 5'UTR and 3'UTR were used. 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. The vertical axis shows this relative expression level, and the horizontal axis shows the complementarity rate of each mRNA, as a scatter plot. Three samples were measured for each mRNA, and the average values ​​are plotted. Furthermore, the standard deviation of these measurements was calculated and shown as an error bar on the plot. The 5'UTR of the GAPDH gene sequence is shown in black, and the Pfizer sequence is shown in white. The complementarity rate of the mRNA with the highest expression level was 75% to 80%. [Figure 11]Figure 11 shows the correlation between the free energy change in the interaction between 5'UTR and 3'UTR and the expression level of each mRNA for mRNAs whose 3'UTR is partially complementary to the 5'UTR. The expression level was calculated by setting the expression level of mRNA whose 5'UTR and 3'UTR are both Pfizer sequences as 1. The free energy change was calculated using the secondary structure prediction program Mfold (Michael Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res. 2003 Jul 1;31(13):3406-15). The left, center, and right of the figure show the results when the 5'UTR is the GAPDH gene, the HSD17B4 gene, and the Pfizer sequence, respectively. For each 5'UTR, the measured values ​​obtained were fitted to a four-coefficient logistic curve y=d+(ad) / (1+(x / c)^b), which is a type of sigmoid curve, and the coefficients a, b, c, d, and correlation coefficient were calculated. The coefficient c is the free energy change value at the inflection point on the sigmoid curve. In the graph, multiple samples were measured for each mRNA, and the average values ​​are plotted. The resulting sigmoid curve is shown as a solid line, and the free energy change value at the inflection point is shown as a dotted line. [Figure 12]FIG. 12 shows possible values ​​of free energy change in the interaction between the 5'UTR and 3'UTR of the corresponding mRNA in the mRNA with the maximum expression level. The left, center, and right of the figure show the results when the 5'UTR is the GAPDH gene, the HSD17B4 gene, and the Pfizer sequence, respectively. The minimum free energy change was determined by Mfold for the mRNA with a 100% complementarity rate between the 5'UTR and 3'UTR. The maximum free energy change was determined by Mfold for the mRNA in which all bases in the 3'UTR with a 100% complementarity rate between the 5'UTR and 3'UTR were substituted to mismatch with the 5'UTR. The relative free energy change was calculated using the increase rate (right shift) from the inflection point as an index, and the difference between the inflection point value and the maximum value as 100%. The expression level of mRNA was highest when the free energy change rate from the inflection point was between 10% and 50%. [Figure 13] Figure 13 shows a method for predicting the expression level of mRNA from the 5'UTR and 3'UTR sequences. The maximum and minimum values ​​of the free energy change were obtained in the same manner as in Figure 12. It was found that the average value of the maximum and minimum values ​​of the free energy coincides with the value of the free energy change at the inflection point of the sigmoid curve. By using this average value, it was possible to predict the inflection point with an error of 4.9 kcal / mol or less, or an accuracy of 13.1% or less. As in Figure 12, this error percentage was calculated by setting the difference between the value at the inflection point and the maximum free energy change as 100%. Based on the prediction of this inflection point, the free energy change of the interaction between 5'UTR and 3'UTR in mRNA with the maximum expression level can be predicted. [Figure 14] FIG. 14 shows a schematic diagram of a nucleic acid construct of the present disclosure. [Figure 15]Figure 15 is a graph showing the results of verifying the expression level in the myoblast cell line C2C12 for the mRNAs used in Figure 6. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was taken as the relative expression level of E2Crimson protein for each mRNA. Measurements were performed for 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 16] Figure 16 is a graph showing the results of verifying the expression level in 293 cells for mRNAs with the Pfizer sequence as the 5'UTR, various sequences partially complementary to this 5'UTR as the 3'UTR, SARS CoV2 Spike as the ORF, and E2Crimson fusion protein. 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, and the average values ​​are shown in the bar graph, and the individual values ​​for each sample are shown in black plots. [Figure 17A] FIG. 17 is a schematic diagram of the concept of substitution and removal in the design method of the non-complementary portion. FIG. 17 is shown in FIG. 17A and FIG. 17B. FIG. 17(1) shows a schematic diagram of an mRNA converted to a non-complementary sequence by replacing the base in the complementary sequence. FIG. 17(2) shows a schematic diagram of an mRNA converted to a non-complementary sequence by removing the base in the complementary sequence. FIG. 17(3) shows a schematic diagram of an mRNA converted to a non-complementary sequence by removing the base on the 5'UTR in the complementary sequence. FIG. 17(4) shows a schematic diagram of an mRNA in which the complementary sequence is converted to a non-complementary sequence by replacing the base and the complementary sequence is converted to a non-complementary sequence by removing the base. Figure 17(4-1) shows a schematic diagram of an mRNA in which the above-mentioned base deletions and base substitutions are mixed only on the 3'UTR, Figure 17(4-2) shows a schematic diagram of an mRNA in which the above-mentioned base deletions and base substitutions are mixed only on the 5'UTR, and Figure 17(4-3) shows a schematic diagram of an mRNA in which the above-mentioned base deletions and base substitutions are mixed only on the 5'UTR and both the 3'UTR. [Figure 17B]Figure 17B is a continuation of Figure 17A. Figure 17(5) shows a schematic diagram of an mRNA that has been converted to a non-complementary sequence by adding a base to the 3'UTR. Figure 17(6) shows a schematic diagram of an mRNA that has been converted to a non-complementary sequence by adding a base to the 5'UTR. [Figure 18A] Figure 18 is divided into Figures 18A to 18C, and for (2) to (6) in the schematic diagram shown in Figure 17, the specific 5'UTR is the Pfizer sequence, and the 3'UTR shows the base sequences of the 5'UTR and 3'UTR of various mRNAs that are partially complementary to this 5'UTR. Figure 18(2) shows mRNAs that have been converted to non-complementary sequences by removing bases in the 3'UTR, classified by the number of bases in the non-complementary portion. [Figure 18B] Figure 18B is a continuation of Figure 18A. Figure 18(3) shows an mRNA that has been converted to a non-complementary sequence by removing the base in the 5'UTR. Figure 18(4) shows an mRNA that has been converted to a non-complementary sequence by replacing the base in the complementary sequence, and an mRNA that has been converted to a non-complementary sequence by removing the base. Figure 18(4-1) shows an mRNA that has a mixture of base removal and base substitution in the 3'UTR only, in the 5'UTR only, and in both the 5'UTR and 3'UTR. [Figure 18C] Figure 18C is a continuation of Figure 18B. Figure 18(5) shows mRNAs in which a non-complementary sequence is created by adding bases in the 3'UTR, and the non-complementary portion is 1 base, 2 bases, or 3 bases. Figure 18(6) shows mRNAs in which a non-complementary sequence is created by adding bases in the 5'UTR, and the non-complementary portion is 1 base, 2 bases, or 3 bases. In each sequence, the upper row shows the 5'UTR sequence with the 5' end on the left, and the lower row shows the 3'UTR sequence with the 5' end on the right. In the lower row, the base marked with - indicates that the base was converted to a non-complementary sequence by removing that base in the complementary sequence. The ORF is omitted. The bases that are complementary between the 5'UTR and 3'UTR are enclosed. [Figure 19]Figure 19 is a graph showing the results of examining the expression levels in 293 cells of mRNAs with the Pfizer sequence as the 5'UTR and various sequences partially complementary to the 5'UTR as the 3'UTR. In these 3'UTRs, bases were removed to create non-complementary sequences. 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 taken as 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 20] Figure 20 is a graph showing the results of examining the expression levels in 293 cells of mRNAs that have the Pfizer sequence as the 5'UTR and various sequences as the 3'UTR that are partially complementary to the 5'UTR but have a non-complementary portion of 3 bases. 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, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black plots. [Figure 21] Figure 21 is a graph showing the results of verifying the expression level of the mRNA used in Figure 19 in the myoblast cell line C2C12. 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, and the average values ​​are shown in the bar graph, while the individual values ​​for each sample are shown in black plots. [Figure 22] Figure 22 is a graph showing the results of verifying the expression level in the myoblast cell line C2C12 for the mRNAs used in Figure 20. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was taken as the relative expression level of the E2Crimson protein in each mRNA. Measurements were performed for 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 23] Figure 23 is a graph showing the results of verifying the expression level in 293 cells for mRNAs with the Pfizer sequence as the 5'UTR, various sequences partially complementary to this 5'UTR as the 3'UTR, and SARS CoV2 Spike and E2Crimson fusion protein as ORFs. In these 3'UTRs, bases were removed to create non-complementary sequences. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was taken as the relative expression level of the 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 24] Figure 24 shows the base sequences of the 5'UTR and 3'UTR of various mRNAs, where the 5'UTR is the Pfizer sequence and the 3'UTR is partially complementary to the 5'UTR. Figure 24(1) shows the case where the number of bases in the non-complementary part is the same in the 5'UTR and 3'UTR, and Figure 24(2) shows the case where the number of bases in the non-complementary part is different in the 5'UTR and 3'UTR. In each sequence, the upper row shows the 5'UTR sequence with the 5' end on the left, and the lower row shows the 3'UTR sequence with the 5' end on the right. In the lower row, the bases marked with - indicate that the bases in the complementary sequence have been removed to convert them into non-complementary sequences. The ORF is omitted. The bases that are complementary in the 5'UTR and 3'UTR are enclosed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present disclosure will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will take precedence.

[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" refers to the indicated value plus or minus 10%.

[0012] As used herein, the term "untranslated region (UTR)" refers to a region that is not translated into a protein and is present before or after a coding region that is translated into a protein in a nucleic acid such as an mRNA that contains a portion that codes for a protein. The UTR present on the 5' side of a nucleic acid is called the 5'UTR, and the UTR present on the 3' side is called the 3'UTR.

[0013] As used herein, the terms "mutually" or "complementarity" refer to the specific pairing of adenine and thymine or uracil, and guanine and cytosine or uracil (wobble base pair) in the base sequence of a nucleic acid. "Non-complementary portion" refers to a portion that is not complementary in the base sequence, and "complementary portion" refers to a portion that is complementary in the base sequence. In this specification, complementary portions and non-complementary portions can be specified in units of one base. "The length of each non-complementary portion is all one base" means that when a complementary portion and a non-complementary portion are specified in a portion of a target nucleic acid, if there is a single non-complementary portion, it is one base long, and if there are multiple non-complementary portions, all of the multiple non-complementary portions are one base long. In addition, in the non-complementary portion, either the 5'UTR or the 3'UTR can be 0 bases. In this case, it may be referred to as "removal" in this specification (although when viewed from the other side, it may also be referred to as "addition", "removal" is used in this specification unless otherwise specified, and "removal" in a broad sense is understood to include removal and addition in the narrow sense). In addition, when one or more non-complementary bases are present in the 5'UTR and 3'UTR in the non-complementary portion, it may be referred to 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 part with the same number corresponds to "substitution", and when the number of bases differs, the side with fewer bases corresponds to "removal" relative to the side with more bases. For example, 5'-NNNAGNNN 3'-NNNA0NNN (N is the complementary strand, 0 is no base) In this case, UA 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 is a deletion.

[0014] Also, 5'-NNNAGNNNU0NNN 3'-NNNA0NNNUGNNN In such a case, the AG portion on the 5' side is the same as above, but the portion corresponding to U0 may be considered as "addition", although in the present specification, it may be described as "deletion" in a broad sense.

[0015] In this specification, unless otherwise specified, deletions, substitutions, and additions are determined by comparing nucleic acids in order from the 5' side.

[0016] As used herein, the term "partially complementary" refers to complementary over at least a portion (preferably, two or more bases) of a target base sequence. The number of bases in the complementary portion is preferably 3 or more and 11 or less.

[0017] As used herein, the term "nucleic acid construct" refers to a construct that is at least partially composed of nucleic acid, and typically refers to a (non-natural) nucleic acid molecule (e.g., recombinant nucleic acid) that results 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 that are combined and arranged in a manner not found in nature. A nucleic acid construct can be a "vector" (e.g., a plasmid, a rAAV vector genome, an expression vector, etc.), i.e., a nucleic acid molecule designed to deliver exogenously produced DNA to a host cell.

[0018] As used herein, the term "complementarity (%)" refers to the percentage of identity of the base sequence of a single-stranded nucleic acid to the base sequence of the complementary sequence of a reference sequence. The percentage of 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 are completely identical, the percentage of complementarity is 100%. In the embodiment of the present disclosure in which the non-complementary region is removed, the above-mentioned percentage of identity is calculated assuming that the non-complementary region is present. As used herein, "protein", "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified amino acids. The term also encompasses naturally 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 moiety). In the present disclosure, amino acids may be represented by the commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by commonly accepted one-letter codes. In this specification, nucleotides may be represented as T even in the case of RNA in accordance with the rules of sequence listing, but those skilled in the art will understand that T means U in the case of RNA.

[0019] As used herein, "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. Examples of nucleic acids include DNA, RNA, cDNA, mRNA, rRNA, tRNA, microRNA (miRNA), and lncRNA. This term also includes "polynucleotide derivatives." "Polynucleotide derivatives" refers to polynucleotides that contain nucleotide derivatives or have unusual internucleotide bonds. The term "nucleotide derivative" refers to a nucleotide having a structure different from that of normal nucleotides used in natural DNA or RNA, such as locked nucleic acid (LNA), ethylene nucleic acid such as 2'-O,4'-C-ethylene bridged nucleic acid (ENA), other bridged nucleic acid (BNA), hexitol nucleic acid (HNA), amide-bridged nucleic acid (AmNA), morpholino nucleic acid, tricyclo-DNA (tcDNA), polyether nucleic acid (see, for example, U.S. Pat. No. 5,908,845), cyclohexene nucleic acid (CeNA), etc. Examples of internucleotide bonds that are different from normal ones include interoligonucleotide bonds in which the phosphodiester bond is converted to a phosphorothioate bond, interoligonucleotide bonds in which the phosphodiester bond is converted to an N3'-P5' phosphoramidate bond, and interoligonucleotide bonds in which the ribose and phosphodiester bond are converted to a peptide nucleic acid bond.

[0020] 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), controlling the production of a polypeptide, protein, or non-protein-coding functional RNA, specifically binding to a particular protein, or controlling the cleavage or replication of a nucleic acid.

[0021] As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., nucleic acid construct, instructions, etc.) are provided, usually divided 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 and used immediately before use. Such a kit is advantageously provided with instructions or instructions, preferably describing how to use the parts provided or how to handle the reagents. When the kit is used as a reagent kit in this specification, the kit usually includes instructions describing how to use the nucleic acid construct, etc.

[0022] As used herein, the term "instructions" refers to instructions that explain to a user how to use the present disclosure. The instructions contain text that instructs how to use the nucleic acid construct of the present disclosure. The instructions are usually provided in paper form, but are not limited thereto, and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, e-mail).

[0023] In this specification, "(Gibbs) free energy change (ΔG)" is described thermodynamically and statistically by the following equations (1) and (2).

[0024] ΔG=ΔH-TΔS (1) ΔG=ΔG°+RTlnK (2) Here, K is the equilibrium constant, and ΔG° is the Gibbs free energy change at standard conditions (1 atm, 25°C). When considering the equilibrium constant of a nucleic acid, it can be described as an equilibrium reaction in which two strands A and B (e.g., 3'UTR and 5'UTR) associate in a 1:1 ratio (Equation (3)).

[0025] A+B⇔AB (3) If the molar fraction of the double-stranded state is α and the total concentration of the nucleic acid is C, then the concentrations of A and B when completely dissociated, [A] and [B], are C / 2, and the equilibrium constant can be expressed by the following equation (4).

[0026] K = 2α / ((1-α) 2 ×C) (4) When the nucleic acids are in equilibrium with 50% dissociation, α=1 / 2, ΔG=0 (5) Therefore, substituting equations (4) and (5) into equation (2), we get ΔG°=-RTmln(4 / C) (6) It becomes. By substituting equation (6) into equation (1), we obtain the following equation (7).

[0027] 1 / Tm=(R / ΔH°)ln(Ct / 4)+ΔS° / ΔH°(7) By plotting the measured values ​​as functions of 1 / Tm and ln(C / 4), the enthalpy change ΔH° and entropy change ΔS° at the standard state can be calculated from the intercept and slope.

[0028] (Preferred embodiment) Preferred embodiments of the present disclosure are 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 a person 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.

[0029] In one embodiment, 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 at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, and when each of the non-complementary portions is all 1 base in length, the complementary rate of the 3' UTR to the 5' UTR is higher than 75%.The nucleic acid construct of the present disclosure can control protein expression.Preferably, the nucleic acid construct of the present disclosure can increase protein expression.

[0030] In some embodiments, 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, 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.

[0031] In another embodiment, each of the non-complementary portions may be 1 base in length, 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% and less than 90%, or greater than 81% and less than 89%.

[0032] In some 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, at least 20 complementary portions may 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 a preferred embodiment, 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, at least 20 complementary portions may be 5 to 11 bases. The lengths of the complementary portions may be the same or different, but are preferably the same.

[0033] In some embodiments, the non-complementary portions can be 2 bases in length and the complementary portions can each independently be 5-7 bases in length.

[0034] In some embodiments, the non-complementary portions can be 3 bases in length and the complementary portions can each independently be 8-11 bases in length.

[0035] In one embodiment, the 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 at least one UTR comprises a non-complementary portion (2) and a complementary portion (3) to the other UTR, the non-complementary portion alternating with the complementary portion.

[0036] In one embodiment, the portion other than the alternating portions (also referred to as the fractional portion (4)) is characterized in that it is one or more bases long, or shorter, than the base length of the complementary portion.

[0037] In one embodiment, when the non-complementary portion (2) is one base, the complementary portion (3) is 3 to 6 bases long in the case of base substitution, or 3 to 9 bases long in the case of base deletion.

[0038] In one embodiment, when the non-complementary portion (2) has 2 bases, the complementary portion (3) has a length of 5 to 7 bases in the case of base substitution, or a length of 5 to 11 bases in the case of base deletion.

[0039] In one embodiment, 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.

[0040] In one embodiment, the non-complementary portion (2) typically has 0 to 3 bases, and preferably has 2 or 3 bases.

[0041] Thus, the appropriate base length may vary depending on substitution or deletion, but one skilled in the art can design it appropriately based on the description in this specification.

[0042] In one embodiment, the 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 them to satisfy the above-mentioned preferred base lengths, and it is advantageous for all of them to satisfy the above-mentioned preferred base lengths.

[0043] In one embodiment, the non-complementary portion (2) may be 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 advantageous for all of them to be satisfied.

[0044] 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% to 90%, higher than 75% to 89%, 80% to 90%, or 81% to 89%.

[0045] The lengths of the complementary and non-complementary portions can be appropriately determined depending on the desired complementarity rate, but typically, the length of the non-complementary portions is independently 1 to 3 bases, and the length of the complementary portions can be independently 3 to 11 bases.

[0046] In certain embodiments, when the non-complementary portions are all 1 base, the complementary portions may each independently be 3 to 7 bases in length.

[0047] In certain embodiments, when the non-complementary portions are all 2 bases long, the complementary portions may each independently be 5 to 7 bases long.

[0048] In certain embodiments, when the non-complementary portions are all 3 bases long, the complementary portions may each independently be 8 to 11 bases long.

[0049] In certain embodiments, either the 5'UTR or the 3'UTR may have non-complementary portions removed.

[0050] In some embodiments, the UTR may be selected from the UTRs 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. Other examples of UTRs are disclosed in JP-A-2021-501572, JP-A-2015-517803, and JP-A-2022-164843.

[0051] In another embodiment, the nucleic acid constructs of the present disclosure may be provided as a kit.

[0052] In a further aspect, the disclosure provides a method for producing a nucleic acid construct, the nucleic acid construct comprising a 5′ untranslated region (UTR) and a 3′ UTR that are at least partially complementary to each other, wherein at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, the method comprising the steps of designing a plurality of candidate nucleic acid constructs, calculating a free energy change (ΔG) of a secondary structure of the plurality of designed candidate nucleic acid constructs, determining whether the free energy change is within a predetermined value or a range (e.g., a maximum value (ΔG max ) and minimum value (ΔG min ) (an increase rate of 0% to 60%) relative to the average of the expression levels of the selected candidate nucleic acid constructs in cells, and, if necessary, measuring the expression level of the selected candidate nucleic acid constructs in cells.

[0053] In some embodiments, the free energy change (ΔG(kcal / mol)) can be calculated by a nucleic acid secondary structure prediction program, such as mfold (GCG Software), IPknot, CentroidFold (https: / / www.ncrna.org / ), ViennaRNA (http: / / rna.tbi.univie.ac.at / ), RNALOSS (P Clote, RNALOSS: a web server for RNA locally optimal secondary structures, Nucleic Acids Res. 2005 Jul 1; 33: W600-4.), or RNA Secondary structure prediction (http: / / www.genebee.msu.su / services / rna2_reduced.html).

[0054] Those skilled in the art can appropriately determine the predetermined value or range of the free energy change with reference to Examples 6 and 7. Specifically, by preparing nucleic acid constructs having various complementarity rates and correlating the protein expression level by the nucleic acid construct with the free energy change, the predetermined value or range of the free energy change that maximizes the expression level can be determined.

[0055] In some embodiments, the free energy of the secondary structure of a nucleic acid construct of the present disclosure is: ((ΔG max +ΔG min ) / 2)×t Here, t may be 0.9 to 1.7, preferably t may be 1 to 1.6, and most preferably t may be 1.1 to 1.5.

[0056] In some embodiments, the free energy change can be the free energy change of the interaction between a 3'UTR and a 5'UTR in a nucleic acid construct.

[0057] In some embodiments, the maximum free energy change (ΔG max) is the free energy change for the interaction between all bases of the 3'UTR or 5'UTR and the 5'UTR or 3'UTR sequence with a mismatch substitution in its complementary strand (i.e., a sequence with a complementarity of 0%), and the minimum value of the free energy change (ΔG min ) may be the free energy change for the interaction between the 5'UTR and 3'UTR of a sequence with 100% 5'-3'UTR complementarity.

[0058] In a specific embodiment, the increase rate relative to the average value may be 0% to 60%, and preferably 10% to 50%.

[0059] The present disclosure can be applied in the application field of nucleic acid technology (e.g., medicine). For example, mRNA, which is a nucleic acid construct of the present disclosure, can be used as a drug substance for an mRNA drug. Furthermore, plasmid DNA, which is a nucleic acid construct of the present disclosure, can be used to create a cell bank for use in the production of an mRNA drug substance. Furthermore, linear template DNA, which is a nucleic acid construct of the present disclosure, can be used as a raw material in the production process of an mRNA drug substance.

[0060] 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 "within the range of" two values ​​is specified, the range includes the two values ​​themselves.

[0061] All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0062] 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, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0063] The specific reagents used were those 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.

[0064] (Molecular Biology Experimental Procedures) General DNA, RNA, and gene recombination experimental manipulations 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)).

[0065] (Production Example) (Purification of DNA fragments using low melting point agarose gel) DNA fragments were separated by electrophoresis for 1 hour at 100V in 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 with long-wavelength ultraviolet light (366 nm). The agarose gel containing the DNA fragments was dissolved by incubating at 63°C for 5 minutes in the presence of Thermostable β-Agarase (Nippon Gene) and then at 60°C for 10 minutes, after which TE-saturated phenol (Nacalai) was added and mixed thoroughly. The phenol phase and aqueous phase were separated by centrifugation (20,000×g, 10 minutes), and the aqueous phase was recovered in a new tube. The collected aqueous phase was mixed thoroughly with 1-butanol and then centrifuged (20,000×g, 10 min) 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 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, mixing thoroughly, and then centrifuging (20,000×g, 10 min). The precipitated DNA fragments were washed with 70% ethanol and then dissolved in TE buffer (Nacalai).

[0066] (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), then cooled gradually 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.

[0067] (Ligation, E. coli transformation, plasmid preparation) 1 μl of 1-20 ng / μl restriction enzyme treated plasmid solution, 1 μl of 0.1-20 ng / μl DNA fragment solution or 1 μl of 0.5 μM double-stranded oligonucleotide solution, and DNA Ligation Kit<Mighty Mix> (Takara Bio) and 2 μl of competent cells were mixed and incubated at 16°C for 1 to 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 cultured at 37°C for 1 hour at a rotation speed of 30 rpm using a small rotary incubator RT-50 (Taitec). After cultivation, the E. coli was smeared on an LB medium 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 with a medium-sized thermostatic shaker BR-53FP (Taitec) at a rotation speed of 220 rpm. After culture, the E. coli were collected by centrifugation (20,000 × g, 1 minute), and plasmids were prepared using the QIAprep Spin Miniprep Kit and the QIAcube nucleic acid extraction and purification device (Qiagen) according to the manufacturer's instructions.

[0068] (Construction of a plasmid serving as a template for RNA synthesis) The DNA fragment consisting of the T7 promoter sequence, Kozak sequence, termination codon, and 101-base-long polyA 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, which was made double-stranded by annealing synthetic oligonucleotides shown in SEQ ID NOs: 1 and 2, which have partially complementary 3' ends, and polymerase extension reaction from 5' to 3' direction of KOD-Plus-Ver.2. This DNA fragment has a BsaI site for inserting 5'UTR between the T7 promoter sequence and Kozak sequence, a BspQI site for inserting translation region (ORF) between the Kozak sequence and termination codon, a PaqCI site for inserting 3'UTR between the termination codon and polyA sequence, and a BsmBI site downstream of the polyA sequence for linearization of template DNA. This amplified DNA fragment was purified using a 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).

[0069] The DNA fragments encoding the fluorescent protein E2Crimson were amplified by PCR using primers Common-F and Common-R (SEQ ID NOs: 3 and 4) and KOD-Plus-Ver.2 (Toyobo) as templates, 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 polymerase extension reaction from 5' to 3' direction of KOD-Plus-Ver.2. The amplified DNA fragments were inserted into the XcmI site of pBR322_ΔtypeIIS plasmid to construct pBR_E2Crimson plasmid. The E2Crimson DNA fragment obtained by treating pBR_E2Crimson with BspQI was inserted into the PaqCI site of pT7_TL_pA100 to construct pT7_TL_E2Crimson_pA100 plasmid. The DNA fragment encoding the SARS CoV2 Spike protein was the sequence described in Messenger RNA encoding the full-length SARS-CoV-2 spike glycoprotein Sept. 2020 document 11889; 19th Meeting of the Vaccination and Vaccine Subcommittee of the Health Science Council (https: / / www.mhlw.go.jp / stf / shingi2 / 0000192554_00004.html). The pT7_TL_Spk_E2Crimson_pA100 plasmid was constructed by inserting this DNA fragment upstream of the region encoding E2Crimson in the pT7_TL_E2Crimson_pA100 plasmid.

[0070] The Pfizer 5'UTR sequence was obtained by annealing synthetic oligonucleotides shown in SEQ ID NOs: 13 and 14. The DNA fragment was inserted into the BsaI site of pT7_TL_E2Crimson_pA100 or pT7_TL_Spk_E2Crimson_pA100 to construct the pT7_TL_5Pf_E2Crimson_pA100 or pT7_TL_5Pf_Spk_E2Crimson_pA100 plasmid. The Pfizer 3'UTR sequence was obtained by annealing synthetic oligonucleotides shown in SEQ ID NOs: 11 and 12, which are partially complementary at the 3' end, and amplifying the single-stranded portion of the oligonucleotide by polymerase extension reaction from 5' to 3' with KOD-Plus-Ver.2 (Toyobo) to make it double-stranded, using the product as a template, by PCR using primers Common-F and Common-R (SEQ ID NOs: 3 and 4) and KOD-Plus-Ver.2. The 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 the pBR-3Pf plasmid with BsaI was inserted into the PaqCI site of pT7_TL_5Pf_E2Crimson_pA100 or pT7_TL_5Pf_Spk_E2Crimson_3Pf_pA100 (SEQ ID NO: 136) or pT7_TL_5Pf_Spk_E2Crimson_3Pf_pA100 (SEQ ID NO: 300) to construct pT7_TL_5Pf_E2Crimson_3Pf_pA100.

[0071] 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 combination of the constructed plasmids are shown in Table 1 below.

[0072] [Table 1]

[0073] The 3'UTR sequence, which is partially complementary to the Pfizer 5'UTR sequence, was obtained by annealing the synthetic oligonucleotides described below. The 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 plasmids are described below.

[0074] [Table 2-1]

[0075] [Table 2-2]

[0076] [Table 2-3]

[0077] [Table 2-4]

[0078] 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.

[0079] [Table 3]

[0080] (In vitro transcription with T7 RNA polymerase) In order to terminate RNA synthesis by dissociating RNA polymerase from the template DNA and to make the 3' end of the synthesized RNA an A nucleotide sequence, the plasmid serving as the template DNA was linearized with the restriction enzyme BsmBI (New England Biolab). After BsmBI treatment, the plasmid was added with TE-saturated phenol, mixed thoroughly, and then centrifuged (20,000×g, 10 min) to separate into an organic phase and an aqueous phase, and the aqueous phase was collected in a new tube. The collected aqueous phase was added with 1-butanol, mixed thoroughly, and then centrifuged (20,000×g, 10 min) to separate into a 1-butanol phase and an aqueous phase, and the separated 1-butanol phase was removed. This operation was repeated three times to completely remove phenol and reduce the volume of the aqueous phase. The aqueous phase was added with 3M sodium acetate solution (pH 5.2) (Nacalai) and ethanol, mixed thoroughly, and then centrifuged (20,000×g, 10 min) to precipitate the DNA fragments. The precipitated DNA fragment was washed with 70% ethanol and then dissolved in distilled water to obtain linear template DNA. The solution consisted of 25 ng / μl linear template DNA, 1× 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-methyl pseudo-UTP (TriLink or Yamasa Shoyu), 1 U / μl Recombinant RNase Inhibitor (Takara Bio), and 2 U / ml inorganic pyrophosphatase (New In vitro transcription reaction was performed by incubating at 42°C for 3 hours under the conditions of 2.5U / μl T7 RNA polymerase (Takara Bio) and 2.5U / μl DNase I (Takara Bio) at a final concentration of 0.125U / μl to remove the remaining linear template DNA. After this reaction, Recombinant DNase I (Takara Bio) was added to a final concentration of 0.125U / μl, and further incubated at 37°C for 15 minutes. After the reaction, the solution containing the RNA was added with a 1:1 mixture of citric acid saturated phenol (pH 4.3) (Nacalai) and chloroform (Nacalai) and mixed thoroughly, and then centrifuged (20,000×g, 10 minutes) to separate it into an organic phase and an aqueous phase. Chloroform was added to the collected aqueous phase and mixed thoroughly, and then centrifuged (20,000×g, 10 minutes) to separate it into a chloroform phase and an aqueous phase. This operation was repeated twice to completely remove the phenol contained in the aqueous phase. To this aqueous phase, 3M sodium acetate solution (pH 5.2) and ethanol were added, mixed thoroughly, and centrifuged (20,000×g, 10 min) to precipitate RNA. The precipitated RNA was washed with 70% ethanol and dissolved in 50 μl of distilled water. This RNA solution was applied to a MicroSpin S-200 Column (Cytiva) that had been previously centrifuged at 700×g for 1 min, and centrifuged at 700×g for 2 min., after which 50 μl of distilled water was added and centrifuged at 700×g for 2 min to remove unreacted nucleotides contained in this RNA solution. To this eluate, 3M sodium acetate solution (pH 5.2) and ethanol were added, mixed thoroughly, and centrifuged (20,000×g, 10 min) to precipitate RNA. The precipitated RNA was washed with 70% ethanol and dissolved in 20 μl of distilled water. RNA concentration was quantified using a microspectrophotometer Nano drop One (Thermofisher) and Qubit RNA Broad Range Assay Kit (Thermofisher) according to the manufacturer's instructions.

[0081] Quantitative Example (RNA transfection into cultured cells and quantification of fluorescent protein expression levels) 293 cells were cultured in DMEM (Thermofisher or Nacalai) supplemented with 10% FBS (Thermofisher) and a penicillin-streptomycin mixed solution (Nacalai) at 37°C in the presence of 5% CO2. 1 × 10 cells were cultured in 1.5 ml of medium per well of a 6-well culture plate. 5 The cells were seeded and cultured at 37°C for 24 hours, after which RNA transfection was performed. A mixture of 48.5μl of Opti-MEM (Thermofisher) and 1.5μl of Lipofectamine messengerMAX (Thermofisher) per well and 50μl of Opti-MEM containing 50ng of RNA were prepared in advance, and then mixed and incubated at room temperature for 10 minutes. This solution was added to the culture wells and cultured at 37°C for another 24 hours. After RNA transfection, the 293 cells were washed with 1ml of DPBS (Thermofisher) per well, and then dissociated from the bottom of the culture plate with 0.5ml of trypsin-EDTA (Nacalai). After dissociation, the cells were transferred to a 1.5ml tube with the addition of 1ml of DPBS, and then collected by centrifugation at 3000×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 microplate, flat bottom, black (Greiner). The relative amount of E2Crimson protein in each well was calculated by measuring the fluorescence at an excitation wavelength of 606 nm and an emission wavelength of 651 nm using a plate reader Infinite 200 PRO (TECAN). After the above fluorescence measurement, 3 μl of DPBS containing 5 μg / ml Calcein-AM (Dojindo Chemical) was added to the cell suspension dispensed into each well, mixed, and incubated at 37°C for 30 minutes. The relative number of live cells in each well was calculated by measuring the fluorescence at an excitation wavelength of 480 nm and an emission wavelength of 533 nm of calcein, which is a hydrolysis product of calcein-AM by intracellular esterase. It was confirmed separately that the fluorescence of E2Crimson protein does not interfere with the fluorescence of calcein. 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.

[0082] (Example 1: Improving expression efficiency by complementarity between 5'-3' untranslated regions: usefulness of non-complementary 2 and 3 bases) In this example, the expression levels of mRNAs whose 5'UTR is the GAPDH gene sequence, the Pfizer sequence, and the HSD17B4 gene sequence were examined. In each case, mRNAs whose 5'UTR and 3'UTR are both Pfizer sequences were used as a comparison. The constructions were produced and the expression levels were quantified based on the above production examples and quantification examples.

[0083] (result) The results are shown in Figure 1. The ratio of E2Crimson fluorescence measurements to Calcein fluorescence measurements was taken as 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. When (number of complementary bases) / (number of non-complementary bases)=3, and the 5'UTR was GAPDH or HSD17B4, the expression level was higher when there were 2 or 3 non-complementary bases. When the 5'UTR was Pfizer, the results were roughly equivalent for 1 to 3 non-complementary bases.

[0084] Figure 2 shows the results of verifying the expression level of mRNAs in which the 5'UTR is a GAPDH gene sequence and the 3'UTR is a sequence that has 100%, 94%, or 75% complementarity to the 5'UTR. 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, and the average values ​​are shown in the bar graph, and the individual values ​​for each sample are shown in black plots. The highest expression level was observed when the non-complementary portion was 2 bases (#2), and the expression levels were equivalent when the non-complementary portion was 1 base and 4 bases. When the complementarity exceeded 90%, the expression level decreased. Furthermore, when the non-complementary portion exceeded 4 bases, the expression level decreased.

[0085] (Example 2: Improving expression efficiency by complementarity between 5'-3' untranslated regions: 5'UTR GAPDH) In this example, the expression levels of mRNAs having the GAPDH gene sequence as the 5'UTR and various sequences partially complementary to the 5'UTR as the 3'UTR were examined in 293 cells. The constructions were produced and quantified according to the above production example and quantification example.

[0086] (result) FIG. 3 shows the base sequences of the 3'UTR and 5'UTR used in this example. The results are shown in FIG. 4. For comparison, mRNAs with Pfizer sequences for both 5'UTR and 3'UTR were used. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was taken as the relative expression level of the E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in bar graphs and individual values ​​for each sample in black plots. When the non-complementary portion was 1 base, a high expression level was observed when the complementarity rate was 75 to 89% and the complementary portion was 3 to 7 bases. When the complementary portion exceeded 7 bases, the expression level decreased. In addition, when the non-complementary portion was 2 bases and the complementary portion was 6 bases, and when the non-complementary portion was 3 bases and the complementary portion was 11 bases, a high expression level was observed.

[0087] (Example 3: Expression efficiency improved by complementarity between 5'-3' untranslated regions: 5'UTR Pfizer sequence) In this example, the expression levels of mRNAs having the Pfizer sequence as the 5'UTR and various sequences partially complementary to this 5'UTR as the 3'UTR were examined in 293 cells. The constructions were produced and quantified according to the above production example and quantification example.

[0088] (Results) Figure 5 shows the base sequences of the 3'UTR and 5'UTR used in this example. The results are shown in Figure 6. For comparison, mRNAs with Pfizer sequences 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 taken as 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 bar graphs, and the individual values ​​for each sample are shown in black plots. Complementary-non-complementary: The expression level was enhanced in 4-1, 3-1, 6-2, 5-2, and 9-3 mRNAs compared to the Pfizer sequence, increasing by up to about 50%. The expression level tended to be higher when the number of complementary bases was shorter.

[0089] (Example 4: Improving expression efficiency by complementarity between 5'-3' untranslated regions: 5'UTR HSD17B4 sequence) In this example, the expression levels of mRNAs having the HSD17B4 gene sequence as the 5'UTR and various sequences partially complementary to this 5'UTR as the 3'UTR were examined in 293 cells. The constructions were produced and quantified according to the above production example and quantification example.

[0090] (result) FIG. 7 shows the base sequences of the 3'UTR and 5'UTR used in this example, and the results are shown in FIG. 8. For comparison, mRNAs in which both the 5'UTR and 3'UTR were Pfizer sequences were used. 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, and the average values ​​are shown in the bar graph, and the individual values ​​for each sample are shown in black plots. Complementary-non-complementary: Expression levels were enhanced in 4-1, 3-1, 6-2, 5-2, 9-3, and 8-3 mRNAs compared to the Pfizer sequence.

[0091] In summary, when the non-complementary portion is 1 base, the complementary portion is preferably 3 to 7 bases; when the non-complementary portion is 2 bases, the complementary portion is preferably 5 to 7 bases; and when the non-complementary portion is 3 bases, the complementary portion is preferably 8 to 11 bases.

[0092] (Example 5: Expression efficiency improvement by complementation between 5'-3' untranslated regions: Critical significance at a complementation rate of 75-80%) In this example, the expression levels of mRNAs having a GAPDH gene sequence as the 5'UTR and various sequences partially complementary to this 5'UTR as the 3'UTR were examined in 293 cells. The constructions were produced and quantified based on the above production examples and quantification examples. The sequence numbers of the synthetic oligonucleotides and the combinations of the constructed plasmids are as follows:

[0093] [Table 4]

[0094] (result) FIG. 9 shows the base sequences of the 3'UTR and 5'UTR used in this example, and the results are shown in FIG. 10. For comparison, mRNAs in which the 5'UTR and 3'UTR were both Pfizer sequences were used. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was taken as the relative expression level of the E2Crimson protein in each mRNA. Three samples were measured for each mRNA, and the average values ​​are plotted. FIG. 10 shows that the complementarity rate was maximum between 75% and 80%, and that a complementarity rate of 75% or higher is preferable.

[0095] (Example 6: Improving expression efficiency by complementarity between 5'-3' untranslated regions: Free energy change) In this example, the correlation between the free energy change in the interaction between 5'UTR and 3'UTR and the expression level of each mRNA was examined for mRNAs in which the 3'UTR is partially complementary to the 5'UTR. The construction was produced and quantified based on the above production example and quantification example.

[0096] (result) The results are shown in Figure 11. The expression level was calculated by setting the mRNA with the Pfizer sequence for both 5'UTR and 3'UTR as 1. The free energy change was calculated using the secondary structure prediction program Mfold (Michael Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res. 2003 Jul 1;31(13):3406-15). The left, center, and right figures show the results when the 5'UTR was the GAPDH gene, the HSD17B4 gene, and the Pfizer sequence, respectively. For each 5'UTR, the actual measured values ​​obtained were fitted to a four-coefficient logistic curve y=d+(ad) / (1+(x / c)^b), which is a type of sigmoid curve. Figure 11 shows that the free energy change correlates with the expression level, and it became clear that the expression level can be predicted to some extent from the free energy change.

[0097] FIG. 12 shows the maximum free energy change. The maximum free energy change was determined by Mfold for mRNA in which base substitutions were made to mismatch all bases in the 3'UTR, where the complementarity between the 5'UTR and the 3'UTR is 100%. The relative free energy change was calculated using the increase rate (right shift) from the inflection point as an index, with the difference between the inflection point and the maximum value as 100%. The expression level was maximum for mRNA in which the increase from the inflection point was between 10% and 50%.

[0098] Figure 13 shows a method for predicting the expression level of mRNA from the 5'UTR and 3'UTR sequences. The maximum and minimum values ​​of the free energy change were obtained in the same manner as in Figure 12. It was found that the average value of the maximum and minimum values ​​of the free energy coincides with the value of the free energy change at the inflection point of the sigmoid curve. By using this average value, it was possible to predict the inflection point with an error of 4.9 kcal / mol or less, or an accuracy of 13.1% or less. As in Figure 12, this error percentage was calculated by setting the difference between the value at the inflection point and the maximum free energy change as 100%. Based on the prediction of this inflection point, the free energy change of the interaction between 5'UTR and 3'UTR in mRNA with the maximum expression level can be predicted.

[0099] (Example 7: Prediction of expression level using free energy change) 1. Design candidate sequences for the 5'UTR. 2. Design candidate sequences for 3'UTR that are partially complementary (complementarity rate 40-90%) to 5'UTR so that they have 1, 2, or 3 non-complementary bases and 4-14 complementary bases. In this case, the number of complementary bases should be consecutive. For example, if 7-10 complementary bases are selected, design all sequences with complementary bases of 7, 8, 9, and 10 bases. Design 10-20 types for each type of 5'UTR. 3. For the designed sequence, the free energy change is calculated using the secondary structure prediction program Mfold. 4. The average of the maximum and minimum free energy changes is used as the reference value. Here, the maximum free energy change is based on a sequence in which all bases in the 3'UTR have been mismatched, and the minimum free energy change is based on a sequence with 100% 5'-3'UTR complementarity. 5. Candidate sequences having free energy change values ​​with an increase rate of 0% to 60% relative to the reference value are selected. 6. If necessary, check the protein expression levels of the selected candidate sequences and select sequences with high expression levels.

[0100] (Example 8: Improving expression efficiency by complementation between 5'-3' untranslated regions: myoblast cell line C2C12) In this example, the expression levels of mRNAs having the Pfizer sequence as the 5'UTR and various sequences partially complementary to this 5'UTR as the 3'UTR were examined in C2C12 cells. The constructions were produced and quantified according to the above production example and quantification example.

[0101] (result) FIG. 5 shows the base sequences of the 3'UTR and 5'UTR used in this example. The results are shown in FIG. 15. For comparison, mRNAs with both the 5'UTR and 3'UTR Pfizer sequences were used. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was used as the relative expression level of the E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in bar graphs, and the individual values ​​for each sample are shown in black plots. Complementary-non-complementary: The expression level was enhanced in 4-1, 3-1, 6-2, 5-2, and 9-3 mRNAs compared to the Pfizer sequence, increasing by up to about 60%. Since the results were equivalent to those in 293 cells shown in FIG. 6, it became clear that the effect of partially complementary 5' untranslated region (UTR) and 3'UTR is not limited to cell types.

[0102] (Example 9: Improving expression efficiency by complementarity between 5'-3' untranslated regions: long mRNA) In this example, the expression levels of mRNAs having the Pfizer sequence as the 5'UTR, various sequences partially complementary to this 5'UTR as the 3'UTR, and the SARS CoV2 Spike and E2Crimson fusion proteins as ORFs were examined in 293 cells. The constructions were produced and quantified according to the above production examples and quantification examples.

[0103] (result) FIG. 5 shows the base sequences of the 3'UTR and 5'UTR used in this example. The results are shown in FIG. 16. For comparison, mRNAs with both the 5'UTR and 3'UTR Pfizer sequences were used. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was used as the relative expression level of the E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, and the individual values ​​for each sample are shown in black plots. Complementary-non-complementary: 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 showed enhanced expression levels compared to the Pfizer sequence, increasing by up to about 2 times. The results were equivalent to those of the mRNA with the E2Crimson protein as the ORF shown in FIG. 6. The ORFs of the SARS CoV2 Spike and E2Crimson fusion proteins are 4.5 kbp, and the ORF of the E2Crimson protein is 0.7 kbp, indicating that the effect of the partially complementary 5' untranslated region (UTR) and 3'UTR is not limited by the type and length of the ORF.

[0104] (Example 10: Improving expression efficiency by complementarity between 5'-3' untranslated regions: Removal of bases from non-complementary regions) In this example, the expression levels of mRNAs having the Pfizer sequence as the 5'UTR and various sequences partially complementary to the 5'UTR as the 3'UTR were examined in 293 cells. The 3'UTRs were made non-complementary by removing bases. The constructions were produced and quantified according to the above production examples and quantification examples.

[0105] (result) FIG. 17(2) shows a schematic diagram of the mRNA used in this example. FIG. 18 shows the base sequences of the 3'UTR and 5'UTR used in this example. The results of the mRNA in which the non-complementary site on the 3'UTR shown in FIG. 18(2) was removed are shown in FIG. 19 and FIG. 20. For comparison, mRNA in which the 5'UTR and 3'UTR were both Pfizer sequences was used. The ratio of the E2Crimson fluorescence measurement value to the Calcein fluorescence measurement value was taken as the relative expression level of the E2Crimson protein in each mRNA. Measurements were performed on multiple samples for each mRNA, and the average values ​​are shown in the bar graph, and the individual values ​​for each sample are shown in black plots. Complementary-non-complementary: The expression level was 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, increasing by up to about 60%. In the case where the non-complementary portion shown in FIG. 6 was replaced by base substitution, the expression level was enhanced in the mRNAs of complementary-non-complementary: 4-1, 3-1, 6-2, 5-2, 9-3, and 8-3 compared to the Pfizer sequence, and it was found that in the case of base removal, the expression level tends to be higher in the mRNA with a long complementary strand and a high complementarity rate. Furthermore, the expression levels of the mRNA in which the non-complementary portion on the 5'UTR is removed as shown in FIG. 18(3), the mRNA in which base substitution and base removal are mixed as shown in FIG. 18(4), the mRNA in which a non-complementary sequence is created by adding bases in the 3'UTR as shown in FIG. 18(5), and the mRNA in which a non-complementary sequence is created by adding bases in the 5'UTR as shown in FIG. 18(6) are also confirmed by the same method. Sequences of the plasmids used for mRNA synthesis are shown in SEQ ID NOs: 301-315 and 334-339. The expression level can be controlled in the same manner as the mRNA in which the non-complementary portion on the 3'UTR is removed as shown in FIG. 18(2).

[0106] [Table 5-1]

[0107] [Table 5-2]

[0108] (Example 11: Improving expression efficiency by complementarity between 5'-3' non-translated regions: Deletion of bases in non-complementary regions - Myoblast cell line C2C12) In this example, the expression levels in C2C12 cells of mRNAs having the Pfizer sequence as the 5'UTR and various sequences partially complementary to this 5'UTR as the 3'UTR were examined. The 3'UTRs were made non-complementary by removing bases. The constructions were produced and quantified according to the above production examples and quantification examples.

[0109] (result) The results obtained with the mRNA used in Figures 19 and 20 are shown in Figures 21 and 22. For comparison, mRNA with Pfizer sequences in both 5'UTR and 3'UTR was used. The ratio of E2Crimson fluorescence measurements to Calcein fluorescence measurements was taken as 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. Expression levels were enhanced compared to the Pfizer sequence in the complementary-non-complementary 4-1, 3-1, 8-2, 7-2, 6-2, 5-2, 11-3, 10-3, 9-3, and 8-3, increasing by up to about 50%. When the non-complementary portion shown in Figure 15 was substituted with a base, the expression level was increased in mRNAs with complementary-non-complementary sequences: 4-1, 3-1, 6-2, 5-2, and 9-3 compared to the Pfizer sequence. As seen in the case of 293 cells, when bases were removed, the expression level tended to be higher in mRNAs with longer complementary strands and higher complementarity rates.

[0110] (Example 12: Improving expression efficiency by complementarity between 5'-3' non-translated regions: Deletion of non-complementary bases in myoblast cell line C2C12 long mRNA) In this example, the expression levels in C2C12 cells of mRNAs with the Pfizer sequence as the 5'UTR and various sequences partially complementary to the 5'UTR as the 3'UTR were examined. The 3'UTRs were made non-complementary by removing bases. The ORFs were SARS CoV2 Spike and E2Crimson fusion proteins. The constructions were produced and quantified based on the above production and quantification examples.

[0111] (result) The results are shown in Figure 23. For comparison, mRNAs with Pfizer sequences in both 5'UTR and 3'UTR were used. The ratio of E2Crimson fluorescence measurements to Calcein fluorescence measurements was taken as 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. Complementary-non-complementary: Expression levels were enhanced compared to the Pfizer sequence in 9-1, 6-1, 5-1, 11-2, 10-2, 9-2, 8-2, 7-2, 11-3, 10-3, and 8-3, increasing by up to approximately 40%. When the non-complementary portions shown in Figure 16 were replaced by base substitutions, the expression levels were increased in complementary-non-complementary mRNAs: 6-1, 11-2, 8-2, 7-2, 6-2, 5-2, 12-3, 11-3, 10-3, 9-3, and 8-3 compared to the Pfizer sequence. This indicates that in long-chain mRNAs as well, when bases are removed, the expression levels tend to be higher in mRNAs with longer complementary strands and higher complementarity rates.

[0112] (Example 13: Improving expression efficiency by complementarity between 5'-3' untranslated regions: When non-complementary regions with different numbers of bases are present) FIG. 24 shows the base sequences of the 3'UTR and 5'UTR used in this example. SEQ ID NOs: 316-333 show the sequences of the plasmids used for mRNA synthesis. FIG. 24(1) shows the case where all non-complementary portions are replaced, i.e., the number of bases in the non-complementary portions is the same in the 5'UTR and 3'UTR. Protein expression is confirmed for these by the method described in the above example. The expression level can be controlled in the same way as the mRNA shown in FIG. 5. Furthermore, FIG. 24(2) shows the case where the bases in the non-complementary portions are replaced or removed, i.e., the number of bases in the non-complementary portions is different between the 5'UTR and 3'UTR. Protein expression is confirmed for these by the method described in the above example. The expression level can be controlled in the same way as the mRNA shown in FIG. 18(2) where the non-complementary portions on the 3'UTR are removed.

[0113] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the scope of the claims. It is understood that the patents, patent applications and literature cited in this specification should be incorporated by reference to this specification in the same manner as if the contents themselves were specifically described in this specification. This application claims priority to Patent Application No. 2023-013290 filed with the Japan Patent Office on January 31, 2023, the contents of which are all cited as constituting this specification as necessary in this application. [Industrial Applicability]

[0114] The present disclosure may be applied in the application field of nucleic acid technology (e.g., medicine). [Sequence List Free Text]

[0115] SEQ ID NOs: 1 to 339: See Table 1 in the specification.

Claims

1. A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR which are at least partially complementary to each other, wherein at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, and the length of each of the non-complementary portions is a single base substitution, and the complementarity of the 3' UTR to the 5' UTR is greater than 75%.

2. The nucleic acid construct of claim 1 , wherein bases in the non-complementary portions have been substituted or removed.

3. A nucleic acid construct as described in claim 2, wherein all of the non-complementary portions are removed.

4. The nucleic acid construct of claim 1 , wherein at least one of the non-complementary portions is two or more bases in length.

5. The nucleic acid construct of claim 1 , wherein at least one of the non-complementary portions is 2 or 3 bases in length.

6. The nucleic acid construct of claim 1 , wherein at least one of the complementary portions is 5 or more bases in length.

7. 2. The nucleic acid construct of claim 1, wherein at least one of the complementary portions has a length of 5 to 11 bases.

8. 2. The nucleic acid construct according to claim 1, wherein the non-complementary portions are 2 bases in length, and the complementary portions are each independently 5 to 7 bases in length.

9. 2. The nucleic acid construct according to claim 1, wherein the non-complementary portions have a length of 3 bases, and the complementary portions each have a length of 8 to 11 bases, independently.

10. The nucleic acid construct of claim 1 , wherein each of the non-complementary portions is at least one base in length, and the rate of complementarity is greater than 75%.

11. The nucleic acid construct according to claim 10, wherein the complementarity is between 80% and 90%.

12. The nucleic acid construct according to claim 10, wherein the complementarity is between 81% and 89%.

13. The nucleic acid construct of claim 1 , wherein at least one of the non-complementary portions is a single base.

14. The nucleic acid construct according to claim 10, wherein the length of each of the complementary portions is independently 3 to 11 bases.

15. The nucleic acid construct according to claim 10, wherein the non-complementary portion is 1 base, and the complementary portions are each independently 3 to 7 bases in length.

16. The nucleic acid construct according to claim 10, wherein the non-complementary portions have a length of 2 bases, and the complementary portions have a length of independently 5 to 7 bases.

17. The nucleic acid construct according to claim 10, wherein the non-complementary portions have a length of 3 bases, and the complementary portions each have a length of 8 to 11 bases, independently.

18. A nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR which are at least partially complementary to each other, characterized in that at least one UTR comprises non-complementary and complementary portions to the other UTR, and the non-complementary and complementary portions alternate.

19. The nucleic acid construct according to claim 18, wherein 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.

20. The nucleic acid construct according to claim 18, wherein when the non-complementary portion is 3 bases, 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.

21. The nucleic acid construct of claim 1 , wherein either the 5′UTR or the 3′UTR does not contain a non-complementary portion.

22. The nucleic acid construct of claim 1 , wherein the free energy change (ΔG) is a predetermined value or range.

23. The nucleic acid construct according to claim 22, wherein the predetermined value or range thereof is a value or range of a free energy change (ΔG) having an increase rate of 0% to 60% relative to the average value of the maximum and minimum values ​​of the free energy change.

24. The nucleic acid construct according to claim 23, wherein the predetermined value or range thereof is a value or range of a free energy change having an increase rate of 10% to 50% relative to the average value of the maximum and minimum values ​​of the free energy change (ΔG).

25. A method for producing a nucleic acid construct, the nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR that are at least partially complementary to each other, wherein at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, the method comprising the steps of designing a plurality of candidate nucleic acid constructs, calculating a free energy change (ΔG) for the plurality of designed candidate nucleic acid constructs, selecting a candidate nucleic acid construct whose free energy change (ΔG) is a predetermined value or within a predetermined range, and, if necessary, measuring the expression level of the selected candidate nucleic acid construct in a cell.

26. A method for producing a nucleic acid construct, the nucleic acid construct comprising a 5' untranslated region (UTR) and a 3' UTR which are at least partially complementary to each other, and at least one UTR comprises a non-complementary portion and a complementary portion to the other UTR, the method comprising the steps of designing a plurality of candidate nucleic acid constructs, calculating a free energy change (ΔG) for the plurality of designed candidate nucleic acid constructs, selecting a candidate nucleic acid construct having a free energy change value with an increase rate in the range of 0% to 60% relative to the average value of the maximum and minimum values ​​of the free energy change (ΔG), and, if necessary, measuring the expression level of the selected candidate nucleic acid construct in a cell.

27. 27. The method of claim 26, wherein the increase relative to the average value is between 10% and 50%.

28. The nucleic acid construct according to claim 1, produced according to the method according to any one of claims 25 to 27.

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