DNA molecules, cell, RNA molecule, DNA coding sequence, poly(A) tail sequence, use of DNA coding sequence, use of poly(A) tail sequence, library, method for regulating protein expression, and, DNA-RNA hybrid molecule

Abstract

An engineered DNA molecule capable of being replicated in a cell, comprising a poly(A) tail coding sequence that makes the engineered DNA molecule more conservative when replicated in cells, particularly in prokaryotic cells, while adjusting the expression level of RNA in eukaryotic cells. Also provided are an RNA comprising the poly(A) tail and a use thereof.

Description

DNA molecules, cell, RNA molecule, DNA coding sequence, poly(A) tail sequence, use DNA coding sequence, use of poly(A) tail sequence, library, method for regulating a Protein expression, and DNA-RNA hybrid molecule - Technical field

[001] This application relates to the field of biotechnology, specifically to an RNA comprising a poly(A) tail. The poly(A) tail makes the replication of the RNA-encoding DNA in the prokaryotic system more stable and can be used to regulate the level of RNA expression in a eukaryotic cell. BACKGROUND

[002] The primary structure of a translatable mRNA drug molecule consists of a 5' cap structure, a 5' non-coding region (5' UTR), a coding region, a 3' non-coding region, and a polyadenosine tail (poly(A) tail). The known functions of the poly(A) tail include maintaining the in vivo stability of mRNA molecules and participating in the initiation of protein translation, the latter of which is achieved through the interaction between the poly(A) tail-binding protein (PABP) and the translation initiation complex. In a eukaryotic cell, the poly(A) tail is synthesized by a post-transcriptional modification under the action of typical poly(A) polymerase.

[003] The first step in preparing mRNA drugs in vitro is that mRNA drugs are synthesized via in vitro transcription (IVT) using a linearized plasmid containing the designed product sequence as a template, and the poly(A) tail is usually added downstream of the 3' UTR in a cotranscriptional manner. To achieve the cotranscriptional addition of poly(A), the corresponding poly(dA:dT) sequence needs to be included in the template plasmid. However, the Petition 870250073560, dated 08 / 20 / 2025, page 15 / 89 / 61 The poly(dA:dT) repeat sequence in the plasmid is unstable during replication in E. coli, with deletion mutations frequently occurring in this sequence, leading to poly(dA:dT) shortening. This phenomenon is not conducive to the process of preparing model transcription plasmids in vitro through large-scale fermentation, and poly(A) truncation has a significant impact on the in vivo stability and biological activity of the mRNA. SUMMARY

[004] The present application provides a novel poly(A) tail to improve its preservation during the in vitro preparation process. In addition, a method for regulating the RNA expression level in a eukaryotic cell based on the poly(A) tail is also provided.

[005] Specifically, the first aspect of the present application provides a manipulated DNA molecule capable of being replicated in a cell, comprising a polyadenosine tail coding sequence (Poly A tail), wherein the poly(A) tail coding sequence comprises: a single element ae at least one element be at least one element c; a single element ae with at least one element be and at least one element d; or a single element ae with at least one element be and at least one element c and at least one element d, in the poly(A) tail coding sequence, the element a consists of a plurality of consecutive adenine(A) nucleotides, and the length range of the element a is > 20 nt; The b element consists of a plurality of consecutive A nucleotides, and the length range of the b element is 3 nt < b < 20 nt; The element c consists of a non-A nucleotide, and the Petition 870250073560, dated 08 / 20 / 2025, p. 16 / 89 / 61 nucleotide is selected from among T, C and G nucleotides; The d element consists of any two or more consecutive nucleotides, and the nucleotides are selected from A, T, C, and G nucleotides, wherein the nucleotides at the 5' and 3' ends of the d element are not A nucleotides, and the d element does not comprise 3 or more consecutive A nucleotides; and the length range of the d element is 2 nt < d < 20 nt; given that element a and element b are not adjacent, and element c and element d are not adjacent, and the poly(A) tail encoding sequence does not comprise any two elements b that are adjacent to each other, does not comprise any two elements c that are adjacent to each other, and does not comprise any two elements d that are adjacent to each other.

[006] In some embodiments, the poly(A) tail coding sequence additionally comprises a single element e, wherein the element e consists of one or two consecutive A's, which are situated at the 3' termination of the poly(A) tail coding sequence and are in a position adjacent to the element d or the element c.

[007] In some embodiments, the poly tail coding sequence (A) comprises no other elements except element a, element b, element c and element d.

[008] In some embodiments, the poly tail (A) encoding sequence comprises no other elements except element a, element b, element c, element d, and element e.

[009] In some embodiments, the poly(A) tail coding sequence comprises at least 2 d elements. In some embodiments, the poly(A) tail coding sequence comprises at least 2 c elements. In some embodiments, the tail coding sequence Petition 870250073560, dated 20 / 08 / 2025, p. 17 / 89 / 61 poli (A) comprises at least one element of an element c.

[0010] In some forms, the number of element b is from 2 to 10, for example, 3, 4, 5, 6, 7, 8 or 9.

[0011] In some forms, the element c number is from 0 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0012] In some forms, the number of element d is from 0 to 5, for example, 1, 2, 3 or 4.

[0013] In some forms, when element c and element d exist simultaneously, the total number of elements c and elements d is from 2 to 15, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0014] In some forms, the number of element a is 1, the number of element b is 3, the number of element c is 2, and the number of element d is 1.

[0015] In some forms, the number of element a is 1, the number of element b is 4, the number of element c is 4, and the number of element d is 1.

[0016] In some forms, the number of element a is 1, the number of element b is 5, the number of element c is 4, and the number of element d is 1.

[0017] In some forms, the number of element a is 1, the number of element b is 3, the number of element c is 3, and the number of element d is 1.

[0018] In some forms, the number of element a is 1, the number of element b is 3, the number of element c is 2, and the number of element d is 1.

[0019] In some embodiments, the length range of element a is <80 nt. In some embodiments, element a has 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, Petition 870250073560, of 08 / 20 / 2025, p. 18 / 89 / 61 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt or 79 nt.

[0020] In some embodiments, element b has 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt or nt of length.

[0021] In some embodiments, the length range of element d is 2 nt <d < 20 nt, 3 a 18 nt, 5 a 16 nt, 4 a 10 nt ou 6 a 12 nt, por exemplo, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt ou 20 nt, de preferência, 6 nt.

[0022] Em algumas modalidades, o comprimento da sequência codificadora de cauda poli (A) é maior que 40 nt, por exemplo, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt, 83 nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 175 nt, 176 nt, 177 nt, 178 nt, 179 nt, 185 nt, 186 nt, 187 nt, 188 nt, 189 nt, 195 nt, 196 nt, 197 nt, 198 nt, 199 nt, 205 nt, 206 nt, 207 nt, 208 nt, 209 nt, 215 nt, 216 nt, 217 nt, 218 nt, 219 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 160 nt, 161 nt, 162 nt, 163 nt, 164 nt, 170 nt, 171 nt, 172 nt, 173 nt, 174 nt, 180 nt, 181 nt, 182 nt, 183 nt, 184 nt, 190 nt, 191 nt, 192 nt, 193 nt, 194 nt, 200 nt, 201 nt, 202 nt, 203 nt, 204 nt, 210 nt, 211 nt, 212 nt, 213 nt, 214 nt, 220 nt, 221 nt, 222 nt, 223 nt, 224 nt, Petição 870250073560, de 20 / 08 / 2025, pág. 19 / 89 / 61 225 nt, 226 nt, 227 nt, 228 nt, 229 nt, 230 nt, 231 nt, 232 nt, 233 nt, 234 nt, 235 nt, 236 nt, 237 nt, 238 nt, 239 nt, 240 nt, 241 nt, 242 nt, 243 nt, 244 nt, 245 nt, 246 nt, 247 nt, 248 nt, 249 nt, 250 nt, 251 nt, 252 nt, 253 nt, 254 nt, 255 nt, 256 nt, 257 nt, 258 nt, 259 nt, 260 nt, 261 nt, 262 nt, 263 nt, 264 nt, 265 nt, 266 nt, 267 nt, 268 nt, 269 nt, 270 nt, 271 nt, 272 nt, 273 nt, 274 nt, 275 nt, 276 nt, 277 nt, 278 nt, 279 nt, 280 nt, 281 nt, 282 nt, 283 nt, 284 nt, 285 nt, 286 nt, 287 nt, 288 nt, 289 nt, 290 nt, 291 nt, 292 nt, 293 nt, 294 nt, 295 nt, 296 nt, 297 nt, 298 nt, 299 nt, 300 nt, 301 nt, 302 nt, 303 nt, 304 nt, 305 nt, 306 nt, 307 nt, 308 nt, 309 nt, 310 nt, 311 nt, 312 nt, 313 nt, 314 nt, 315 nt, 316 nt, 317 nt, 318 nt, 319 nt, 320 nt, 321 nt, 322 nt, 323 nt, 324 nt, 325 nt, 326 nt, 327 nt, 328 nt, 329 nt, 330 nt, 331 nt, 332 nt, 333 nt, 334 nt, 335 nt, 336 nt, 337 nt, 338 nt, 339 nt, 340 nt, 341 nt, 342 nt, 343 nt, 344 nt, 345 nt, 346 nt, 347 nt, 348 nt, 349 nt, 350 nt, 351 nt, 352 nt, 353 nt, 354 nt, 355 nt, 356 nt, 357 nt, 358 nt, 359 nt, 360 nt, 361 nt, 362 nt, 363 nt, 364 nt, 365 nt, 366 nt, 367 nt, 368 nt, 369 nt, 370 nt, 371 nt, 372 nt, 373 nt, 374 nt, 375 nt, 376 nt, 377 nt, 378 nt, 379 nt, 380 nt, 381 nt, 382 nt, 383 nt, 384 nt, 385 nt, 386 nt, 387 nt, 388 nt, 389 nt, 390 nt, 391 nt, 392 nt, 393 nt, 394 nt, 395 nt, 396 nt, 397 nt, 398 nt, 399 nt ou 400 nt, etc.

[0023] In some embodiments, 50% or more of the α-element polynucleotides are located in the 5' or 3' portion of the poly(A) tail coding sequence. In some embodiments, 50% or more of the α-element polynucleotides are located in the 5' portion of the poly(A) tail coding sequence. In some embodiments, 50% or more of the α-element polynucleotides are located in the 3' portion of the poly(A) tail coding sequence. In some embodiments, the number of α-element nucleotides located in the 3' portion of the poly(A) tail coding sequence is equal to the number of nucleotides located in the 5' portion of the poly(A) tail coding sequence.

[0024] In some forms, the element c is G, C, or T. Petition 870250073560, dated 08 / 20 / 2025, p. 20 / 89 / 61

[0025] In some embodiments, element d comprises a palindromic sequence. Element d is a palindromic sequence. In some embodiments, element d comprises a sequence selected from the group consisting of GATATC (SEQ ID NO: 15), GTATAC (SEQ ID NO: 16), GAATCT (SEQ ID NO: 17), GCATATGACT (SEQ ID NO: 18) and GATATCGTATAC (SEQ ID NO: 19). In some embodiments, element d is a sequence selected from the group consisting of GATATC (SEQ ID NO: 15), GTATAC (SEQ ID NO: 16), GAATCT (SEQ ID NO: 17), GCATATGACT (SEQ ID NO: 18) and GATATCGTATAC (SEQ ID NO: 19). In some embodiments, the d element comprises a polynucleotide sequence represented by SEQ ID NO: 15. In some embodiments, the polynucleotide sequence of the d element is represented by SEQ ID NO: 15.

[0026] In some embodiments, the nucleotide at the 3' end of the poly(A) tail coding sequence is A. In some embodiments, the nucleotide at the 3' end of the poly(A) tail coding sequence is G. In some embodiments, the nucleotide at the 3' end of the poly(A) tail coding sequence is C. In some embodiments, the nucleotide at the 3' end of the poly(A) tail coding sequence is T.

[0027] In some embodiments, the 3' portion of the poly(A) tail coding sequence comprises one or more non-A nucleotides. In some embodiments, 1 / 2 of the poly(A) tail coding sequence near the 3' terminus comprises one or more non-A nucleotides. In some embodiments, 1 / 3 of the poly(A) tail coding sequence near the 3' terminus comprises one or more non-A nucleotides. In some embodiments, 1 / 4 of the poly(A) tail coding sequence near the 3' terminus comprises one or more non-A nucleotides.

[0028] In some embodiments, the structure of the poly(A) tail coding sequence is: Petition 870250073560, dated 08 / 20 / 2025, p. 21 / 89 / 61 element a-element c-element b-element c-element b-element c-element b; element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b; element b-element c-element b-element c-element b-element d-element a-element c; element a-element d-element b-element c-element b; or element b-element c-element b-element c-element d-element a.

[0029] In some embodiments, the structure of the poly(A) tail coding sequence is: element a-element c-element b-element c-element b-element c-element b-element c-element b; specifically, 60A-G-19AG-19A-G-19A-G-3A.

[0030] In some embodiments, the structure of the poly(A) tail coding sequence is: 7A-C-18A-G-60A-GG-7A-C-18A-G-14A, 19A-G-19A-G-19A-element d-60A-G, 60A-element d-19A-G-19A-G-17A, 19A-G-19A-G-19A-element d-60A, 19A-G-19A-G-19A-element d-60A, 19A-C-19A-C-19A-element d-60A, 19A-T-19A-T-19A-element d-60A, 19A-G-19A-G-19A-element d-60A or 19A-G-19A-G-19A-element d-60A; and given that the d element consists of 6 or 12 nucleotides.

[0031] In some embodiments, the poly tail (A) encoding sequence is represented by any of the SEQ ID NOs: 1 to 10. Petition 870250073560, dated 08 / 20 / 2025, p. 22 / 89 / 61

[0032] In some embodiments, the poly(A) tail encoding sequence is represented by SEQ ID NO: 3 or SEQ ID NO: 4.

[0033] In some embodiments, the manipulated DNA molecule is additionally connected to a fragment of the gene of interest on the 5' end of its poly(A) tail coding sequence, wherein the gene fragment of interest and the poly(A) tail coding sequence concomitantly encode RNA. In some embodiments, the manipulated DNA molecule is additionally connected to a fragment of the gene of interest on the 5' end of its poly(A) tail coding sequence, wherein the gene fragment of interest and the poly(A) tail coding sequence concomitantly encode mRNA. In some embodiments, the gene fragment of interest comprises a protein-coding sequence or a non-protein-coding sequence, such as a functional RNA-coding sequence.In some embodiments, the gene fragment of interest additionally comprises a 5' UTR coding sequence on the 5' end side of the protein coding sequence or the functional RNA coding sequence. In some embodiments, the gene fragment of interest additionally comprises a 3' UTR coding sequence on the 3' end side of the protein coding sequence or the functional RNA coding sequence. In some embodiments, the gene fragment of interest additionally comprises a 3' UTR coding sequence on the 3' end side of the protein coding sequence or the functional RNA coding sequence and additionally comprises a 5' UTR coding sequence on the 5' end side of the protein coding sequence or the functional RNA coding sequence.In some embodiments, the manipulated DNA molecule additionally comprises a replicon, for example, an origin of replication, such as an ORI. In some embodiments, the DNA molecule... Petition 870250073560, dated 08 / 20 / 2025, page 23 / 89 / 61, the manipulated DNA molecule additionally comprises a marker gene to facilitate screening of cells containing the manipulated DNA molecule, and the marker gene is selected from, for example, antibiotic resistance genes, fluorescent proteins, and the like. In some embodiments, the DNA molecule additionally comprises a promoter, which initiates RNA transcription encoded concomitantly by the gene fragment of interest and the poly(A) tail coding sequence. In some embodiments, the promoter is a prokaryotic promoter. In some embodiments, the promoter is a eukaryotic promoter. In some embodiments, the DNA molecule additionally comprises a replicon, for example, an origin of replication, a promoter, a 5' UTR coding sequence, a protein coding sequence, and a 3' UTR coding sequence.In some embodiments, the DNA molecule additionally comprises a replicon, for example, an origin of replication, a resistance gene, a 5' UTR coding sequence, a protein coding sequence, and a 3' UTR coding sequence. In some embodiments, the DNA molecule additionally comprises a replicon, for example, an origin of replication, a resistance gene, a promoter, a 5' UTR coding sequence, a protein coding sequence, and a 3' UTR coding sequence. In some embodiments, the protein coding sequence encodes a viral antigen protein of HPV. In some embodiments, the HPV protein is derived from HPV type 16 and / or type 18. In some embodiments, the protein coding sequence encodes the E2, E6, or E7 protein of HPV. In some embodiments, the protein coding sequence encodes a fusion protein of the E6 and E7 proteins of HPV.In some embodiments, the protein-coding sequence encodes a fusion protein of HPV E2, E6, and E7 proteins. In some embodiments, the polypeptide fragments of the fusion protein are derived from HPV type 16 and / or type 18. Petition 870250073560, dated 08 / 20 / 2025, page 24 / 89 / 61 In some embodiments, the polypeptide fragments of the fusion protein are derived from HPV type 16 and / or type 18 proteins E2, E6, and E7. In some embodiments, the protein coding sequence encodes a polypeptide represented by SEQ NO: 26 or a conservatively substituted variant thereof.

[0034] In some embodiments, the manipulated DNA molecule comprises a polynucleotide sequence represented by any of the SEQ ID NOs: 22 to 25 or a synonymous mutant thereof or a polynucleotide sequence that shares 85% or more sequence identity with the polynucleotide sequence represented by any of the SEQ ID NOs: 22 to 25 or a synonymous mutant thereof.

[0035] In some embodiments, the DNA molecule is a DNA plasmid. In some embodiments, the DNA molecule is a linear plasmid or a circular plasmid. In some embodiments, the DNA molecule is single-stranded or double-stranded. In some embodiments, the plasmid is a pUC, pTZ, pMB1, or pCoIE1-based plasmid. In some embodiments, the plasmid is a pUC57 vector-based plasmid.

[0036] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a recA bacterium. In some embodiments, the cell is Escherichia coli. In some embodiments, the E. coli is selected from the group consisting of K-12 and strains derived therefrom and strain B and strains derived therefrom. In some embodiments, the E. coli is selected from the group consisting of MG1655, DH5 or DH5a, DH10B, BL21, DB3.1, HB101, JM109, JM110, MC1061, MG1655, Pir1, Stbl2, Stbl3, Top10, XL1 Blue, XL10 Gold, BLR, HMS174, Tuner, Rostetta2, Lemo21, T7Express and Origami2.

[0037] The second aspect of the present application reveals a cell comprising the DNA molecule of the first aspect. In some Petition 870250073560, dated 08 / 20 / 2025, page 25 / 89 / 61 embodiments, the DNA molecule of the first aspect mentioned above can be replicated and / or transcribed in the cell. In some embodiments, the cell is a prokaryotic cell, and the DNA molecule of the first aspect mentioned above can be replicated in the prokaryotic cell. In some embodiments, the cell is a recA bacterium. In some embodiments, the cell is E. coli. In some embodiments, the prokaryotic cell is a competent cell. In some embodiments, the prokaryotic cell is a manipulated cell. In some embodiments, the prokaryotic cell is a manipulated prokaryotic cell. In some embodiments, the cell is E. coli, and the E. coli is selected from the group consisting of K-12 and derivatives thereof and strain B and derivatives thereof. In some strains, E. coli is selected from the group consisting of MG1655, DH5 or DH5a, DH10B, BL21, DB3.1, HB101, JM109, JM110, MC1061, MG1655, Pir1, Stbl2, Stbl3, Top10, XL1 Blue, XL10 Gold, BLR, HMS174, Tuner, Rostetta2, Lemo21, T7 Express, and Origami2. In some embodiments, the cell is a eukaryotic cell, and the DNA molecule of the first aspect mentioned above can be transcribed in the eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is selected from the group consisting of a yeast or a mold.

[0038] In a third aspect of the present application, a poly(A) tail is provided. The poly(A) tail is: (1) obtained by transcribing the DNA molecule manipulated in accordance with the first aspect; (2) obtained by chemical synthesis and has the same polynucleotide sequence as the poly(A) tail obtained by transcribing the DNA molecule manipulated in accordance with the first aspect; or (3) obtained by further modification of the poly(A) tail in item (1) or (2) above. Petition 870250073560, dated 08 / 20 / 2025, p. 26 / 89 / 61

[0039] In some embodiments, the additional modification comprises the substitution of one or more ribonucleotides in the poly(A) tail obtained by item (1) or (2) above by one or more deoxyribonucleotides. In some embodiments, one or more ribonucleotides are replaced by deoxyribonucleotides to which they correspond, for example, one or more A ribonucleotides of the poly(A) tail are replaced by A deoxyribonucleotides, one or more U ribonucleotides are replaced by T deoxyribonucleotides, one or more C ribonucleotides are replaced by C deoxyribonucleotides, one or more G ribonucleotides are replaced by G deoxyribonucleotides, or one or more G ribonucleotides are replaced by I (inosine) ribonucleotides or I deoxyribonucleotides. In some embodiments, the modification is a chemical modification. In some embodiments, the modification is base editing.In some embodiments, the modification is the deamination treatment of one or more ribonucleotides in the poly(A) tail obtained by item (1) or (2) above.

[0040] In some embodiments, the poly(A) tail comprises a polynucleotide sequence selected from any one of the SEQ ID NOs: 1 to 10. In some embodiments, the polynucleotide sequence of the poly(A) tail is represented by any one of the SEQ ID NOs: 1 to 10.

[0041] The present application also provides for the use of the aforementioned poly(A) tail. In some embodiments, the use of the poly(A) tail is provided to make an RNA molecule more stable, wherein the poly(A) tail is located at the 3' end of the RNA, and wherein the more stable means are more stable outside a cell, inside a cell or in an animal body compared to an RNA molecule comprising another poly(A) tail. In some embodiments, the use of the poly(A) tail is provided to reduce the stability of an RNA molecule, being Petition 870250073560, dated 20 / 08 / 2025, p. 27 / 89 / 61, states that the poly(A) tail is located at the 3' end of RNA, and reduced stability refers to reduced stability outside a cell, inside a cell, or in an animal body relative to an RNA molecule comprising another poly(A) tail. In some embodiments, use of the poly(A) tail is provided to increase the expression level of an RNA molecule in the same period, wherein increased expression level refers to an increased expression level outside a cell, inside a cell, or in an animal body relative to an RNA molecule comprising another poly(A) tail.In some embodiments, the use of the poly(A) tail is provided to reduce the expression level of an RNA molecule within the same time frame, wherein expression level reduction refers to a reduced expression level outside a cell, inside a cell, or in an animal body relative to an RNA molecule comprising another poly(A) tail. In some embodiments, the use of the poly(A) tail is provided to prolong the expression time of an RNA molecule, wherein expression time prolongation refers to a prolonged expression time outside a cell, inside a cell, or in an animal body relative to an RNA molecule comprising another poly(A) tail.In some embodiments, the use of the poly(A) tail is provided to shorten the expression time of an RNA molecule, wherein the shortening of the expression time refers to a shortened expression time outside a cell, inside a cell, or in an animal body relative to an RNA molecule comprising another poly(A) tail. In some embodiments, the use of the poly(A) tail is provided to extend the half-life of an RNA molecule, wherein the extension of the half-life refers to a prolonged half-life outside a cell, inside a cell, or in an animal body relative to an RNA molecule comprising another poly(A) tail. In some embodiments, the use of the poly(A) tail is provided to shorten the half-life. Petition 870250073560, dated 08 / 20 / 2025, page 28 / 89 / 61 of an RNA molecule, whereby the shortening of the half-life refers to a shortened half-life outside a cell, inside a cell or in an animal body in relation to an RNA molecule comprising another poly(A) tail.

[0042] In some embodiments, the RNA molecule is an mRNA molecule. In some embodiments, the poly(A) tail and the other poly(A) tail are two different poly(A) tails belonging to the poly(A) tail according to the third aspect of the present application. In some embodiments, the poly(A) tail is the poly(A) tail according to the third aspect of the present application, and the other poly(A) tail is a poly(A) tail different from the poly(A) tail according to the third aspect of the present application. In some embodiments, the interior of a cell means inside a host cell, and the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell.

[0043] The present application also provides a DNA fragment or a DNA-RNA hybrid molecule fragment to encode the poly(A) tail according to the third aspect of the present application, and an use of the DNA fragment or the DNA-RNA hybrid molecule fragment to make the replication of the DNA molecule or the DNA-RNA hybrid molecule to encode RNA more conservative in a host cell, and in said use, the DNA fragment or the DNA-RNA hybrid molecule fragment to encode the poly(A) tail according to the third aspect of the present application is situated on the 3' end side of the RNA coding sequence in the DNA molecule or the DNA-RNA hybrid molecule. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a recA bacterium. In some embodiments, the host cell is E. coli. In some embodiments, the E. coli is selected from the group consisting of K-12 Petition 870250073560, dated 08 / 20 / 2025, p. 29 / 89 / 61 and strains derived from the same and strain B and strains derived from the same. In some embodiments, E. coli is selected from the group consisting of MG1655, DH5 or DH5a, DH10B, BL21, DB3. 1, HB101, JM109, JM110, MC1061, MG1655, Pir1, Stbl2, Stbl3, Top10, XL1Blue, XL10Gold, BLR, HMS174, Tuner, Rostetta2, Lemo21, T7Express and Origami2.

[0044] The fourth aspect of the present application also provides an RNA molecule, and it comprises the poly(A) tail according to the third aspect. In some embodiments, the RNA molecule is an mRNA molecule. In some embodiments, the RNA molecule is: (1) obtained by transcribing the DNA molecule manipulated in accordance with the first aspect; (2) obtained by chemical synthesis and has the same polynucleotide sequence as the RNA molecule in item (1) above; or (3) obtained by further modification of the RNA molecule in item (1) or (2) above.

[0045] In some embodiments, the further modification comprises the substitution of one or more ribonucleotides in the RNA molecule obtained by (1) or (2) above by one or more deoxyribonucleotides. In some embodiments, the one or more ribonucleotides are substituted by deoxyribonucleotides to which they correspond, for example, one or more A ribonucleotides of the RNA molecule are substituted by A deoxyribonucleotides, one or more U ribonucleotides are substituted by T deoxyribonucleotides, one or more C ribonucleotides are substituted by C deoxyribonucleotides, one or more G ribonucleotides are substituted by G deoxyribonucleotides, or one or more G ribonucleotides are substituted by I (inosine) ribonucleotides or I deoxyribonucleotides. In some embodiments, the modification is a chemical modification. In some modes, the modification is the base edit. In some modes, the modification is the deamination treatment. Petition 870250073560, dated 20 / 08 / 2025, page 30 / 89 / 61 of one or more ribonucleotides in the RNA molecule obtained by (1) or (2) above. In some embodiments, the additional modification is a post-transcriptional modification. In some embodiments, the additional modification comprises capping treatment. In some embodiments, the additional modification comprises splicing. In some embodiments, the additional modification comprises capping and splicing treatment.

[0046] In some embodiments, the RNA molecule comprises coding RNA or non-coding RNA (ncRNA). In some embodiments, the RNA molecule is a pre-mRNA. In some embodiments, the RNA is mature mRNA. In some embodiments, the RNA molecule is a long non-coding RNA (lncRNA). In some embodiments, the RNA molecule additionally comprises a 5'-cap structure. In some embodiments, the polynucleotide sequence of the RNA molecule is represented by any of the SEQ ID NOs: 22 to 25. In some embodiments, the RNA molecule comprises a polynucleotide sequence represented by any of the SEQ ID NOs: 22 to 25.

[0047] In addition, the present application also provides a hybrid DNA and RNA molecule that carries the same genetic information as the DNA molecule manipulated according to the first aspect, the same genetic information as the poly(A) tail according to the third aspect, or the same genetic information as the RNA molecule according to the fourth aspect.

[0048] In addition, the present application also provides a library of nucleic acid molecules. In some embodiments, the nucleic acid molecule library comprises the DNA molecule manipulated according to the first aspect, the poly(A) tail according to the third aspect, the DNA fragment or the DNA-RNA hybrid molecule fragment to encode the poly(A) tail according to the third aspect of the present application, or the RNA molecule according to the fourth aspect. Petition 870250073560, dated 08 / 20 / 2025, p. 31 / 89 / 61

[0049] Furthermore, the present application also provides a method for regulating protein expression comprising introducing a plurality of nucleic acid molecules into the aforementioned nucleic acid molecule library into cells of interest at different times and / or at different quantity ratios. In some embodiments, the nucleic acid molecule is the DNA molecule manipulated according to the first aspect mentioned above. In some embodiments, the nucleic acid molecule is the RNA molecule according to the fourth aspect mentioned above.

[0050] It should be understood that the aspects and embodiments of this application described herein include the aspects and embodiments comprising, consisting of, and essentially consisting of the same. The preferred embodiments of this application are described in detail above; however, this application is not limited to these. Within the technical concept of this application, a variety of simple modifications may be made to the technical solutions of this application, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and belong to the scope of protection of this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 shows the replication stability of 10 poly (A)s in the present application in E. coli DH5a in Example 2; Figure 2 shows the base 10 poly(A)s deletion statistics in the present application in E. coli DH5a in Example 2; Figure 3 shows the replication stability of poly(A) P1, P2, P3, P4 and poly(A) controls C1 and C2 in E. coli DH5a under a plasmid system comprising HPV antigen sequences in Example 3; Petition 870250073560, dated 08 / 20 / 2025, page 32 / 89 / 61 Figure 4 shows the base deletion statistics of poly(A) P3, P4 and poly(A) controls C1 and C2 in E. coli DH5a under a plasmid system comprising an HPV antigen sequence in Example 3; Figure 5 shows a comparison of replication stability between poly(A) P1, P2, P3 and control poly(A) C1 under two temperature conditions of 30 °C and 37 °C in E. coli DH5a in a plasmid system comprising an HPV antigen sequence in Example 3; Figure 6 shows an example of a universal vector plasmid DNA profile in the Examples. Figure 7 shows the animal imaging results of luciferase expression levels with poly(A) P3, P4, P5, P8, P9 and control C2 in mice in Example 4; Figure 8 shows the quantitative results of fluorescence intensity after imaging animals of luciferase expression levels with poly(A) P3, P4, P5, P8, P9 and control C2 in mice in Example 4 (ns, no significant difference; ★★, significant difference, p<0.01). DETAILED DESCRIPTION

[0052] The present application provides, firstly, a method for stably amplifying a poly(A) tail transcription template DNA in vitro, so as to reduce the mutation frequency of the poly(A) tail transcription template sequence when the DNA is replicated in large quantities in a cell. Thus, a large quantity of RNA comprising a poly(A) tail with a defined sequence is obtained based on the DNA. Based on this, RNA with a poly(A) tail that is manipulated to have a specific function, such as mRNA, can be produced on a large scale through in vitro fermentation.

[0053] In addition, the present application also provides a DNA sample that Petition 870250073560, dated 20 / 08 / 2025, p. 33 / 89 / 61 comprises a poly(A) tail transcription model that can be stably amplified in vitro and an RNA transcribed from DNA. Furthermore, under the premise of satisfying stable in vitro amplification, the present application additionally provides a group of poly(A) tails with different regulatory effects on RNA stability and / or expression efficiency, an RNA comprising the poly(A) tail, a DNA comprising the poly(A) tail coding sequence, and a library consisting of the poly(A) tail, RNA, or DNA.

[0054] In addition, the present application also provides uses of the poly(A) tail, RNA, DNA and library mentioned above. Terms

[0055] As used herein, element a, element b, element c, element d, and element e are types of elements comprised in poly(A). Element a consists of a plurality of consecutive adenine (A) nucleotides, and the length range of element a is > 20 nt; element b consists of a plurality of consecutive A nucleotides, and the length range of element b is 3 nt < b < 20 nt; element c consists of a non-A nucleotide, and the nucleotide is selected from among T, C, and G nucleotides; The d element consists of any two or more consecutive nucleotides, and the nucleotides are selected from A, T, C, and G nucleotides, provided that the nucleotides at the 5' and 3' ends of the d element are not A nucleotides, and the d element does not comprise 3 or more consecutive A nucleotides, and the length range of the d element is 2 nt. <d < 20 nt.The element e consists of one or two consecutive A's, and the element e is located at the 3' end of the poly(A) tail encoding sequence and is adjacent to the element d or to the element c when it exists. When poly(A) comprises two or more elements b, elements c, and elements d, the sequences of each two elements b may be the same or different, and the sequences of each two elements c may be the same or different. Petition 870250073560, dated 20 / 08 / 2025, p. 34 / 89 / 61 different, and the sequences of each two elements d may be equal or different, provided that each meets the above definitions of elements a, b, c and d. In the present petition, element a, element b, element c, element d, element e, etc. in the poly(A) tail may be indicated by the term element.

[0056] As used herein, when describing the positional relationship of two or more elements as non-adjacent, this means that the two or more elements are not adjacent to each other. In other words, the two or more elements comprise at least one or more other nucleotides or bases different from the nucleotides of the two elements between each two elements.

[0057] As used herein, coding means i) a DNA sequence comprising genetic information that can be transcribed into an RNA molecule and / or ii) an RNA molecule comprising genetic information that can be translated into an amino acid sequence. As used herein, therefore, the coding sequence refers to a ribonucleotide (RNA) sequence or a fragment thereof in an mRNA precursor or mature mRNA that can be translated into a protein and also refers to a complementary sequence or a fragment thereof of a deoxyribonucleotide (DNA) sequence that serves as a template for transcribing the mRNA precursor or mature mRNA. In addition, the coding sequence of the present application may further comprise polynucleotide sequences encoding proteins, functional nucleic acids or fragments thereof, such as miRNA, shRNA, dsRNA, guide RNA, poly(A) tail, 5' UTR, 3' UTR, etc.Among them, a DNA molecule that comprises genetic information that can be transcribed into an RNA molecule is called the coding nucleic acid of the RNA molecule; and an RNA molecule that comprises genetic information that can be translated into an RNA molecule. Petition 870250073560, dated 08 / 20 / 2025, page 35 / 89 / 61: the amino acid sequence is called the nucleic acid encoding the amino acid sequence.

[0058] In the present application, nucleotides in all polynucleotide sequences are numbered from the 5' end to the 3' end, that is, the nucleotide at the 5' end is the first nucleotide, and the nucleotide at the 3' end is the last nucleotide. Unless otherwise specified, 5' end and 5' end may be used interchangeably; 3' end and 3' end may be used interchangeably. 5' end and 3' end focus on describing the relative positional relationship between nucleotides, between nucleotide sequence segments, or between nucleotides and nucleotide sequence segments in the same nucleic acid sequence; 5' end and 3' end are used to describe the positions of the first and last nucleotides of a nucleic acid sequence or a segment of a nucleic acid sequence, respectively.The 5' end side is used to describe the relative positional relationship between two non-overlapping sequences in the same polynucleotide sequence; describing a sequence located on the 5' end side of another sequence means that the sequence is closer to the 5' end of the polynucleotide sequence compared to the other sequence. Similarly, describing a sequence as located on the 3' end side of another sequence means that the sequence is closer to the 3' end of the polynucleotide sequence compared to the other sequence, provided that the sequence and the other sequence do not comprise any overlapping parts.Specifically, for example, the poly(A) tail DNA coding sequence being located at the 3' end of the RNA coding sequence means that the poly(A) tail DNA coding sequence, as a component of the RNA coding sequence, comprises the nucleotides at the 3' end of the sequence. Petition 870250073560, dated 08 / 20 / 2025, page 36 / 89 / 61, coding RNA. Furthermore, as used herein, the 5' portion refers to that near the midpoint of the 5' end of the polynucleotide sequence, delimited by the central position of the polynucleotide sequence. The 3' portion refers to that near the midpoint of the 3' end of the polynucleotide sequence, delimited by the central position of the polynucleotide sequence. The number of nucleotides from the central position, as described in this application, to the 5' end is equal to the number of nucleotides from the central position, as described in this application, to the 3' end.

[0059] As used herein, when referring to the replication of a nucleic acid molecule, the term conservative means a low probability of mutation during the replication process. In this context, conservative is a relative concept. For example, when describing that the poly(A) tail DNA coding sequence is used to make the replication of the RNA-coding DNA molecule more conservative in the host cell, this refers to the probability that the original RNA-coding DNA molecule will replicate into a progeny DNA molecule.If the RNA molecule comprises the Poly(A) tail, the offspring DNA molecule has a higher probability of 100% sequence identity with the original DNA molecule compared to the coding DNA of an RNA molecule that does not comprise the Poly(A) tail (such as an RNA molecule that comprises some other Poly(A) tail); the average sequence identity between multiple offspring DNA molecules obtained by replicating the original DNA molecule and the original DNA molecule is greater.

[0060] In the present application, when describing regulating the expression of an RNA molecule, regulation means increasing or decreasing the total amount of functional protein or RNA expressed by the RNA molecule within the same time period; or allowing the RNA to express the functional protein or RNA within a longer or shorter time period, and the Petition 870250073560, dated 20 / 08 / 2025, p. 37 / 89 / 61 The increased or decreased, longer or shorter time period is compared with another RNA molecule expressing the same protein or functional RNA. When describing the regulation of protein expression, this means regulating the expression of RNA molecules comprising the protein-coding sequence. The regulatory effect described herein can be achieved by connecting the poly(A) tail of the present application to the 3' terminus of an RNA molecule that does not comprise a poly(A) tail or by replacing the original poly(A) tail of the RNA with the poly(A) tail of the present application.

[0061] As used herein, the percentage of identity, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% identity, refers to the degree of similarity between amino acid sequences or between nucleotide sequences, as determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. For example, after two sequences have identical residues in as many positions as possible through the introduction of gaps, the ratio between the number of positions occupied by identical bases or amino acid residues and the total number of positions is determined. The percentage of identity can be determined using software programs known in the art. Preferably, the alignment is performed using standard parameters. A preferred alignment program is BLAST. Other preferred programs are BLASTN and BLASTP.Details of these programs can be found at the following: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0062] As used herein, nucleic acid complementarity refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity refers to the percentage of residues in a nucleic acid molecule that can form hydrogen bonds. Petition 870250073560, dated 20 / 08 / 2025, p. 38 / 89 / 61 hydrogen (i.e., Watson-Crick base pairing) with another nucleic acid molecule (for example, approximately 5, 6, 7, 8, 9, 10 out of 10 correspond to approximately 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). Completely complementary means that all consecutive residues of one nucleic acid sequence will form a hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, substantially complementary refers to a degree of complementarity of any one of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% across a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.For a single base or a single nucleotide, according to the Watson-Crick base-pairing principle, when A is paired with T or U, and C is paired with G or I, this is called complementary or correlational, and vice versa; any other base pairing is called non-complementary. In the present application, the complementary polynucleotide sequence of a given polynucleotide sequence refers to a polynucleotide sequence that is completely complementary to the given polynucleotide sequence.

[0063] As used herein, a conservative substitution variant of a protein, polypeptide, or amino acid sequence refers to one in which one or more amino acid residues undergo amino acid substitution without altering the overall conformation and function of the protein or enzyme, including, but not limited to, substitution of amino acids in the amino acid sequence of the original protein in the manner described by the aforementioned conservative substitution. Therefore, the similarity between two proteins or amino acid sequences with similar functions may differ. For example, 70% to 99% similarity Petition 870250073560, dated 20 / 08 / 2025, p. 39 / 89 / 61 (identity) based on the MEGALIGN algorithm. Conservative substitution variants also comprise polypeptides or enzymes with 60% or more amino acid identity as determined by the BLAST or FASTA algorithms, preferably 75% or more, more preferably 85% or more, and most preferably even 90% or more, and have properties or functions equal to or substantially similar to those of the native or original protein or enzyme.

[0064] In the context of this application, the terms DNA and RNA refer to single-stranded or double-stranded DNA or RNA molecules. Unless otherwise indicated, the terms DNA and DNA molecule refer to double-stranded DNA molecules composed of A, C, G, and / or T nucleotides, and the terms RNA and RNA molecule refer to single-stranded RNA molecules composed of A, C, G, and / or U nucleotides. In the present invention, the nucleotides A, C, G, T, and U refer to nucleotides comprising adenine, guanine, cytosine, thymine, and uracil as respective nitrogenous bases.

[0065] RNA molecules comprise coding RNA or non-coding RNA (ncRNA), such as pre-mRNA, mature mRNA, or long non-coding RNA (lncRNA).

[0066] As used herein, the DNA-RNA hybrid molecule is a molecule comprising a polynucleotide sequence consisting of deoxyribonucleotides and ribonucleotides. The DNA-RNA hybrid molecule can be obtained by the following method: to replace one or more deoxyribonucleotides in DNA with ribonucleotides; replace one or more ribonucleotides in RNA with deoxyribonucleotides; or synthesize de novo using deoxyribonucleotides and ribonucleotides as raw materials through biological synthesis or Petition 870250073560, dated 08 / 20 / 2025, page 40 / 89 / 61 chemistry. It should be noted that the method of obtaining a DNA-RNA hybrid molecule is not limited to the method above, and a DNA-RNA hybrid molecule obtained by any method belongs to the category of a DNA-RNA hybrid molecule defined in this application.

[0067] As used herein, when describing two nucleic acid molecules as having the same genetic information, this means that the two nucleic acid molecules are complementary or comprise exactly the same base sequence, or a nucleic acid molecule with exactly the same base sequence as another nucleic acid molecule can be obtained by converting one or more thymines in the base sequence of a nucleic acid molecule to uracil. Therefore, any two of DNA, RNA, and a hybrid DNA-RNA molecule can have the same genetic information. In the present invention, the term base sequence refers to the order of arrangement of bases in a polynucleotide molecule.Unless otherwise specified, those skilled in the art should understand that thymine may be indicated by T when the base sequence or polynucleotide sequence described in the present application is used to describe a DNA sequence, while T will be replaced by U (uracil) when the base sequence or polynucleotide sequence is used to describe RNA (such as mRNA). Therefore, whenever a DNA is revealed by a specific sequence number (SEQ ID NO) in the present invention, a complementary or corresponding RNA sequence (e.g., mRNA or poly(A) tail) is also revealed, each T in the DNA sequence being replaced by a U. Poly(A) tail and its uses

[0068] As used herein, the term Poly A tail or Poly(A) sequence refers to an uninterrupted or unbroken sequence of adenylate residues typically located at the 3' end. Petition 870250073560, dated 20 / 08 / 2025, p. 41 / 89 / 61 of an RNA molecule. In RNA, in the presence of a 3'-UTR, the Poly-A sequence is attached to the 3' end of the 3'-UTR. An uninterrupted Poly-A tail is characterized by consecutive adenylate residues. The poly-A tail can be of any length. In some embodiments, the Poly-A tail comprises, or consists of, at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 adenylate (A) nucleotides, in particular about 120 A. Typically, the vast majority of nucleotides in the Poly-A tail are adenosines, with the vast majority referring to at least 75%, at least 80%, at least 85%, at least 90% of the nucleotides, etc., but the remaining nucleotides may be nucleotides other than A (non-A nucleotides), such as U (uridylic acid), G (guanylic acid), or C (cytidylic acid).

[0069] In some embodiments, the in vitro RNA preparation process is a prokaryotic fermentation process, that is, the coding nucleic acid of the RNA molecule comprising the poly(A) tail is introduced into prokaryotic cells, and the prokaryotic cells are amplified to achieve the purpose of amplifying the coding nucleic acid, and then the amplified coding nucleic acid is transcribed into RNA. In some embodiments, the in vitro RNA preparation process is to connect an RNA fragment comprising a protein-coding sequence to a poly(A) tail by homologous recombination, digestion and enzymatic ligation or other non-homologous recombination methods, and the poly(A) tail is prepared by a prokaryotic fermentation process.During the process of prokaryotic fermentation, the coding nucleic acid comprising the poly(A) tail is introduced into prokaryotic cells, and the prokaryotic cells are amplified to achieve the purpose of amplifying the coding nucleic acid; subsequently, the amplified coding nucleic acid is transcribed into an RNA that... Petition 870250073560, dated 08 / 20 / 2025, pp. 42 / 89 / 61, includes the poly(A) tail. In some embodiments, the aforementioned coding nucleic acid is linear. In some embodiments, the aforementioned coding nucleic acid is circular. In some embodiments, the aforementioned coding nucleic acid is a plasmid. In some embodiments, the aforementioned coding nucleic acid is single-stranded or double-stranded. In some embodiments, the aforementioned coding nucleic acid is chemically modified before being introduced into a prokaryotic cell. In some embodiments, the aforementioned coding nucleic acid is chemically synthesized before being introduced into a prokaryotic cell. In some embodiments, the coding nucleic acid is inserted into the nucleoid / karyoid genomic DNA of the prokaryotic cell.In some embodiments, the coding nucleic acid is free in the cytoplasm or outside the nucleoid / karyoid of the prokaryotic cell. In some embodiments, the prokaryotic cell is E. coli.

[0070] Based on this, the present application provides a series of poly(A) tails, which are highly conserved during in vitro RNA preparation. The poly(A) tail comprises one or more non-A nucleotides at one or more positions.

[0071] In some embodiments, the poly(A) tail coding sequence comprises: a single element ae at least one element be at least one element c; a single element ae, at least one element bbe, and at least one element d; or a single element ae, at least one element bbe, and at least one element cce, and at least one element d, where element ae and element b are not adjacent, element cce and element d are not adjacent, and Petition 870250073560, dated 20 / 08 / 2025, p. 43 / 89 / 61 the poly tail (A) encoding sequence does not comprise any two b elements adjacent to each other, does not comprise any two c elements adjacent to each other and does not comprise any two d elements adjacent to each other.

[0072] In some embodiments, the poly(A) tail coding sequence additionally comprises a single element e, wherein the element e consists of one or two consecutive A's, which are situated at the 3' termination of the poly(A) tail coding sequence and are in a position adjacent to the element d or the element c.

[0073] The poly(A) tail in the present invention may be an RNA segment or a hybrid molecule of DNA and RNA.

[0074] The present application also provides a poly(A) tail that can regulate the level of protein expression. Furthermore, the present application also provides a poly(A) tail that can regulate the level of protein expression while remaining highly conservative during the in vitro preparation process. In some embodiments, the poly(A) tail that can regulate the level of protein expression; or the poly(A) tail that can regulate the level of protein expression while remaining highly conservative during the in vitro preparation process, is selected from the group consisting of: element a-element c-element b-element c-element b-element c-element b-element c-element b; element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b; element b-element c-element b-element c-element b-element d-element a-element c; element a-element d-element b-element c-element b; and element b-element c-element b-element c-element b Petition 870250073560, dated 08 / 20 / 2025, p. 44 / 89 / 61 element d-element a.

[0075] In some embodiments, the poly(A) tail that can regulate the level of protein expression; or the poly(A) tail that can regulate the level of protein expression while remaining highly conservative during the in vitro preparation process, is selected from the group consisting of: 60A-G-19A-G-19A-G-19A-G-3A; 7A-C-18A-G-60A-element d-7A-C-18A-G-14A; 60A-element d-19A-G-19A-G-17A; 19A-G-19A-G-19A-element d-60A; 19A-G-19A-G-19A-element d-60A-G; 19A-G-19A-G-19A-element d-60A; 19A-C-19A-C-19A-element d-60A; and 19A-T-19A-T-19A-element d-60A.

[0076] In some embodiments, the poly(A) tail that can regulate the level of protein expression; or the poly(A) tail that can regulate the level of protein expression while remaining highly conservative during the in vitro preparation process, is: 60A-element d19A-G-19A-G-17A or 19A-G-19A-G-19A-element d-60A.

[0077] Specifically, the two elements connected by - are directly connected and there is no nucleotide between the two elements.

[0078] In the previously mentioned poly(A) tail structure, yA represents the number of consecutive A's in element a or element b, where y is a natural number, for example, 19A means that it comprises 19 consecutive A's; 60A means that it comprises 60 consecutive A's.

[0079] In some embodiments, the poly(A) tail can regulate the level of protein expression; or the poly(A) tail can regulate the level of protein expression while remaining highly Petition 870250073560, dated 08 / 20 / 2025, page 45 / 89 / 61 conservative during the in vitro preparation process, is selected from any of the polynucleotide sequences represented by SEQ ID NOs: 1 to 10.

[0080] In addition, the present application also provides the use of the aforementioned poly(A) tail to regulate protein expression, wherein, in said use, the poly(A) tail is located at the 3' end of the mRNA, as the 3' end of the 3' UTR. In some embodiments, regulatory protein expression uses the method to regulate protein expression described below. Manipulated DNA libraries and molecules

[0081] The present application also provides a manipulated DNA molecule that can replicate in a cell, comprising the aforementioned Poly A tail coding sequence or a complementary sequence thereof. Those skilled in the art should understand that, in addition to the Poly A tail coding sequence, the manipulated DNA molecule must also comprise structural elements necessary for the DNA molecule to replicate or replicate efficiently in a cell. The structural elements necessary for the DNA molecule to replicate or replicate efficiently in a cell are known in the art and comprise, for example, an origin of replication (ORI).In some embodiments, the manipulated DNA molecule additionally comprises a marker gene or a fragment thereof, and / or a reporter gene or a fragment thereof, and a single restriction endonuclease site that allows the insertion of DNA elements, preferably a restriction endonuclease site that functions as a multiple cloning site (MCS). The marker gene facilitates the identification of a cell containing a plasmid comprising the marker gene, which can be selected from, for example, an antibiotic resistance gene. Each restriction endonuclease site in the MCS can be... Petition 870250073560, dated 20 / 08 / 2025, page 46 / 89 / 61 specifically recognized by a different restriction endonuclease.

[0082] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term DNA plasmid refers to a plasmid consisting of a double-stranded DNA molecule. In some embodiments, the plasmid is a circular DNA molecule. In some embodiments, the plasmid may also encompass a linear DNA molecule. Specifically, the term plasmid also encompasses molecules obtained by linearizing a circular plasmid, for example, by cleaving a circular plasmid with a restriction endonuclease, thus converting the circular plasmid molecule into a linear molecule, as well as a replicable linear molecule in a prokaryote.A plasmid can be replicated, that is, it can be amplified in a cell independently of the genomic genetic information stored in a nucleoid or karyoid of a prokaryotic cell and can be used for cloning, that is, to amplify genetic information in a bacterial cell. Preferably, the DNA plasmid according to the present application is a medium-copy-number or high-copy-number plasmid, more preferably a high-copy-number plasmid. Examples of such high-copy-number plasmids are vectors based on pUC, pTZ, or any other plasmid comprising an ORI supporting the high copy number of the plasmid (e.g., pMB1, pCoIE1, etc.).

[0083] In some embodiments, the DNA molecule is a DNA molecule or a fragment thereof that constitutes a nucleoid or a karyoid of a prokaryotic organism, that is, the coding sequence comprising the previously mentioned poly(A) tail or a complementary sequence thereof may be replicated along with the prokaryotic genome.

[0084] In some forms, the DNA molecule is Petition 870250073560, dated 20 / 08 / 2025, pp. 47 / 89 / 61 additionally connected to a fragment of the gene of interest on the 5' end of the poly(A) tail coding sequence, and the fragment of the gene of interest and the poly(A) tail coding sequence concomitantly encode RNA. In some embodiments, the fragment of the gene of interest and the poly(A) tail coding sequence concomitantly encode mRNA. The fragment of the gene of interest comprises a coding sequence for a protein, a polypeptide, or a fragment thereof. In some embodiments, the fragment of the gene of interest also comprises a coding sequence for an element that can be used to initiate or regulate the expression of the protein, polypeptide, or a fragment thereof after transcription, and the elements include, but are not limited to, 5' UTR, 3' UTR, etc.In some embodiments, the gene fragment of interest comprises a coding sequence for at least one untranslated region (UTR). In some embodiments, the gene fragment of interest comprises at least the coding sequence of the 5' UTR and the coding sequence of the protein, polypeptide, or a fragment thereof. In some embodiments, the gene fragment of interest comprises, sequentially from 5' to 3', at least: the coding sequence of the 5' UTR, the coding sequence of the protein, polypeptide, or a fragment thereof, and the coding sequence of the 3' UTR. The coding sequence of the protein, polypeptide, or a fragment thereof may be ultimately translated into one or more proteins, or one or more polypeptides, for example, short peptides, oligopeptides, polypeptides, fusion proteins, proteins, and fragments thereof, as parts of known proteins, as functional parts.The functional portion can be, for example, a biologically active portion of a protein, or an antigenic portion that can effectively generate antibodies, such as an antigenic epitope. The two ends of the protein's coding sequence... Petition 870250073560, dated 08 / 20 / 2025, p. 48 / 89 / 61 A polypeptide or a fragment thereof comprises, respectively, a start codon (5' end) and a stop codon (3' end), which are, respectively, the first three nucleotides and the last three nucleotides of the mRNA molecule that can be translated. The 5' UTR usually comprises at least one ribosome binding site (RBS), such as the Shine-Dalgarno sequence in a prokaryote, or at least one translation initiation site, such as the Kozak sequence in a eukaryote. The RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes after translation initiation. The activity can be optimized by varying the length and sequence of a given RBS or translation initiation site, as well as the distance from that RBS or translation initiation site to the start codon.Alternatively or optionally, the 5' UTR comprises an internal ribosome entry site or an IRES. The 3' UTR may comprise one or more regulatory sequences, for example, binding sites for amino acid sequences that increase the stability of the mRNA molecule, binding sites for RNA regulatory molecules (such as miRNA molecules), and / or signal sequences involved in the intracellular transport of mRNA molecules.

[0085] Based on the previous embodiments, in some embodiments, the gene fragment of interest additionally comprises one or more additional regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of mRNA molecules, binding sites for amino acid sequences that enhance the translation of mRNA molecules, regulatory elements (such as riboswitches), binding sites for regulatory RNA molecules (such as miRNA molecules), and / or nucleotide sequences that positively affect translation initiation. Furthermore, preferably within the 5'UTR, there is no functional upstream open reading frame. Petition 870250073560, dated 20 / 08 / 2025, p. 49 / 89 / 61 upstream translation initiation site out of frame, upstream start codon out of frame and / or nucleotide sequences that produce secondary structures that reduce or prevent translation. The presence of such nucleotide sequences in the 5' UTR may adversely affect translation.

[0086] The coding sequence of a protein, polypeptide, or fragment thereof comprises codons that can be translated into an amino acid sequence. All codons included in the coding sequence may be naturally occurring codons that encode amino acids or may be partially or entirely composed of artificially synthesized codons. In some embodiments, some or all codons are subject to codon optimization. In some embodiments, some or all codons encode non-natural amino acids.

[0087] In some embodiments, the DNA molecule additionally comprises structural elements necessary to initiate or regulate RNA transcription on the 5' end of the gene fragment of interest, and the structural elements are known in the art. In some embodiments, the structural element c comprises at least one promoter. Promoters and their sequences are known in the art, including weak promoters, medium-strength promoters, strong promoters, minipromoters, or core promoters, etc. In some specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter can initiate transcription of the gene fragment of interest and / or the poly(A) tail in a prokaryotic cell. In some embodiments, the promoter can initiate transcription of the gene fragment of interest and / or the poly(A) tail in a eukaryotic cell.The promoter comprises at least one transcription recognition site followed by a transcription factor binding site. The recognition and binding sites may interact with an amino acid sequence that mediates or regulates the... Petition 870250073560, dated 20 / 08 / 2025, p. 50 / 89 / 61 transcription. Compared to the recognition site, the binding site is closer to the gene fragment of interest mentioned earlier. The binding site can be, for example, a Pribnow box in a prokaryote or a TATA box in a eukaryote. For example, in some embodiments, when the Pribnow box is used, the transcription recognition site may be located approximately 35 bp upstream of the transcription start site, while the transcription factor binding site may be located approximately 10 bp upstream of the transcription start site.In some embodiments, the promoter comprises at least one additional regulatory element, for example, an upstream AT-rich element located approximately 40 and / or 60 nucleotides before the transcription start site and / or an additional regulatory element to increase promoter activity located between the recognition site and the binding site. In some embodiments, the promoter is a strong promoter, that is, the promoter comprises a sequence to promote transcription of the aforementioned RNA coding sequence. Strong promoters are known to those skilled in the art, such as the OXB18, OXB19, and OXB20 promoters derived from the RecA promoter of E. coli, or they can be identified or synthesized by routine laboratory procedures. In some embodiments, the promoter is a T7 promoter.In some embodiments, the promoter additionally comprises extra regulatory elements, such as an enhancer contained within the DNA plasmid that can promote transcription of the aforementioned RNA coding sequence.

[0088] This application also provides a library comprising the aforementioned manipulated DNA molecules. In some embodiments, the library comprises at least two DNA molecules with different poly(A) tail coding sequences. Petition 870250073560, dated 08 / 20 / 2025, pp. 51 / 89 / 61

[0089] In addition, the present application also provides the use of the aforementioned manipulated DNA molecule to stably amplify the poly(A) tail coding sequence or the RNA coding sequence with a poly(A) tail. In some embodiments, the method for amplifying the poly(A) tail coding sequence or the RNA coding sequence with a poly(A) tail is as described below in the method for stably amplifying the poly(A) tail transcription template DNA in vitro. Libraries and manipulated RNAs

[0090] The present application provides an RNA comprising the aforementioned poly(A) tail and a fragment of the gene of interest on the 5' end of the poly(A) tail coding sequence. In some embodiments, the RNA additionally comprises a 5' cap structure. In some embodiments, the RNA is mRNA.

[0091] As used herein, mRNA (messenger RNA) is any RNA, naturally occurring, unnaturally occurring, or modified, that encodes at least one protein, polypeptide, or fragment thereof, that is enabled to be translated to produce the encoded protein, polypeptide, or fragment thereof in vitro, in vivo, in situ, or ex vivo. Therefore, mRNA can be mature mRNA or premature mRNA, and the elements or structures that the mRNA must or optionally comprise are known in the art. In some embodiments, the mRNA comprises coding sequences for multiple functional elements necessary to express, regulate, or enhance the expression level of the protein, polypeptide, or fragment thereof. Functional elements include, but are not limited to, 5' cap, 5' UTR, 3' UTR, etc. Both the 5' UTR and the 3' UTR are generally transcribed from genomic DNA, which are the elements present in premature mRNA.As mature mRNA, Petition 870250073560, dated 08 / 20 / 2025, pp. 52 / 89 / 61

[0092] The term 5' cap refers to the 5' end of mRNA, which comprises a methylated guanylate that is linked to the 5' end of the mRNA via pyrophosphate to form a 5',5'-triphosphate linkage with its adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), which are called type O, type I, and type II, respectively. Type O means that the ribose of the terminal nucleotide is not methylated, type I means that the ribose of one terminal nucleotide is methylated, and type II means that the ribose of both terminal nucleotides is methylated.In some embodiments, regarding the 5' cap, according to the manufacturer's protocol, a 5'-guanosine cap structure can be produced by completing the 5' capping of a polynucleotide during an in vitro transcription reaction using the following RNA cap chemical analogs: 3'-O-Mem7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA) or m7G(5')ppp(5')(2'-OMeA)pG (CleanCapAG). For example, in some embodiments, 5' capping of the modified RNA can be performed after transcription using the vaccinia virus capping enzyme to produce an O-type cap structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). I-type cap structures can be generated using either the vaccinia virus capping enzyme or 2'-O methyltransferase to produce m7G(5')ppp(5')(2'-OMeA)pG.Type II cap structures can be generated from type I cap structures by subsequent 2'-O-methylation of the third nucleotide from the 5' end using a 2'-O-methyltransferase. Type III cap structures can be generated from type II cap structures by subsequent 2'-O-methylation of the fourth nucleotide from the 5' end using a 2'-O-methyltransferase.

[0093] In some embodiments, some or all of the uridines in the mRNA are chemically modified uridines. Petition 870250073560, dated 08 / 20 / 2025, pp. 53 / 89 / 61

[0094] In some embodiments, some or all of the uridines in the mRNA are pseudouridines or 1-methyl-pseudouridines.

[0095] In some embodiments, some or all of the uracil nucleotides in mRNA are replaced by pseudouridine (ψ) nucleotides or by N1-methyl pseudouridine (m1ψ) nucleotides.

[0096] In some embodiments, the mRNA additionally comprises a stabilizing element. Stabilizing elements may comprise, for example, histone stem-loop structures. In some embodiments, the mRNA comprises a coding region, at least one histone stem-loop structure, and optionally a poly(A) sequence or a polyadenylation signal. The poly(A) sequence or the polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the mRNA comprises a combination of a poly(A) sequence or a polyadenylation signal and at least one histone stem-loop structure; although both have alternative mechanisms in nature, they act synergistically to increase protein expression to a level beyond that observed with each element alone.The synergistic effect of a combination of poly(A) and at least one histone stem-loop structure is independent of the order of the elements or the length of the poly(A) sequence. In some embodiments, the histone stem-loop structure is usually derived from a histone gene and comprises a loop formed by intramolecular base pairing by two adjacent partial or complete reverse complementary sequences separated by a spacer region (composed of a short sequence). The unpaired loop region is typically unable to base pair with either of the stem-loop elements. The stability of the stem-loop structure generally depends on the length, number of non-alignments or overhangs, and base composition of the paired region. In some embodiments, base pairing may be produced. Petition 870250073560, dated 20 / 08 / 2025, p. 54 / 89 / 61 oscillating (non-Watson Crick base pairing). In some embodiments, said at least one histone stem-loop sequence comprises 15 to nucleotides in length.

[0097] In some embodiments, one or more AU-rich sequences can be removed from the mRNA. Such sequences are sometimes called AUREs, which are destabilizing sequences found in the 3' UTR. AUREs can be removed from the mRNA. Alternatively, AUREs can be retained in the mRNA.

[0098] In some embodiments, mRNA is formulated within a lipid nanoparticle (LNP lipid nanoparticle). In some embodiments, lipids are mixed with mRNA to form lipid nanoparticles. In some embodiments, RNA is formulated into lipid nanoparticles. In some embodiments, lipid nanoparticles are first formed as empty lipid nanoparticles and then combined or encapsulated with vaccine mRNA immediately before administration (e.g., within a few minutes to an hour).

[0099] Lipid nanoparticles generally comprise ionizable lipids, non-cationic lipids, sterols, and PEG lipid components and a target nucleic acid, such as the mRNA mentioned above. Lipid nanoparticles according to the present invention can be produced using components, compositions, and methods commonly known in the art; see, for example, the documents... PCT / US2016 / 068300, PCT / US2016 / 047406, PCT / US2016 / 014280, PCT / US2014 / 055394, PCT / US2017 / 037551, PCT / US2016000129, PCT / US2017 / 038426, PCT / US2016 / 52117, PCT / US2016 / 052352, PCT / US2015 / 027400, PCT / US2016 / 014280, PCT / US2014 / 027077, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575 and PCT / US2016 / 069491, all of which are incorporated herein by reference in their entirety.

[00100] This application also provides a library that Petition 870250073560, dated 20 / 08 / 2025, p. 55 / 89 / 61, includes the mRNA molecules mentioned above. The library includes at least two mRNA molecules with different poly(A) tails.

[00101] This application also provides uses of mRNA and mRNA library. At least two or more mRNA molecules with poly(A) tails with different gradients of influence on mRNA expression level can be used to regulate the expression level of the coding sequence of the protein, polypeptide or a fragment thereof mentioned above, for example, by adjusting the ratio of different mRNA molecules in the library comprising said two or more mRNA molecules or by introducing one or more of said two or more mRNA molecules with equal or different content at different times. Cells

[00102] This application also provides cells comprising the aforementioned manipulated DNA molecules, wherein the DNA molecules can be stored and / or amplified in the cells. In some embodiments, the cells are prokaryotic cells in which the DNA molecules can be replicated. In some embodiments, the cells are prokaryotic cells in which the DNA molecules can be replicated and / or transcribed. In some embodiments, the DNA molecules are eukaryotic cells in which the DNA molecules can be replicated. In some embodiments, the DNA molecules can be transcribed and / or replicated in the cells containing DNA.

[00103] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterium, an actinomycete, a cyanobacterium, a mycoplasma, a rickettsia, and a chlamydia. In some embodiments, the cell is selected from the group consisting of Bacillus subtilis, Lactobacillus, Acetobacter, Corynebacterium, Brevibacterium, Petition 870250073560, dated 08 / 20 / 2025, pp. 56 / 89 / 61 Arthrobacter, Pseudomonas, and Pediococcus. In some embodiments, the cell is a recA bacterium. In some embodiments, the cell is E. coli. In some embodiments, the cell is E. coli, which is selected from the group consisting of K-12 and derivatives thereof and strain B and derivatives thereof. In some embodiments, E. coli is selected from the group consisting of MG1655, DH5 or DH5a, DH10B, BL21, DB3.1, HB101, JM109, JM110, MC1061, MG1655, Pir1, Stbl2, Stbl3, Top10, XL1Blue, XL10Gold, BLR, HMS174, Tuner, Rostetta2, Lemo21, T7Express, and Origami2. In some embodiments, the cell is selected from the group consisting of Streptomyces, Micromonospora, and Nocardia. In some embodiments, the cell is a fungus. In some embodiments, the cell is selected from among a yeast or a mold. Methods

[00104] The present application provides a method for stably amplifying a poly(A) tail transcription template DNA in vitro, so as to reduce the mutation frequency of the poly(A) tail transcription template sequence when the DNA is replicated in large quantities in a cell. The method comprises: expanding cells containing the manipulated DNA molecules.

[00105] In some embodiments, prior to cell expansion, the method further comprises the introduction of the manipulated DNA molecule into the cells. In some embodiments, the introduction may involve chemical transformation or electrotransformation. In some embodiments, the introduction is a natural endocytic process of the manipulated DNA molecule performed by the cell.

[00106] In some embodiments, after cell expansion, the method further comprises extracting cellular DNA and synthesizing RNA by in vitro transcription. In some embodiments, after cell expansion, the method further comprises inducing RNA transcription. Petition 870250073560, dated 20 / 08 / 2025, pp. 57 / 89 / 61 in cells and then extract and isolate RNA from them. In some embodiments, the method further comprises extracting cellular DNA and transducing it into a second cell that can transcribe RNA. In some embodiments, transduction comprises administration to a human being, the administration being selected from the group consisting of intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intra-arterial (e.g., via the carotid artery), intramuscular, and intratumoral injection or infusion.

[00107] In addition, the present application also provides a method for regulating protein expression, wherein the method comprises: to introduce two or more of the previously mentioned manipulated DNA molecules into cells of interest at different times and / or in different quantitative ratios; or to introduce two or more of the previously mentioned RNA molecules into cells of interest at different times and / or in different quantitative ratios; whereby the two or more of the previously mentioned manipulated DNA molecules and the two or more of the previously mentioned RNA molecules have different poly(A) tails, and the poly(A) tails have different gradients of influence on the level of RNA expression.

[00108] In some embodiments, the present application also provides a method for regulating protein expression, wherein the method comprises introducing the aforementioned manipulated DNA molecule or the aforementioned RNA molecule into a cell of interest. In some embodiments, the coding sequence of the poly(A) tail encoded by the DNA and the poly(A) tail comprised in the RNA comprises a structure selected from the group consisting of: element a-element c-element b-element c-element b Petition 870250073560, dated 08 / 20 / 2025, p. 58 / 89 / 61 element c-element b-element c-element b; element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b; element b-element c-element b-element c-element b-element d-element a-element c; element a-element d-element b-element c-element b; or element b-element c-element b-element c-element d-element a.

[00109] In some embodiments, the coding sequence of the poly(A) tail encoded by DNA and the poly(A) tail comprised in RNA comprises a structure selected from the group consisting of: 60A-G-19A-G-19A-G-19A-G-3A; 7A-C-18A-G-60A-element d-7A-C-18A-G-14A; 60A-element d-19A-G-19A-G-17A; 19A-G-19A-G-19A-element d-60A; 19A-G-19A-G-19A-element d-60A-G; 19A-G-19A-G-19A-element d-60A; 19A-C-19A-C-19A-element d-60A; and 19A-T-19A-T-19A-element d-60A.

[00110] In some embodiments, the coding sequences of the poly(A) tail encoded by DNA and the poly(A) tail comprised in RNA comprise the following structure: 60A-element d-19A-G-19A-G-17A or 19A-G-19A-G-19A-element d-60A.

[00111] Specifically, the two elements connected by the - are directly connected and there is no nucleotide between the two elements.

[00112] In the previously mentioned poly(A) tail structure, yA represents the number of consecutive A's in element a or element b, and y is a natural number, for example, 19A means that it comprises 19 A's. Petition 870250073560, dated 08 / 20 / 2025, page 59 / 89 / 61 consecutive; 60A means that it comprises 60 consecutive A's.

[00113] In some embodiments, the coding sequences of the DNA-encoded poly(A) tail and the RNA-encoded poly(A) tail comprise or consist of any polynucleotide sequence selected from the polynucleotide sequences represented by SEQ ID NOs: 1 to 10.

[00114] It should be understood that the present application covers various aspects, modalities, and combinations of aspects and / or modalities described herein. The above description and the following examples are intended to illustrate, rather than limit, the scope of the present application. Other aspects, improvements, and modifications within the scope of the present application will be apparent to those skilled in the art. Therefore, those skilled in the art should recognize that the scope of the present application also includes such improvements and modifications to those aspects and modalities. Examples Example 1: Poly tail construction (A)

[00115] The poly(A) tails and the DNA sequences encoding the poly(A) tails shown in Table 1 below were constructed by conventional genetic engineering methods. Table 1. Project Specific Structure Sequence Length P1 60A-G-19A-G-19A-G-19A-G-AAA SEQ ID NO: 1 124 nt P2 7A-C-18A-G-60A-element d-7A-C-18A-G-14A SEQ ID NO: 2 130 nt P3 60A-element d-19A-G-19A-G-17A SEQ ID NO: 3 123 nt P4 19A-G-19A-G-19A-element d-60A SEQ ID NO: 4 125 nt P5 19A-G-19A-G-19A-element d-60A-G SEQ ID NO: 5 126 nt P6 19A-G-19A-G-19A-element d-60A SEQ ID NO: 6 125 nt P7 19A-G-19A-G-19A-element d-60A SEQ ID NO: 7 131 nt P8 19A-C-19A-C-19A-element d-60A SEQ ID NO: 8 125 nt P9 19A-T-19A-T-19A-element d-60A SEQ ID NO: 9 125 nt P10 19A-G-19A-G-19A-element d-60A SEQ ID NO: 10 125 nt C1 A60-element d-A60 SEQ ID NO: 11 130 nt C2 A30-element d-A70 SEQ ID NO: 12 110 nt C3 A60-element c-A60 SEQ ID NO: 13 121 nt C4 A60-element d-A60 SEQ ID NO: 14 126 nt Example 2: Testing the poly(A) function using the luciferase coding sequence as an example. Petition 870250073560, dated 08 / 20 / 2025, pp. 60 / 89 / 61 2.1 Testing the replication stability of DNA molecules encoding mRNA in prokaryotic cells

[00116] Using luciferase as the protein coding region, the stability of different poly(A) variants in E. coli and their effects on luciferase expression in cells were investigated. 1) Construction of a universal vector comprising the coding region of the luciferase protein.

[00117] The E. coli pUC57 cloning vector was used as the vector backbone in this universal vector, and a T7 promoter sequence (5'-TAATACGACTCACTATAAGG-3'), a 5' UTR, a luciferase protein, a 3' UTR, and a poly (dA:dT) polyadenylic acid chain were sequentially arranged between multiple cloning sites: Xba I restriction site and EcoR I restriction site. 2) The poly (dA:dT) polyadenylic acid chain in the universal vector has been replaced by P1-P10 from the present application and controls C1-C4 (A60-10 nt-A60 spacer (control C1), A30-10 nt-A70 spacer (control C2), A60-1 nt-A60 spacer (control C3) or A60-6 nt-A60 spacer (control C4). Among them, C1 and C2 are derived from a literature patent (US Patent No. 10717982B2).

[00118] All primers required for the construction of P1-P10 and control C1-C4 were synthesized, and double digestion was conducted by two restriction endonucleases to remove the poly(dA:dT) from the universal vector constructed in step 1); and then P1-P10 and C1-C4 were ligated to the vector from which the poly(dA:dT) was removed by T4 DNA ligase 1, thus completing the replacement of poly(dA:dT) in the universal vector. 3) Detection of the replication stability of different poly(A) variants in E. coli

[00119] The vector plasmid constructed in step 2) was confirmed as correct by sequencing and then transformed into E. coli DH5a. A Petition 870250073560, dated 08 / 20 / 2025, page 61 / 89 / 61 The transformed plate was cultured at 30 °C, then the plasmid extraction and sequencing were completed. After sequencing was complete, the stability and base deletion of different poly(A) variants were analyzed and calculated based on the sequencing results. Replication stability is expressed as the percentage of clones without any base changes, and the higher the percentage, the greater the plasmid's replication stability in E. coli.

[00120] The results are shown in Figures 1 and 2, and the specific experimental results are as follows: A total of 100 clones were tested for control C1, in which 15 clones had base deletions, representing 15%; and the number of correct clones was 85, representing 85%.

[00121] A total of 50 clones were tested for control C2, in which all clones were correct without any base alterations or deletions, with a correct clone percentage of 100%.

[00122] A total of 50 clones were tested for control C3, in which 9 clones had base deletions, representing 18%; and the number of correct clones was 41, representing 82%.

[00123] A total of 50 clones were tested for control C4, in which 14 clones had base deletions, representing 28%; and the number of correct clones was 36, representing 72%.

[00124] A total of 100 clones were tested for P1, in which 9 clones had base deletions, representing 9%; and the number of correct clones was 91, representing 91%.

[00125] A total of 100 clones were tested for P2, in which 12 clones had base deletions, representing 12%; the number of correct clones was 88, representing 88%.

[00126] A total of 62 clones were tested for P3, of which 5 clones had base deletions, representing 8%; and the number of correct clones Petition 870250073560, dated 08 / 20 / 2025, pages 62 / 89 / 61, was 57%, representing 92%.

[00127] A total of 50 clones were tested for P4, in which 3 clones had base deletions, representing 6%; and the number of correct clones was 47, representing 94%.

[00128] A total of 50 clones were tested for P5, in which 4 clones had base deletions, representing 8%; and the number of correct clones was 46, representing 92%.

[00129] A total of 50 clones were tested for P6, in which 5 clones had base deletions, representing 10%; and the number of correct clones was 45, representing 90%.

[00130] A total of 50 clones were tested for P7, in which 7 clones had base deletions, representing 14%, and the number of correct clones was 43, representing 86%.

[00131] A total of 50 clones were tested for P8, in which 3 clones had base deletions, representing 6%, and the number of correct clones was 47, representing 94%.

[00132] A total of 50 clones were tested for P9, in which 4 clones had base deletions, representing 8%, and the number of correct clones was 46, representing 92%.

[00133] A total of 50 clones were tested for P10, in which 5 clones had base deletions, representing 10%, and the number of correct clones was 45, representing 90%.

[00134] Taking into account the results of the correct clone ratio and the number of bases deleted, the poly(A) variant designed in this application is superior to or comparable to the state of the art in terms of replication stability in E. coli cells. In particular, the replication stability of the poly(A) variants P3, P4, and P8 is the highest. The replication stability of P3, P4, and P8 is equivalent to that of C2, with no statistically significant difference (p>0.05, χ2 test). Furthermore, the Petition 870250073560, dated 08 / 20 / 2025, page 63 / 89 / 61 replication stability of P3, P4 and P8 is better than that of the controls C1, C3 and C4, and the difference is statistically significant (p<0.05, χ2 test). Example 3: Poly(A) function test using the HPV antigen protein coding sequence as an example. 1) Construction of a plasmid with HPV as a protein-coding region

[00135] As described above in Example 1, vectors comprising luciferase-coding genes combined with different poly(A)s were constructed. Based on these vectors, the luciferase-coding gene was replaced by the HPV-coding gene using conventional molecular cloning methods; and the main elements were arranged in the order of T7 promoter sequence (5'TAATACGACTCACTATAAGG-3'), 5' UTR, HPV antigen protein-coding sequence, 3' UTR, and poly(A) coding sequence. 2) Performing small-scale bacterial culture to test the stability of four HPV poly(A) variants in E. coli.

[00136] The HPV vector plasmid comprising P1, P2, P3, and P4 constructed in step 1) was confirmed correct by sequencing and then transformed into E. coli DH5a. The transformed plates were cultured at 30 °C, completing the plasmid extraction and sequencing. After sequencing was complete, the stability and base deletion of different poly(A) variants were analyzed and calculated based on the sequencing results. Stability is expressed as the percentage of clones without any base changes, and the higher the percentage, the more stable it is.

[00137] The results showed (Figures 3 and 4) that when the gene of interest was replaced by the HPV antigen coding sequence, a total of 88 clones were tested for control C1, in which 47 clones had base deletions, representing 53%, and the number of correct clones was 41, representing 47%. Petition 870250073560, dated 08 / 20 / 2025, page. 64 / 89 / 61 A total of 50 clones were tested for control C2, in which 1 clone had a base deletion, representing 2%, and the number of correct clones was 49, representing 98%. A total of 101 clones were tested for P1, in which 14 clones had base deletions, representing 14%, and the number of correct clones was 87, representing 86%. A total of 100 clones were tested for P2, in which 12 clones had base deletions, representing 12%, and the number of correct clones was 88, representing 88%. A total of 70 clones were tested for P3, in which 4 clones had base deletions, representing 6%, and the number of correct clones was 66, representing 94%. A total of 50 clones were tested for In P4, 9 clones had a plurality of base deletions, representing 18%, and the number of correct clones was 41, representing 82%.

[00138] Considering the mutation rate and the average number of base deletions, when the luciferase protein-coding gene in Example 1 was replaced by the HPV antigen protein, the different poly(A) variants of the present application still maintain high replication stability; in particular, P3 and C2 have comparable stability, with no statistically significant difference (p>0.05, χ2 test), and, compared with new variants in other groups of the present application, cloning stability is ideal. The probability of large fragment deletion in C2 is 1 / 50=2%, while the probability of large fragment deletion in P3 is 1 / 70=1.4%. Since large poly(A) fragment deletion will affect the in vivo expression and efficacy of mRNA products, P3 is more in line with product requirements.The results above indicate that the poly(A) variants designed in the present application are universally applicable in examples involving different... Petition 870250073560, dated 08 / 20 / 2025, pp. 65 / 89 / 61 protein coding regions.

[00139] As described above, in Example 1 and Example 2, plates transformed with E. coli were cultured in a biochemical incubator at 30 °C overnight, and the resulting clones were sequenced to assess replication stability. Furthermore, the culture temperature of E. coli affects the DNA replication rate, which further affects replication stability. Regarding Example 3, after plates transformed with E. coli were cultured in a biochemical incubator at 37 °C, sequencing detections were also compared in the present application. The results show (Figure 5) that culture at 37 °C significantly increases the base deletion ratio of the C1 control, from 53% to 98% when cultured at 30 °C.Unlike the control, there is no significant difference in the mutation rates of P1, P2, and P3 under the two temperature conditions, indicating that the poly(A) variants designed in the present application still have high replication stability in the samples with respect to different E. coli culture temperature conditions, resulting in a truly universal application. 3) Detection of the stability of the three poly(A) variants in large-scale fermentation and in different generations.

[00140] The production of mRNA drugs must rely on large-scale fermentation to prepare sufficient template plasmids, and plasmid stability during fermentation (in this application, plasmid stability refers particularly to poly dA:dT stability) is crucial for the production of mRNA drugs with uniform quality. On the other hand, to meet the stability requirements of different production batches, it is necessary to establish a strain library comprising the plasmid of interest, including strain libraries from different generations, such as a primary library and a secondary library. Therefore, it is necessary to evaluate the plasmid stability in E. coli for different generations. In Petition 870250073560, dated 20 / 08 / 2025, p. 66 / 89 / 61, in response to the two problems above, the present application detected the stability of P1 and P3 between different generations of the fermentation process in Example 3. According to the sequencing results, 4 correct E. coli clones were selected for each poly(A) variant to perform the passage culture through fermentation, respectively. The results show that, in the 3rd, 5th, 7th and 9th inoculation passages, the plasmids of the four P1 and P3 clones remain stable, without base changes. Example 4: Detection of luciferase mRNA expression levels in mice

[00141] In eukaryotic cells, a poly(A) tail of a certain length is essential to protect the 3' end of mRNA, maintain mRNA stability, and promote protein expression. Affected by physiological or environmental factors in the body, the poly(A) gradually becomes shorter, thus initiating mRNA degradation. This application investigated the effects of different poly(A) variants on protein expression levels. In vivo expression assays were performed in mice to evaluate the effects of different poly(A) variants on luciferase activity. During the implementation of this application, luciferase mRNA-LNPs comprising control C2, as well as P3, P4, P5, P8, and P9 were injected intramuscularly into mice. Imaging of animals was performed 6 hours after injection, and fluorescence intensity was quantitatively measured to compare the effects of different poly(A)s on luciferase activity in vivo.The specific experimental process is as follows:

[00142] luciferase mRNA was synthesized by in vitro transcription, and linearized DNA was obtained by digestion with BspQ I type II restriction endonuclease. The 3' end of the linearized DNA was displayed as different poly(A): control C2, P3, P4, P5, P8 or P9. The linearized DNA was used as a template for in vitro transcription. A 100 pL reaction system no. Petition 870250073560, dated 08 / 20 / 2025, pp. 67 / 89 / 61, which reaction buffer 1X; 5 mM (final concentration) of ATP, CTP, N1MUTP, and GTP, respectively; 4 mM (final concentration) of CleanCap AG; and 5 pL of in vitro transcriptase were included. After thoroughly mixing the reaction mixture, the reaction was conducted at 37 °C for 3 h. The in vitro transcribed mRNA was collected by LiCl precipitation and finally dissolved in enzyme-free water.

[00143] Animal experiment: The luciferase mRNA synthesized in vitro was encapsulated in LNPs, and the resulting mRNA stock solution was dispersed in 20 mM acetic acid solution (pH 5.0) to obtain an RNA solution with an mRNA concentration of 200 pg / mL. The lipid mixture was prepared by mixing ionizable fat, cholesterol, DSPC, and DMG-PEG2000 with a molar ratio of ionizable fat:cholesterol:DSPC:DMG-PEG2000 = 50:38.5:10:1.5. The mRNA and lipid mixture were mixed by controlling the flow rates of the aqueous and oil phases through the mixture T, and the injection pump was started to mix the mRNA solution with the lipid mixture to form LNPs. Next, the solution was diluted 10 times with diluent, then concentrated by centrifugation in an ultrafiltration tube, followed by three replacement cycles.The solution obtained above was added with aqueous Tris solution to adjust the pH to 7.0–8.0 to obtain a solution of mRNA encapsulated in LNP, where LNP stands for lipid nanoparticle. The concentration and particle size of the LNP-encapsulated mRNA were determined using the Ribogreen RNA quantification kit (Invitrogen, R11490) and the Darwin ZetaSizer particle size analyzer, respectively. In the 4-component LNP, the molar ratio of each component is given as SM102:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5. After encapsulation, quality control was conducted on the LNP by measuring particle size, encapsulation efficiency, PDI, and other indicators. The results of the quality control... Petition 870250073560, dated 08 / 20 / 2025, pp. 68 / 89 / 61, quality data shows that the prepared LNPs meet the standards for particle size range of 50 nm to 150 nm, PDI <0.3 and encapsulation efficiency >90%, which could be used for subsequent experiments. The mRNA content in LNP was determined by the ribogreen method, and then the LNP was diluted to an mRNA content of 100 ng / pL.

[00144] BALB / c mice were randomly divided into groups according to body weight and administered after 2 to 3 days of adaptive feeding. Each of the controls C2, P3, P4, P5, P8, and P9 was injected into five mice, and each mouse received 100 pL (10 pg of mRNA) by intramuscular injection. In addition, five mice injected with PBS were used as a control group. Imaging of animals was performed 6 h after injection, and then fluorescence values ​​were calculated. The results show (Figures 7 to 8) that, compared to the control C2, the expression activity of P3 is significantly increased by 1.8 times, with a statistically significant difference (p<0.01, Student's t-test). The expression levels of P4, P5, P8, and P9 are comparable to those of C2, with no significant difference (p>0.05, Student's t-test).

[00145] The sequences used in the examples above in this application are shown in the sequence listing below. It should be understood that the following sequences are only exemplary sequences of the embodiments of this application and do not represent any limitation of the embodiments of this application. The nucleic acid sequences in the sequence listing below may represent DNA sequences or RNA sequences, and T represents a uridine when they represent RNA sequences. Petition 870250073560, dated 08 / 20 / 2025, pp. 69 / 89 / 61 List of sequences: SEQ ID NO. Name Sequence Nucleic acid sequence 1 P1 AAAAA GAAAAAAAAAAAAAAAAAAGATATCGTATACAAAAAAAAA AAAAAAAAAAAAAAAAAA 8 P8 AAAAAAAAAAAAAAAAACAAAAAAAAAAAAAAAAAA CAAAAAAAAAAAAAAAAGATATCAAAAAAAAAAAA AAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 15 Element d, exemplo 1 GATATC Petition 870250073560, dated 08 / 20 / 2025, pp. 70 / 89 / 61 SEQ ID NO. Name Sequence Nucleic acid sequence 16 Element d, example 2 GTATAC 17 Element d, example 3 GAATCT 18 Element d, example 4 GCATATGACT 19 Element d, example 5 GATATCGTATAC 20 3' UTR CTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTT CCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGC CTTGAGCATCTGGATTCTGCCTGCTCGCTTTCTTGCTGTCCA ATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTA AACTGGTTGACCATGTTGATTGATTGAGTTGCTGCTGCTGCTGTCCA ATTTCTATTAA GCCT 21 5' UTR ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAA CAGACACC 22 Sequence of the gene encoding the luciferase ATGGAAGACGCCAAAAACATTAAGAAGGGCCCAGCGCCAT TCTACCCACTCGAAGACGGGACCCGGCGACCAGGCGAGCCACC AAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGC CTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCG AGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAG CGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAG CGAGAATAGCTTGCTTGGTTGGCCTT GTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACA ACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCC CACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCC TCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCAT GTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGA GTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCA TCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCC AAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATT CAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCC CCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGC TTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTT CGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTT GCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGG TGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCG ACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGC GGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCA AACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTG ACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGG ACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTC GAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGG GTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATG ATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGC TCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCG CCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGG CTCAAAAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCC AGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACCGACGATGCCGGC GAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAAC CATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAG GTTACAACCGCCAAGAAGCTGGCGGTGGTGTTGTGTTCGT GGACGAGGGAAGGCCGAGCCGAGCCGAGCCG CGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCG GAAAGATCGCCGTGTAA 23 Sequence of the gene encoding ATGGATGCTATGAAACGGGGCCTGTGCTGCGTGCTGCTCCT GTGCGGCGCTGTTTGTGAGCCCTAGCATCACCCAGGACT Petition 870250073560, of 20 / 08 / 2025, p. 71 / 89 / 61 SEQ ID NO. Nome Sequência Sequência de ácidos nucleicos proteína do HPV GCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCA AAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCA GTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGG GGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGC GGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTG GAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGC CTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAA CATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGG CGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGC CTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGCCACC CCCATGGAGTCCCCGGCCCCTGGAGGCCACAGCCCCGACAG CCCCGGGCGGCGGCAGCGGCGATATGCACCAGAAGAGAAC CGCCATGTTCCAGGACCCTCAGGAGAGACCTAGGAAGCTGC CTCACCTGTGTACAGAGCTCCAGACAACCATCCACGACATC ATCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGAAG AGAGGTGTACGACTTCGCCTTCAGAGACCTGTGCATCGTGT ACAGAGACGGCAACCCTTACGCCGTGTGCGATAAGTGTCTG AAGTTCTATTCCAAAATCTCCGAATATAGGTACATGCACGG CGACACCCCTACCCTGCACGAGTACATGCTGGACCTCCAGC CTGAGACCACAGACCTGTACTGCTACGAGCAGCTGAACGAC AGCTCTGAGGAAGAGGACGAGATTGACGGACCTGCTGGCC AGGCCGAGCCTGACAGAGCCCACTACAATATCGTGACATTCTGTTGCAAATGCGACTCCACACTGGACAAGTGCCTGAAGTT CTACAGCAAGATCTCTGAGTACAGATACTACTGCTACTCTG TGTACGGCACCACACTGGAGCAGCAGTACAACAAGCCTCTG TGCGACCTCCTGATCCGCTGCATCAACTGCCAGAAGCCTCT GTGCCCTGAGGAGAAGCAGAGACACCTGGACAAGAAGCAG CGGTTCCACAACATCAGAGGCAGATGGACCGGCAGGTGCAT GTCCTGCTGTAGATCCTCCAGAACCAGACGGGAGACCCAGC TGCACTACAACATCGTGACCTTCTGCTGCAAGTGCGACTCT ACCCTGAGACTGTGCGTGCAGTCTACCCACGTGGACATCAG AACCCTGGAGGACCTGCTGATGGGCACCCTGGGCATCGTGT GCCCTATCTGCTCTCAGAAGCCTATGGCCAGGTTCGAGGAC CCTACCAGAAGACCCTACAAGCTGCCTGACCTGTGCACCGA GCTGAACACCTCTCTGCAAGACATCGAGATCACCTGCGTGT ACTGCAAGACCGTGCTGGAGCTGACCGAGGTGTTCGAGTTC GCCTTCAAGGACCTGTTCGTGGTGTACAGAGACAGCATCCC TCACGCTGCCTGCCACAAGTGCATCGACTTCTATTCCAGGAT CAGGGAGCTGCGCTATTACTCCGACTCTGTGATGTACGGCC CCAAGGCCACCCTCCAGGACATCGTGCTGCACCTGGAGCCT CAGAACGAGATCCCCGTGGACCTGCTGTGCCACGAGCAGCT GTCTGACTCTGAAGAGGAGAACGACGAGATCGACGGCGTG AACCACCAGCACCTGCCTGCCAGGAGAGCTGAACCCCAGCG GCATACCATGCTGTGTATGTGCTTCTACTCTAGGATCAGAG AGCTGAGGTACTACTCTGACTCTGTGTACGGCGACACCCTGGAGAAGCTGACCAACACCGGCCTGTACAACCTGCTGATCCG GTGCCTGAGGTGCCAGAAGCCTCTGAACCCTGCCGAGAAGC TGAGACACCTGAACGAGAAGAGAAGATTCCACAAGATCGC TGGCCACTACAGAGGCCAGTGCCACTCTTGCTGCAACAGAG CCAGACAGGAGAGACTCCAGCGGAGAAGGGAGACCCAGGT GGCCAGGAGAGCCGAGCCTCAGAGACACACCATGCTGTGC ATGTGCTGCAAGTGCGAGGCCAGAATCGAGCTGGTGGTGGA GAGCTCTGCCGACGACCTGAGAGCCTTCCAGCAGCTGTTCC TGTCTACCCTGAGCTTCGTGTGCCCTTGGTGCGCCTCTCAGC AGTAA 24 5’ UTR-proteína do HPV que codifica gene-3’ UTR-poli (A) P3 ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAA CAGACACCGGATCCGCCACCATGGATGCTATGAAACGGGGC CTGTGCTGCGTGCTGCTCCTGTGCGGCGCTGTGTTTGTGAGC CCTAGCATCACCCAGGACTGCTCCTTCCAACACAGCCCCAT Petição 870250073560, de 20 / 08 / 2025, pág. 72 / 89 / 61 SEQ ID NO. Nome Sequência Sequência de ácidos nucleicos CTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTA CCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGC AGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTG GCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTC CAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATA CACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGT CTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAG ACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCG CCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCG ACTCCTCAACCCTGCCACCCCCATGGAGTCCCCGGCCCCTG GAGGCCACAGCCCCGACAGCCCCGGGCGGCGGCAGCGGCG ATATGCACCAGAAGAGAACCGCCATGTTCCAGGACCCTCAG GAGAGACCTAGGAAGCTGCCTCACCTGTGTACAGAGCTCCA GACAACCATCCACGACATCATCCTGGAGTGCGTGTACTGTA AGCAGCAGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTC AGAGACCTGTGCATCGTGTACAGAGACGGCAACCCTTACGC CGTGTGCGATAAGTGTCTGAAGTTCTATTCCAAAATCTCCG AATATAGGTACATGCACGGCGACACCCCTACCCTGCACGAG TACATGCTGGACCTCCAGCCTGAGACCACAGACCTGTACTG CTACGAGCAGCTGAACGACAGCTCTGAGGAAGAGGACGAG ATTGACGGACCTGCTGGCCAGGCCGAGCCTGACAGAGCCCA CTACAATATCGTGACATTCTGTTGCAAATGCGACTCCACACTGGACAAGTGCCTGAAGTTCTACAGCAAGATCTCTGAGTACA GATACTACTGCTACTCTGTGTACGGCACCACACTGGAGCAG CAGTACAACAAGCCTCTGTGCGACCTCCTGATCCGCTGCAT CAACTGCCAGAAGCCTCTGTGCCCTGAGGAGAAGCAGAGA CACCTGGACAAGAAGCAGCGGTTCCACAACATCAGAGGCA GATGGACCGGCAGGTGCATGTCCTGCTGTAGATCCTCCAGA ACCAGACGGGAGACCCAGCTGCACTACAACATCGTGACCTT CTGCTGCAAGTGCGACTCTACCCTGAGACTGTGCGTGCAGT CTACCCACGTGGACATCAGAACCCTGGAGGACCTGCTGATG GGCACCCTGGGCATCGTGTGCCCTATCTGCTCTCAGAAGCC TATGGCCAGGTTCGAGGACCCTACCAGAAGACCCTACAAGC TGCCTGACCTGTGCACCGAGCTGAACACCTCTCTGCAAGAC ATCGAGATCACCTGCGTGTACTGCAAGACCGTGCTGGAGCT GACCGAGGTGTTCGAGTTCGCCTTCAAGGACCTGTTCGTGG TGTACAGAGACAGCATCCCTCACGCTGCCTGCCACAAGTGC ATCGACTTCTATTCCAGGATCAGGGAGCTGCGCTATTACTCC GACTCTGTGATGTACGGCCCCAAGGCCACCCTCCAGGACAT CGTGCTGCACCTGGAGCCTCAGAACGAGATCCCCGTGGACC TGCTGTGCCACGAGCAGCTGTCTGACTCTGAAGAGGAGAAC GACGAGATCGACGGCGTGAACCACCAGCACCTGCCTGCCAG GAGAGCTGAACCCCAGCGGCATACCATGCTGTGTATGTGCT TCTACTCTAGGATCAGAGAGCTGAGGTACTACTCTGACTCT GTGTACGGCGACACCCTGGAGAAGCTGACCAACACCGGCCTGTACAACCTGCTGATCCGGTGCCTGAGGTGCCAGAAGCCTC TGAACCCTGCCGAGAAGCTGAGACACCTGAACGAGAAGAG AAGATTCCACAAGATCGCTGGCCACTACAGAGGCCAGTGCC ACTCTTGCTGCAACAGAGCCAGCAGAGAGAGACTCCAGCG GAGAAGGGAGACCAGCCAGGGAGGAGGCCAGGCCAG AGACACACCATGCTGTGCATGTGCTGCAAGTGCGAGGCCAG AATCGAGCTGGTGGTGGAGAGCTCTGCCGACGACCTGAGAG CCTTCCAGCAGCTGTTCCTGTCTACCCTGAGCTTCGTGTGCC CTTGGTGCGCCTCTCAGCAGTAAGGCGCCGCTCGCTCTCTCTGCTTTAAGTTAAGTCTTTAGTCTTTAGTCTTTCTT CAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCAT CTGGATTCTGCCTGCTCGCTTTCTTGCTGTCCAATTTCTATTA AAGGTTCCTTTGTTCCCTAAGTCCAACTACTAACTGGGGG ATATTATGAAGGGCCTTGAGCATCTGGATTCTGAATTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATT Petition 870250073560, of 20 / 08 / 2025, p. 73 / 89 / 61 SEQ ID NO. Name Sequence Nucleic acid sequence AAAAAAAAAAAAAAAAAAAAGATATCAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 25 5' UTR-protein of HPV encoding gene-3' UTR-poly (A) P4 ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAA CAGACACCGGATCCGCCACCATGGATGCTATGAAACGGGGC CTGTGCTGCGTGCTGCTCGTGCGGCGCTGTGTTTGAGC CCTAGCATCACCCAGGACTGCTCCTTCCAACAGCCCCAT CTCCTCCGACTGACTGACTGACTGACTGACTGACT CCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAAACCTGC AGGACGAGGAGCTCTGCGGGGCCTCTGGCGGCTGGTCCTG GCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTC CAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGGATA CTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAG ACCTCCGAGCAGCTGGTGGGCGCTGAAGCCCTGGATCACTCG CCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCG ACTCCTCAACCCTGCCACCCATGGAGTCCCCGGCCCCTG GAGGCCCAGCCAGGCGCGCGCGCGCGC ATATGCACCAGAAGAGAACCGCCATGTTCCAGGACCCTCAG GAGAGACCTAGGAAGCTGCCTCACCTGTGTACAGAGCTCCA GACAACCATCCACGACATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTC AGAGACCTGTGCATCGTGTACAGAGACGGCAACCCTTACGC CGTGTGCGATAAGTGTCTGAAGTTCTATTCCAAAATCTCCG AATATAGGTACATGCACGGCGACACCCCTACCCTGCACGAG TACATGCTGGACCTCCAGCCTGAGACCACAGACCTGTACTG CTACGAGCAGCTGAACGACAGCTCTGAGGAAGAGGACGAG ATTGACGGACCTGCTGGCCAGGCCGAGCCTGACAGAGCCCA CTACAATATCGTGACATTCTGTTGCAAATGCGACTCCACACT GGACAAGTGCCTGAAGTTCTACAGCAAGATCTCTGAGTACA GATACTACTGCTACTCTGTGTACGGCACCACACTGGAGCAG CAGTACAACAAGCCTCTGTGCGACCTCCTGATCCGCTGCAT CAACTGCCAGAAGCCTCTGTGCCCTGAGGAGAAGCAGAGA CACCTGGACAAGAAGCAGCGGTTCCACAACATCAGAGGCA GATGGACCGGCAGGTGCATGTCCTGCTGTAGATCCTCCAGA ACCAGACGGGAGACCCAGCTGCACTACAACATCGTGACCTT CTGCTGCAAGTGCGACTCTACCCTGAGACTGTGCGTGCAGT CTACCCACGTGGACATCAGAACCCTGGAGGACCTGCTGATG GGCACCCTGGGCATCGTGTGCCCTATCTGCTCTCAGAAGCC TATGGCCAGGTTCGAGGACCCTACCAGAAGACCCTACAAGC TGCCTGACCTGTGCACCGAGCTGAACACCTCTCTGCAAGAC ATCGAGATCACCTGCGTGTACTGCAAGACCGTGCTGGAGCT GACCGAGGTGTTCGAGTTCGCCTTCAAGGACCTGTTCGTGG TGTACAGAGACAGCATCCCTCACGCTGCCTGCCACAAGTGCATCGACTTCTATTCCAGGATCAGGGAGCTGCGCTATTACTCC GACTCTGTGATGTACGGCCCCAAGGCCACCCTCCAGGACAT CGTGCTGCACCTGGAGCCTCAGAACGAGATCCCCGTGGACC TGCTGTGCCACGAGCAGCTGTCTGACTCTGAAGAGGAGAAC GACGAGATCGACGGCGTGAACCACCAGCACCTGCCTGCCAG GAGAGCTGAACCCCAGCGGCATACCATGCTGTGTATGTGCT TCTACTCTAGGATCAGAGAGCTGAGGTACTACTCTGACTCT GTGTACGGCGACACCCTGGAGAAGCTGACCAACACCGGCCT GTACAACCTGCTGATCCGGTGCCTGAGGTGCCAGAAGCCTC TGAACCCTGCCGAGAAGCTGAGACACCTGAACGAGAAGAG AAGATTCCACAAGATCGCTGGCCACTACAGAGGCCAGTGCC ACTCTTGCTGCAACAGAGCCAGACAGGAGAGACTCCAGCG GAGAAGGGAGACCCAGGTGGCCAGGAGAGCCGAGCCTCAG AGACACACCATGCTGTGCATGTGCTGCAAGTGCGAGGCCAG Petition 870250073560, dated 08 / 20 / 2025, pp. 74 / 89 / 61 SEQ ID NO. Name Sequence Nucleic acid sequence AATCGAGCTGGTGGTGGAGAGCTCTGCCGACGACCTGAGAG CCTTCCAGCAGCTGTTCCTGTCTACCCTGAGCTTCGTGTGCC CTTGGTGCGCCTCTCAGCAGTAAGGCGCGCCGCTCGCTTTCTGCTGTCCAATTTTTAGTTTAGTTTAGTTTCTTTAGTCCCTT CAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCAT CTGGATTCTGCCTGCTCGCTTTCTTGCTGTCCAATTTCTATTA AAGGTTCCTTTGTTCCCTAAGTCCAACTACTAACTGGGGG ATATTATGAAGGGCCTTGAGCATCTGGATTCTGAATTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTGATTCTGATTGAATTC HPV protein sequence MDAMKRGLCCVLLLCGAVFVSPSITQDCSFQHSPISSDFAVKIR ELSDYLLQDYPVCGLQLQLWRLWLQLWLQLQLWLQLQLQLQLQLQLQLQLQLQLQLQLQLQAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATATCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 26 Protein sequence of HPV MDAMKRGLCCVLLLCGAVFVSPSITQSFQHSPISSDFAVKIR TVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLL QETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLE ATAPTAPGGGSGDMHQKRTAMFQDPQERPRKLPHLCTELQTT IHDIILECVYCKQQLLREVYRDVYCDVYCDVYCDVLCLD CLKFYSKISEYRYMHGDTPTLHEYMLDLQPETTDLYCYEQLN DSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLDKCLKFY SKISEYRYYCYSVYGTTLEQYNKPLCDLLIRCINCQKPLCPEE KQRHLDKQRFHCMSSCRYCRHRYWTRIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPM ARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVF EFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRYYSDSVMYGP KATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQH LPARRAEPQRHTMLCMCFYSRIRELRYYSDSVYGDTLEKLTNT GLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHKIAGHYRGQCH SCCNRARQERLQRRRETQVARRAEPQRHTMLCMCCKCEARIE LVVESSADDLRAFQQLFLSTLSFVCPWCASQQ

Claims

1. A manipulated DNA molecule capable of being replicated in a cell, characterized in that it comprises a polyadenosine tail coding sequence (poly A tail), wherein the poly (A) tail coding sequence comprises: a single a element and at least one b element and at least one c element and / or at least one d element: the a element consists of a plurality of consecutive adenine (A) nucleotides, and the length range of the a element is >20 nt; the b element consists of a plurality of consecutive A nucleotides, and the length range of the b element is 3 nt < b <20 nt; the c element consists of a non-A nucleotide, and the nucleotide is selected from among T, C and G nucleotides;The d element consists of any two or more consecutive nucleotides, and the nucleotides are selected from A, T, C, and G nucleotides, provided that the nucleotides at the 5' and 3' ends of the d element are not A nucleotides, and the d element does not comprise 3 or more consecutive A nucleotides; the length range of the d element is 2 nt. <d<20 nt; sendo que o elemento a e o elemento b não são adjacentes, e o elemento c e o elemento d não são adjacentes, e a sequência codificadora de cauda poli (A) não compreende quaisquer dois elementos b que sejam adjacentes um ao outro, não compreende quaisquer dois elementos c que sejam adjacentes um ao outro e não compreende quaisquer dois elementos d que sejam adjacentes um ao outro.; 2. DNA molecule according to claim 1, characterized in that the length of the poly(A) tail coding sequence is 101 to 200 nt, 101 to 150 nt, 120 to 150 nt, 130 to 140 nt, 120 to 135 nt or 123 to 125 nt.

3. DNA molecule according to claim 1 or 2, characterized in that the 3' terminus of the poly(A) tail coding sequence is either an A nucleotide or a non-A nucleotide.

4. DNA molecule according to any one of claims 1 to 3, characterized in that the length of the element is <80 nt.

5. DNA molecule according to any one of claims 1 to 3, characterized in that the alpha element is 30 to 70 nt, 35 to 65 nt, 40 to 60 nt or 45 to 55 nt long, preferably 60 nt long.

6. DNA molecule according to any one of claims 1 to 5, characterized in that 50% or more of the a-element polynucleotides are located in the 5' or 3' portion of the poly(A) tail coding sequence.

7. DNA molecule according to any one of claims 1 to 6, characterized in that element b has a length of 3 to 10 nt, 10 to 19 nt, 12 to 15 nt, 14 to 17 nt or 16 to 19 nt, preferably 19 nt in length.

8. DNA molecule according to any one of claims 1 to 7, characterized in that the number of b elements is from 2 to 10, preferably 2 to 5 and, more preferably, 3.

9. DNA molecule according to any one of claims 1 to 8, characterized in that element c is G.

10. DNA molecule according to any one of claims 1 to 9, characterized in that the number of c elements is from 2 to 10, 3 to 8, 4 to 6 or 2 to 5, preferably 2.

11. DNA molecule according to any one of claims 1 to 10, characterized in that the element d Petition 870250073560, dated 08 / 20 / 2025, page 77 / 89 3 / 8 comprises a palindromic sequence.

12. DNA molecule according to any one of claims 1 to 11, characterized in that the d element has a length of 3 to 18 nt, 5 to 16 nt, 4 to 10 nt or 6 to 12 nt, preferably 6 nt in length.

13. DNA molecule according to any one of claims 1 to 12, characterized in that the d element is any one or more sequences selected from the following sequences: GATATC (SEQ ID NO: 15), GTATAC (SEQ ID NO: 16), GAATCT (SEQ ID NO: 17), GCATATGACT (SEQ ID NO: 18) and GATATCGTATAC (SEQ ID NO: 19).

14. DNA molecule according to any one of claims 1 to 13, characterized in that the d element is any one or more sequences selected from the following nucleotide sequences: SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO:

17.

15. DNA molecule according to any one of claims 1 to 14, characterized in that the nucleotide sequence of element d is represented by SEQ ID NO:

15.

16. DNA molecule according to any one of claims 1 to 15, characterized in that the number of element d is from 0 to 5, preferably 1 to 3 and, more preferably, 1.

17. DNA molecule according to any one of claims 1 to 16, characterized in that, when element c and element d exist simultaneously, the total number of element c and element d is from 2 to 15, preferably 3 to 5 and, more preferably, 3.

18. DNA molecule according to any one of claims 1 to 17, characterized in that the 3' portion of the poly(A) tail coding sequence, preferably the 1 / 2 portion of the poly(A) tail coding sequence near the 3' terminus, comprises one or more non-A nucleotides.

19. DNA molecule according to any one of claims 1 to 18, characterized in that the structure of the poly(A) tail coding sequence is: element a-element c-element b-element c-element b-element c-element b-element c-element b; element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b; element b-element c-element b-element c-element b-element d-element a-element c; element a-element d-element b-element c-element b-element c-element b; or element b-element c-element b-element c-element b-element d-element a.

20. DNA molecule according to any one of claims 1 to 19, characterized in that the structure of the poly(A) tail coding sequence is: element a-element d-element b-element c-element b; wherein element a is 60 nt long, element b is 16 to 19 nt long, and element d is 6 nt long.

21. DNA molecule according to any one of claims 1 to 20, characterized in that the structure of the poly(A) tail coding sequence is: element b-element c-element b-element c-element b-element d-element a; and wherein element a is 60 nt long, element b is 16 to 19 nt long, and element d is 6 nt long.

22. DNA molecule according to any of the claims 1 to 21, characterized in that the poly(A) tail coding sequence is represented by any of the SEQ ID Nos: 1 to 10.

23. DNA molecule according to any one of claims 1 to 22, characterized in that the poly(A) tail coding sequence is represented by SEQ ID NO: 3 or SEQ ID NO:

4.

24. DNA molecule according to any one of claims 1 to 23, characterized in that it is additionally connected to a fragment of the gene of interest at the 5' end of the poly(A) tail coding sequence, and the fragment of the gene of interest and the poly(A) tail coding sequence concomitantly encode RNA.

25. DNA molecule according to any one of claims 1 to 24, characterized in that it additionally comprises a replicon.

26. DNA molecule according to any one of claims 1 to 25, characterized in that it additionally comprises a resistance gene.

27. DNA molecule according to any one of claims 1 to 26, characterized in that it additionally comprises a promoter for initiating RNA transcription.

28. DNA molecule according to any one of claims 1 to 27, characterized in that the gene fragment of interest comprises a 5' UTR coding sequence.

29. DNA molecule according to any one of claims 1 to 28, characterized in that the gene fragment of interest comprises a protein-coding sequence or a non-protein-coding sequence.

30. DNA molecule according to any one of claims 1 to 29, characterized in that the gene fragment of interest comprises a 3' UTR coding sequence.

31. DNA molecule according to any one of claims 1 to 30, characterized in that it comprises a replicon, an antibiotic resistance gene, a promoter, a 5' UTR coding sequence, a protein coding sequence and a 3' UTR coding sequence.

32. DNA molecule according to any one of claims 1 to 31, characterized in that the protein-coding sequence encodes a protein from HPV (human papillomavirus), preferably the HPV protein is derived from HPV type 16 and / or type 18.

33. DNA molecule according to any one of claims 1 to 32, characterized in that the protein-coding sequence encodes HPV protein E2, E6 or E7, a fusion protein of E6 and E7 protein polypeptide fragments or a fusion protein of E2, E6 and E7 protein polypeptide fragments, preferably the HPV protein being derived from HPV type 16 and / or type 18.

34. DNA molecule according to any one of claims 1 to 33, characterized in that the protein-coding sequence encodes the polypeptide represented by SEQ NO:

26.

35. DNA molecule according to any one of claims 1 to 34, characterized in that it comprises a polynucleotide sequence represented by any one of the SEQ ID NOs: 22 to 25, or a synonymous mutant of the polynucleotide sequence represented by any one of the SEQ ID NOs: 22 to 25, or a polynucleotide sequence that shares more than 85% sequence identity with the polynucleotide sequence represented by any one of the SEQ ID NOs: 22 to 25 or a synonymous mutant thereof.

36. DNA molecule according to any one of claims 1 to 35, the DNA molecule being characterized by the suit of Petition 870250073560, dated 08 / 20 / 2025, p. 81 / 89 7 / 8 being a DNA plasmid.

37. Cell, characterized in that it comprises the DNA molecule as defined in any one of claims 1 to 36.

38. Cell according to claim 37, the cell being characterized in that it is a prokaryotic cell.

39. Cell according to claim 37, the cell being characterized in that it is Escherichia coli.

40. RNA molecule, characterized in that it is encoded by the DNA molecule as defined in any one of claims 1 to 36.

41. RNA molecule according to claim 40, characterized in that it further comprises a 5'-cap structure, and / or some or all of the uridines in the RNA are chemically modified uridines; preferably, some or all of the uridines in the RNA are pseudouridines or 1-methyl-pseudouridines.

42. DNA coding sequence, characterized in that it is for the poly(A) tail as defined in any one of claims 1 to 36.

43. Poly(A) tail sequence, characterized in that it is encoded by the DNA coding sequence for the poly(A) tail as defined in any one of claims 1 to 36.

44. Use of the DNA coding sequence for a poly(A) tail as defined in claim 42, characterized in that it is intended to make the replication of a DNA molecule encoding RNA more conservative in a host cell, wherein the poly(A) tail is located at the 3' end of the RNA.

45. Use according to claim 44, characterized in that the host cell is a prokaryotic cell, preferably E. coli.

46. ​​Use of the poly(A) tail sequence according to Petition 870250073560, dated 20 / 08 / 2025, page 82 / 89 8 / 8 claim 43, characterized in that it is intended to regulate the expression of an RNA molecule in a host cell, wherein the poly(A) tail is located at the 3' end of the RNA.

47. Use according to claim 46, characterized in that the host cell is a eukaryotic cell, preferably a mammalian cell and, more preferably, a human cell.

48. Library, characterized in that it comprises the DNA molecule as defined in any one of claims 1 to 36.

49. Library, characterized in that it comprises an RNA molecule encoded by the DNA molecule as defined in any one of claims 1 to 36.

50. A method for regulating protein expression, characterized in that it comprises: introducing a plurality of DNAs into the DNA library as defined in claim 48 into target cells at different times and / or at different quantitative ratios; or introducing a plurality of RNAs into the RNA library as defined in claim 49 into target cells at different times and / or at different quantitative ratios.

51. A hybrid molecule of DNA and RNA, characterized in that it comprises the same genetic information as the DNA molecule as defined in any one of claims 1 to 36, the same genetic information as the poly(A) tail as defined in claim 42, or the same genetic information as the RNA molecule as defined in claim 40.

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