HRP20211745T1 - Postupci za dobivanje jednolančane rnk - Google Patents
Postupci za dobivanje jednolančane rnk Download PDFInfo
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- HRP20211745T1 HRP20211745T1 HRP20211745TT HRP20211745T HRP20211745T1 HR P20211745 T1 HRP20211745 T1 HR P20211745T1 HR P20211745T T HRP20211745T T HR P20211745TT HR P20211745 T HRP20211745 T HR P20211745T HR P20211745 T1 HRP20211745 T1 HR P20211745T1
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- 238000000034 method Methods 0.000 title claims 16
- 239000000463 material Substances 0.000 claims 52
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims 42
- 239000000872 buffer Substances 0.000 claims 39
- 238000002360 preparation method Methods 0.000 claims 21
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 20
- 150000003839 salts Chemical class 0.000 claims 12
- 238000002156 mixing Methods 0.000 claims 10
- 229920002678 cellulose Polymers 0.000 claims 9
- 239000001913 cellulose Substances 0.000 claims 9
- 239000000203 mixture Substances 0.000 claims 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims 8
- 239000007791 liquid phase Substances 0.000 claims 7
- 238000001914 filtration Methods 0.000 claims 6
- 230000005484 gravity Effects 0.000 claims 6
- 239000007790 solid phase Substances 0.000 claims 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims 4
- 239000012928 buffer substance Substances 0.000 claims 4
- 239000002738 chelating agent Substances 0.000 claims 4
- 239000007788 liquid Substances 0.000 claims 4
- 239000011780 sodium chloride Substances 0.000 claims 4
- 238000005406 washing Methods 0.000 claims 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 4
- 238000000926 separation method Methods 0.000 claims 3
- 239000000243 solution Substances 0.000 claims 3
- 239000006228 supernatant Substances 0.000 claims 3
- 229920003043 Cellulose fiber Polymers 0.000 claims 2
- 238000000338 in vitro Methods 0.000 claims 2
- 102000039446 nucleic acids Human genes 0.000 claims 2
- 108020004707 nucleic acids Proteins 0.000 claims 2
- 150000007523 nucleic acids Chemical class 0.000 claims 2
- 238000000746 purification Methods 0.000 claims 2
- 239000000725 suspension Substances 0.000 claims 2
- 238000013518 transcription Methods 0.000 claims 2
- 230000035897 transcription Effects 0.000 claims 2
- 108020005544 Antisense RNA Proteins 0.000 claims 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 claims 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 claims 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 claims 1
- 108020004459 Small interfering RNA Proteins 0.000 claims 1
- 101710137500 T7 RNA polymerase Proteins 0.000 claims 1
- 238000003916 acid precipitation Methods 0.000 claims 1
- 108010028263 bacteriophage T3 RNA polymerase Proteins 0.000 claims 1
- 239000011324 bead Substances 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 239000003184 complementary RNA Substances 0.000 claims 1
- 238000006731 degradation reaction Methods 0.000 claims 1
- 239000003480 eluent Substances 0.000 claims 1
- 238000010828 elution Methods 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 claims 1
- 108020004999 messenger RNA Proteins 0.000 claims 1
- 108091070501 miRNA Proteins 0.000 claims 1
- 239000002679 microRNA Substances 0.000 claims 1
- 238000004810 partition chromatography Methods 0.000 claims 1
- 239000002924 silencing RNA Substances 0.000 claims 1
- 238000000108 ultra-filtration Methods 0.000 claims 1
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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Claims (15)
1. Postupak za dobivanje jednolančane RNK (ssRNA), koji obuhvaća:
(i) proizvodnju preparata RNK koji sadrži ssRNA in vitro transkripcijom;
(ii) dovođenje u kontakt preparata RNK sa celuloznim materijalom pod uvjetima koji omogućavaju vezivanje dvolančane RNK (dsRNA) za celulozni materijal, a ne omogućavaju vezivanje ssRNA za celulozni materijal; i
(iii) odvajanje ssRNA od celuloznog materijala pod uvjetima koji omogućavaju vezivanje dsRNA za celulozni materijal, a ne omogućavaju vezivanje ssRNA za celulozni materijal,
gdje je
u koraku (ii) preparat RNK osiguran kao tekućina koja sadrži ssRNA i prvi pufer i/ili je celulozni materijal osiguran kao suspenzija u prvom puferu, pri čemu prvi pufer sadrži vodu, etanol i sol u koncentraciji koja omogućava vezivanje dsRNA za celulozni materijal, a ne omogućava vezivanje ssRNA za celulozni materijal; i
koncentracija etanola u prvom puferu je 14 do 20% (zapr./zapr.), a koncentracija soli u prvom puferu je 15 do 70 mM.
2. Postupak za dobivanje jednolančane RNK (ssRNA), koji obuhvaća:
(i) proizvodnju preparata RNK koji sadrži ssRNA in vitro transkripcijom;
(ii) dovođenje u kontakt preparata RNK sa celuloznim materijalom pod uvjetima koji omogućavaju vezivanje dvolančane RNK (dsRNA) i ssRNA za celulozni materijal; i
(iii) odvajanje ssRNA od celuloznog materijala pod uvjetima koji omogućavaju vezivanje dsRNA za celulozni materijal, a ne omogućavaju vezivanje ssRNA za celulozni materijal,
gdje
korak (iii) obuhvaća:
(1) miješanje celuloznog materijala za koji su vezane dsRNA i ssRNA sa prvim puferom uz mućkanje i/ili miješanje, gdje prvi pufer sadrži vodu, etanol i sol u koncentraciji koja omogućava vezivanje dsRNA za celulozni materijal, a ne omogućava vezivanje ssRNA za celulozni materijal; i
(2) odvajanje tekuće faze koja sadrži ssRNA od celuloznog materijala;
i
koncentracija etanola u prvom puferu je 14 do 20% (zapr./zapr.), a koncentracija soli u prvom puferu je 15 do 70 mM.
3. Postupak prema patentnom zahtjevu 1, gdje korak (ii) obuhvaća miješanje preparata RNK koji sadrži ssRNA sa celuloznim materijalom uz mućkanje i/ili miješanje, poželjno tokom najmanje 5 min, poželjnije tokom najmanje 10 min.
4. Postupak prema patentnom zahtjevu 3, gdje
(a) sol sadržana u prvom puferu je natrij klorid; i/ili
(b) koncentracija etanola u prvom puferu je 14 do 16% (zapr./zapr.); i/ili
(c) koncentracija soli u prvom puferu je 20 do 60 mM; i/ili
(d) prvi pufer dodatno sadrži pufersku supstancu, poželjno tris(hidroksimetil)aminometan (TRIS), i/ili kelirajući agens, poželjno EDTA; i/ili
(e) u koraku (iii)
(α) mješavina preparata RNK, celuloznog materijala i prvog pufera je osigurana u epruveti i korak (iii) obuhvaća (1) primjenu gravitacije ili centrifugalne sile na epruvetu tako da se tekuća i čvrsta faza razdvoje; i (2) ili sakupljanje supernatanta koji sadrži ssRNA ili uklanjanje celuloznog materijala; ili
(β) mješavina preparata RNK, celuloznog materijala i prvog pufera je osigurana u spin-koloni ili uređaju za filtraciju i korak (iii) obuhvaća (1’) primjenu gravitacije, centrifugalne sile, pritiska, ili vakuuma na spin-kolonu ili uređaj za filtraciju tako da se tekuća i čvrsta faza razdvoje; i (2’) sakupljanje propuštene frakcije koja sadrži ssRNA.
5. Postupak prema bilo kojem od patentnih zahtjeva 1, 3 i 4, gdje se koraci (ii) i (iii) ponavljaju jednom ili dva ili više puta, pri čemu se preparat ssRNA dobiven nakon koraka (iii) jednog ciklusa koraka (ii) i (iii) koristi kao preparat RNK u koraku (ii) sljedećeg ciklusa, i u koraku (ii) svakog ciklusa koraka (ii) i (iii) upotrebljava se svjež celulozni materijal.
6. Postupak prema patentnom zahtjevu 2, gdje korak (ii) obuhvaća
(1) miješanje preparata RNK koji sadrži ssRNA sa celuloznim materijalom uz mućkanje i/ili miješanje, poželjno tokom najmanje 5 min, poželjnije tokom najmanje 10 min; i
(2) odvajanje celuloznog materijala za koji su vezane dsRNA i ssRNA od ostatka.
7. Postupak prema patentnom zahtjevu 6, gdje je u koraku (ii) preparat RNK osiguran kao tekućina koja sadrži ssRNA i drugi pufer i/ili je celulozni materijal osiguran kao suspenzija u drugom puferu, gdje drugi pufer sadrži vodu, etanol i sol, poželjno natrij klorid, u koncentraciji koja omogućava vezivanje dsRNA i ssRNA za celulozni materijal.
8. Postupak prema patentnom zahtjevu 7, gdje
(a) koncentracija etanola u drugom puferu je najmanje 35% (zapr./zapr.), poželjno 38 do 42% (zapr./zapr.); i/ili
(b) koncentracija soli u drugom puferu je 15 do 70 mM, poželjno 20 do 60 mM; i/ili
(c) drugi pufer dodatno sadrži pufersku supstancu, poželjno tris(hidroksimetil)aminometan (TRIS), i/ili kelirajući agens, poželjno EDTA; i/ili
(d) u koraku (ii)(2)
(α) mješavina preparata RNK i celuloznog materijala dobivena u koraku (ii)(1) je osigurana u epruveti i korak (ii)(2) obuhvaća (2a) primjenu gravitacije ili centrifugalne sile na epruvetu tako da se tekuća i čvrsta faza razdvoje; i (2b) ili uklanjanje supernatanta ili sakupljanje celuloznog materijala za koji su vezane dsRNA i ssRNA; ili
(β) mješavina preparata RNK i celuloznog materijala dobivena u koraku (ii)(1) je osigurana u spin-koloni ili uređaju za filtraciju i korak (ii)(2) obuhvaća (2a’) primjenu gravitacije, centrifugalne sile, pritiska, ili vakuuma na spin-kolonu ili uređaj za filtraciju tako da se tekuća i čvrsta faza razdvoje; i (2b’) odbacivanje propuštene frakcije; i/ili
(e) korak (ii) dalje obuhvaća (3) dodavanje alikvota drugog pufera u celulozni materijal za koji su vezane dsRNA i ssRNA; (4) inkubaciju dobivene mješavine uz mućkanje i/ili miješanje, poželjno tokom najmanje 5 min, poželjnije tokom najmanje 10 min; i (5) odvajanje celuloznog materijala za koji su vezane dsRNA i ssRNA od tekuće faze; i izborno (6) ponavljanje koraka (3) do (5) jednom ili dva ili više puta.
9. Postupak prema bilo kojem od patentnih zahtjeva 2 i 6 do 8, gdje
(a) u koraku (iii)(1) celulozni materijal za koji su vezane dsRNA i ssRNA se miješa sa prvim puferom uz mućkanje i/ili miješanje tokom najmanje 5 min, poželjno tokom najmanje 10 min; i/ili
(b) sol sadržana u prvom puferu je natrij klorid; i/ili
(c) koncentracija etanola u prvom puferu je 14 do 16% (zapr./zapr.); i/ili
(d) koncentracija soli u prvom puferu je 20 do 60 mM; i/ili
(e) prvi pufer dodatno sadrži pufersku supstancu, poželjno tris(hidroksimetil)aminometan (TRIS), i/ili kelirajući agens, poželjno EDTA; i/ili
(f) u koraku (iii)
(α) mješavina celuloznog materijala i prvog pufera je osigurana u epruveti i korak (iii)(2) obuhvaća (2a) primjenu gravitacije ili centrifugalne sile na epruvetu tako da se tekuća i čvrsta faza razdvoje; i (2b) ili sakupljanje supernatanta koji sadrži ssRNA ili uklanjanje celuloznog materijala; ili
(β) mješavina celuloznog materijala i prvog pufera je osigurana u spin-koloni ili uređaju za filtraciju i korak (iii)(2) obuhvaća (2a’) primjenu gravitacije, centrifugalne sile, pritiska, ili vakuuma na spin-kolonu ili uređaja za filtraciju; i (2b’) sakupljanje propuštene frakcije koja sadrži ssRNA; i/ili
(g) koraci (ii) i (iii) se ponavljaju jednom ili dva ili više puta, pri čemu se preparat ssRNA dobiven nakon koraka (iii) jednog ciklusa koraka (ii) i (iii) upotrebljava kao preparat RNK u koraku (ii) sljedećeg ciklusa, i u koraku (ii) svakog ciklusa koraka (ii) i (iii) koristi se svjež celulozni materijal.
10. Postupak prema patentnom zahtjevu 2, gdje je u koraku (ii) celulozni materijal osiguran u koloni, korak (ii) obuhvaća nanošenje preparata RNK na kolonu pod uvjetima koji omogućavaju vezivanje dsRNA i ssRNA za celulozni materijal, i korak (iii) obuhvaća eluiranje ssRNA sa celuloznog materijala pod uvjetima koji omogućavaju vezivanje dsRNA za celulozni materijal, a ne omogućavaju vezivanje ssRNA za celulozni materijal.
11. Postupak prema patentnom zahtjevu 10, gdje je u koraku (ii) preparat RNK osiguran i nanijet na kolonu kao tekućina koja sadrži ssRNA i drugi pufer, pri čemu drugi pufer sadrži vodu, etanol i sol, poželjno natrij klorid, u koncentraciji koja omogućava vezivanje dsRNA i ssRNA za celulozni materijal.
12. Postupak prema patentnom zahtjevu 11, gdje
(a) koncentracija etanola u drugom puferu je najmanje 35% (zapr./zapr.), poželjno 38 do 42% (zapr./zapr.); i/ili
(b) koncentracija soli u drugom puferu je 15 do 70 mM, poželjno 20 do 60 mM; i/ili
(c) drugi pufer dodatno sadrži pufersku supstancu, poželjno tris(hidroksimetil)aminometan (TRIS), i/ili helirajući agens, poželjno EDTA; i/ili
(d) korak (iii) se provodi uporabom prvog pufera kao eluenta, pri čemu je prvi pufer poželjno prvi pufer definiran u patentnom zahtjevu 9(b), patentnom zahtjevu 9(c), patentnom zahtjevu 9(d), i/ili patentnom zahtjevu 9(e).
13. Postupak prema bilo kojem od patentnih zahtjeva 1 do 12, gdje
(a) preparat RNK se proizvodi uporabom RNK polimeraze izabrane iz grupe koja se sastoji od RNK polimeraza T3, T7 i SP6; i/ili
(b) prije koraka (ii) preparat RNK se podvrgava najmanje jednom tretmanu pred-pročišćavanja, pri čemu najmanje jedan tretman pred-pročišćavanja poželjno uključuje jedno ili više od sljedećeg: precipitaciju nukleinskih kiselina; vezivanje nukleinskih kiselina za magnetne perle; ultrafiltraciju; i degradaciju DNK; i/ili
(c) ssRNA je iRNK ili inhibitorna RNK, kao što je antisens RNK, siRNA, ili miRNA; i/ili
(d) ssRNA ima dužinu od najmanje 2700 nt, poželjno najmanje 3000 nt, poželjnije najmanje 3500 nt, poželjnije najmanje 4500 nt; i/ili
(e) celulozni materijal sadrži celulozna vlakna, poželjno celulozna vlakna odgovarajućeg kvaliteta za uporabu kao reagens za particijsku kromatografiju; i/ili
(f) prije dovođenja u kontakt sa preparatom RNK u koraku (ii), celulozni materijal je osiguran kao oprani celulozni materijal.
14. Postupak prema patentnom zahtjevu 13(f), gdje pranje celuloznog materijala uključuje
(I) miješanje celuloznog materijala sa otopinom za pranje uz mućkanje i/ili miješanje, poželjno tijekom najmanje 5 min; i
(II) ili uklanjanje tekućine ili sakupljanje celuloznog materijala; i izborno
(III) ponavljanje koraka (I) i (II) jednom ili dva ili više puta.
15. Postupak prema patentnom zahtjevu 14, gdje
(A) ako se korak (ii) provodi pod uvjetima koji omogućavaju vezivanje dsRNA za oprani celulozni materijal, a ne omogućavaju vezivanje ssRNA za oprani celulozni materijal, otopina za pranje ima sastav prvog pufera definiran u patentnom zahtjevu 1, patentnom zahtjevu 4(a), patentnom zahtjevu 4(b), patentnom zahtjevu 4(c), i/ili patentnom zahtjevu 4(d), ili
(B) ako se korak (ii) provodi pod uvjetima koji omogućavaju vezivanje dsRNA i ssRNA za oprani celulozni materijal, otopina za pranje ima sastav drugog pufera definiran u patentnom zahtjevu 7, patentnom zahtjevu 8(a), patentnom zahtjevu 8(b), i/ili patentnom zahtjevu 8(c).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP2016059056 | 2016-04-22 | ||
| EP17722695.8A EP3445850B1 (en) | 2016-04-22 | 2017-04-19 | Methods for providing single-stranded rna |
| PCT/EP2017/059293 WO2017182524A1 (en) | 2016-04-22 | 2017-04-19 | Methods for providing single-stranded rna |
Publications (1)
| Publication Number | Publication Date |
|---|---|
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