JPWO2021030533A5

JPWO2021030533A5 -

Info

Publication number: JPWO2021030533A5
Application number: JP2022509126A
Authority: JP
Publication date: 2023-08-21

Claims

below,
(a) desalting a mixture comprising ribonucleic acid (RNA) to produce a low-salt RNA composition having a salt concentration of less than 20 mM;
(b) heating the low-salt RNA composition to a temperature greater than 60° C. to produce denatured RNA;
(c) in-line mixing a composition comprising said denatured RNA with a high salt buffer to produce a composition comprising denatured RNA and salt at a concentration of at least 50 mM;
(d) binding the denatured RNA of the composition produced in (c) to an oligo-dT resin at a temperature below 40°C;
(e) eluting the RNA from the oligo-dT resin;
A method, including

below,
(a) desalting a mixture comprising ribonucleic acid (RNA) to produce a low-salt RNA composition having a conductivity of less than 2 mS/cm;
(b) heating the low-salt RNA composition to a temperature greater than 60° C. to produce denatured RNA;
(c) in-line mixing a composition comprising said denatured RNA with a high salt buffer to produce a composition comprising denatured RNA and a conductivity of at least 5 mS/cm;
(d) binding the denatured RNA of the composition produced in (c) to an oligo-dT resin at a temperature below 40°C;
(e) eluting the RNA from the oligo-dT resin;
A method, including

3. The method of claim 1 or 2, wherein said mixture of (a) is an in vitro transcription reaction.

(i) said salt of (a) and/or (c) comprises NaCl, and/or
(ii) the high salt buffer has a salt concentration of 100 mM to 1000 mM and/or a conductivity of 5 mS/cm to 85 mS/cm, and/or
(iii) the low-salt RNA composition has a salt concentration of 1-20 mM and/or a conductivity of 0.1-2 mS/cm, and/or
(iv) the low-salt RNA composition is heated in the presence of a denaturant molecule;
Optionally, said denaturant molecule is dimethylsulfoxide, guanidine, or urea.
The method according to any one of claims 1-3.

The desalting comprises binding the RNA to a hydrophobic interaction chromatography (HIC) resin and eluting the RNA from the HIC resin to produce the low salt RNA composition. ,
Optionally, the HIC resin is a (poly)styrene-divinylbenzene (PS-DVB) R150 bead resin with 2000 angstrom pores .

The heating of (b) is
(i) less than 1 minute, at least 10 seconds, or 10-60, 10-30, 20-40, or 30-60 seconds , and/or
(ii) at a temperature between 60°C and 90°C
be done,
The method according to any one of claims 1-5 .

(i) in-line cooling the composition comprising said denatured RNA to a temperature below 60°C or below 40°C , and/or
(ii) storing the composition comprising the denatured RNA in a break tank;
further comprising between (b) and (c);
optionally, the composition comprising said denatured RNA is stored in said break tank at 2-8° C. for 1-5 days ;
A method according to any one of claims 1-6 .

said in-line mixing of (c) is performed in less than 1 minute; and/or
the in-line mixing of (c) is performed in parallel with binding the mixture onto the oligo-dT resin ;
A method according to any one of claims 1-7 .

(i) the composition comprising the denatured RNA produced in (c) has a salt concentration of 50-500 mM and/or a conductivity of 5-85 mS/cm, and/or
(ii) at least 90% of the total RNA in the composition of (c) comprises denatured RNA;
The method according to any one of claims 1-8 .

(i) said bonding of (d) is performed at a temperature of 4° C. to 25° C. and/or in less than 20 minutes , and/or
(ii) the oligo-dT resin is a poly-dT-derivatized (poly)styrene-divinylbenzene (PS-DVB) bead resin with 2000 Angstrom pores, and/or
(iii) the RNA eluted from the oligo-dT resin comprises at least 90% or at least 95% poly A chain-containing mRNA;
A method according to any one of claims 1-9 .

the secondary structure of said RNA is monitored before and after (b) using ultraviolet detection, and/or
denaturation of the RNA in (b) is monitored using ultraviolet detection;
A method according to any one of claims 1-10 .

below,
mixing a high salt buffer in-line with a low salt denatured RNA composition comprising denatured ribonucleic acid (RNA) to produce a high salt composition comprising denatured RNA;
binding the denatured RNA to an oligo-dT resin;
method including.

(i) the in-line mixing is performed for less than 1 minute prior to binding the denatured RNA to the oligo-dT resin, and/or
(ii) said binding occurs in less than 20 minutes;
13. The method of claim 12 .

further comprising eluting the RNA from the oligo-dT resin in a low salt buffer;
optionally, said RNA eluted from said oligo-dT resin comprises at least 90% or at least 95% poly A strand containing mRNA.
14. A method according to claim 12 or 13 .

(i) heating a composition comprising RNA and a salt concentration of less than 20 mM to a temperature above 60° C. to produce a denatured RNA composition prior to in-line mixing with the high salt buffer ; or
and/ or,
(ii) desalting the RNA-containing mixture prior to in-line mixing with the high salt buffer to produce an RNA composition having a salt concentration of less than 20 mM; and
heating the RNA composition having a salt concentration of less than 20 mM to a temperature greater than 60° C. to produce the denatured RNA composition;
15. The method of any one of claims 12-14 , further comprising