Method of single-stranded RNA purification
Inventors
Peršič Debenjak, Špela • Černigoj, Urh • DOLENC, Darko • Gagnon, Peter S.
Assignees
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Publication Number
US-12227738-B2
Publication Date
2025-02-18
Expiration Date
Abstract
A method of single-stranded RNA purification comprising the steps applying a sample containing single-stranded RNA to a solid phase bearing dominantly or exclusively primary amino groups on its surface, at a pH value sufficient to bind at least predominantly the single-stranded RNA,eluting the single-stranded RNA from the surface of the solid phase by exposing the surface of the solid phase to increasing pH.
Core Innovation
The invention provides a method of single-stranded RNA purification in which a sample comprising single-stranded RNA is applied to a solid phase having a surface bearing amino groups. The amino groups comprise at least 50% primary amino groups, and the sample is applied at a first pH value in a range sufficient to bind the single-stranded RNA. The method then elutes the single stranded RNA from the surface by exposing the surface to an ascending pH gradient having a range of pH values higher than the first pH value, with the single-stranded RNA collected as a purified fraction.
The purification is directed to removing double-stranded RNA from mixtures that also contain single-stranded RNA, including mRNA in a specified base-length range. The approach relies on binding of single-stranded RNA at the first pH to the amino-functional solid phase and selective removal of double-stranded RNA during elution, attributed to differences in desorption behavior at lower versus higher pH.
In additional variations, double-stranded RNA and other contaminants such as DNA and proteins are removed before the pH gradient using optional wash steps at ionic strength conditions that differ from the elution buffer. The described method includes an ascending pH gradient, washing buffers having a higher ionic strength than the elution buffer's ionic strength, and, in some embodiments, a washing pH higher than the first pH value to keep single-stranded RNA adsorbed while desorbing remaining double-stranded RNA.
Claims Coverage
The independent claim defines a single-stranded RNA purification method based on selective binding to a primary-amino solid phase at a first pH and recovery by elution with an ascending pH gradient. Dependent claims further add inventive constraints and selectivity features, including defined single-stranded RNA size limits, wash-before-elution conditions using higher ionic strength and/or pH relationships, defined elution-buffer pH ranges, and behavior when double-stranded RNA is present in the sample.
Primary-amino solid phase binding at a first pH
Applying a sample comprising single-stranded RNA to a solid phase having a surface bearing amino groups, wherein the amino groups comprise at least 50% primary amino groups, at a first pH value in a range sufficient to bind the single-stranded RNA.
Ascending pH gradient elution of bound single-stranded RNA
Eluting the single stranded RNA from the surface of the solid phase by exposing the surface of the solid phase to an ascending pH gradient having a range of pH values that are higher than the first pH value, wherein the single-stranded RNA is collected as a purified fraction.
Single-stranded RNA length range purification
The method wherein the single-stranded RNA has a length between 1000 and 25,000 bases.
Wash-before-elution using higher ionic strength than elution
Washing the solid phase one or more times after applying the sample and before eluting the single-stranded RNA, using a washing buffer with a higher ionic strength than the elution buffer's ionic strength.
Wash pH higher than first pH to preserve ssRNA while removing dsRNA
Washing a solid phase one or more times after sample application and before eluting single-stranded RNA, using a washing buffer at a pH higher than the first pH value to keep single-stranded RNA adsorbed while desorbing remaining double-stranded RNA.
Defined elution-buffer pH range
Eluting single-stranded RNA from a solid phase using an elution buffer with a specified pH range.
Selective adsorption behavior when double-stranded RNA is present
The method for a sample that contains both double-stranded RNA and single-stranded RNA, where the single-stranded RNA selectively adsorbs to the solid phase during the applying step.
Across the claim set, protection centers on binding single-stranded RNA to a solid phase with a surface having dominantly primary amino groups at a first pH, followed by recovering purified single-stranded RNA using an ascending pH gradient to elute at higher pH. Dependent claims further specify ssRNA length constraints, wash-before-elution schemes based on ionic strength and/or washing pH to manage adsorption and desorption behavior, including in the presence of double-stranded RNA, and defined elution-buffer pH ranges.
Stated Advantages
Selectively removes double-stranded RNA from mixtures with single-stranded RNA during a pH-gradient elution approach on a primary-amino solid phase.
Uses an ascending pH gradient approach rather than requiring burdens associated with prior anion-exchange, reverse-phase, and low-capacity cellulose affinity approaches.
Improves removal of double-stranded RNA and other contaminants such as DNA and proteins by optional washing steps using ionic-strength and pH relationships relative to the elution buffer.
Documented Applications
Purification of single-stranded RNA from mixtures containing double-stranded RNA, including mRNA (noted as 1,000–25,000 bases in the partial content).
Purification workflows implemented in device formats including monolith, columns, membranes, and hydrogel, as described in the partial content.
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