Abstract: The present disclosure describes methods for analyzing mRNA poly(A) tail sequences with a high resolution to determine poly(A) tail length and heterogeneity. The method involves digesting an mRNA molecule to liberate a poly(A) tail, preparing a chromatographic sample comprising the mRNA poly(A) tails, preparing a second chromatographic sample comprising a reference sequence comprising a mRNA poly(A) tails having a predetermined length, separating the first and second samples by a chromatography method, which result in one or more chromatograms, and determining a sequence length of the mRNA poly(A) tails by comparing the chromatograms of the first and second samples. Chromatography methods for analyzing the poly(A) tail may include ultraviolet size-exclusion chromatography (SEC UV), ultraviolet ion-pair reversed-phase liquid chromatography (IP RP LC UV), ultra high performance liquid chromatography (UPHLC), and combinations thereof.

Abstract: The present disclosure describes methods, kits, and systems for digesting polyribonucleotides. The method involves selectively forming oligonucleotide (e.g., DNA:RNA or RNA:RNA) duplexes with single-stranded target RNA and then using sequence-specific nucleases that only act on RNA within duplexes to selectively cleave the target RNA into smaller fragments. Additional sequence-specific ribonucleases may be used to provide additional cuts of the target RNA at predetermined sites. By forming duplexes to increase the availability of nucleases that may be applied to cleave the single-stranded target RNA and selectively control where the target RNA is cleaved, the target RNA may be digested into fragments within controllable size ranges that are optimal for polynucletide analysis, such as by liquid chromatography and mass spectrometry.

Abstract: A device for separating analytes is disclosed. The device has a sample injector, sample injection needle, sample reservoir container in communication with the sample injector, chromatography column downstream of the sample injector, and fluid conduits connecting the sample injector and the column. The interior surfaces of the fluid conduits, sample injector, sample reservoir container, and column form a flow path having wetted surfaces. A portion of the wetted surfaces of the flow path are coated with an alkylsilyl coating that is inert to at least one of the analytes. The alkylsilyl coating has the Formula I: R1, R2, R3, R4, R5, and R6 are each independently selected from (C1-C6)alkoxy, —NH(C1-C6)alkyl, —N((C1-C6)alkyl)2, OH, ORA, and halo. RA represents a point of attachment to the interior surfaces of the fluidic system. At least one of R1, R2, R3, R4, R5, and R6 is ORA. X is (C1-C20)alkyl, —O[(CH2)2O]1-20, -(C1-C10)[NH(CO)NH(C1-C10)]1-20-, or -(C1-C10)[alkylphenyl(C1-C10)alkyl]1-20-.

Abstract: The present disclosure discusses a method of separating and/or purifying polynucleotides. The method includes injecting a sample into a chromatographic column that is packed with a porous sorbent having a pore size that substantially excludes the polynucleotides from the sorbent. This restricted access to the sorbent allows separation of large polynucleotides from each other and from smaller molecular weight impurities.

Abstract: A device for separating analytes is disclosed. The device has a sample injector, sample injection needle, sample reservoir container in communication with the sample injector, chromatography column downstream of the sample injector, and fluid conduits connecting the sample injector and the column. The interior surfaces of the fluid conduits, sample injector, sample reservoir container, and column form a flow path having wetted surfaces. A portion of the wetted surfaces of the flow path are coated with an alkylsilyl coating that is inert to at least one of the analytes. The alkylsilyl coating has the Formula I: R1, R2, R3, R4, R5, and R6 are each independently selected from (C1-C6)alkoxy, —NH(C1-C6)alkyl, —N((C1-C6)alkyl)2, OH, ORA, and halo. RA represents a point of attachment to the interior surfaces of the fluidic system. At least one of R1, R2, R3, R4, R5, and R6 is ORA. X is (C1-C20)alkyl, —O[(CH2)2O]1-20—, —(C1-C10)[NH(CO)NH(C1-C10)]1-20—, or —(C1-C10)[alkylphenyl(C1-C10)alkyl]1-20-.

Abstract: Methods of purifying targeted oligonucleotides within a reaction mixture using hydrophilic interaction liquid chromatography (HILIC) is disclosed. One of the methods in accordance with the present disclosure includes screening the targeted oligonucleotides within the reaction mixture with HILIC to create an initial reaction mixture profile; determining an elution percentage for the targeted oligonucleotides; focusing a HILIC elution gradient around the elution percentage of the targeted oligonucleotides; and purifying the targeted oligonucleotides with HILIC using the focused elution gradient at room temperature. Some embodiments can utilize mass triggering for fraction collection of the targeted oligonucleotides. Some embodiments can utilize UV triggering when the mass falls outside of the mass range of the MS detector.

Abstract: A device for separating analytes is disclosed. The device has a sample injector, sample injection needle, sample reservoir container in communication with the sample injector, chromatography column downstream of the sample injector, and fluid conduits connecting the sample injector and the column. The interior surfaces of the fluid conduits, sample injector, sample reservoir container, and column form a flow path having wetted surfaces. A portion of the wetted surfaces of the flow path are coated with an alkylsilyl coating that is inert to at least one of the analytes. The alkylsilyl coating has the Formula I: R1, R2, R3, R4, R5, and R6 are each independently selected from (C1-C6)alkoxy, —NH(C1-C6)alkyl, —N((C1-C6)alkyl)2, OH, ORA, and halo. RA represents a point of attachment to the interior surfaces of the fluidic system. At least one of R1, R2, R3, R4, R5, and R6 is ORA. X is (C1-C20)alkyl, —O[(CH2)2O]1-20—, —(C1-C10)[NH(CO)NH(C1-C10)]1-20-, or —(C1-C10)[alkylphenyl(C1-C10)alkyl]1-20-.