Nucleic Acid Therapeutics Applications Notebook Table of Contents

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Nucleic Acid Therapeutics Applications Notebook Table of Contents Nucleic acid therapeutics applications notebook Table of Contents Introduction .....................................................................................................................................................................................3 Analysis of Nucleic Acids ................................................................................................................................................................5 Analysis of Phosphorothioate Oligonucleotides ...............................................................................................................................6 Analysis of ss- and dsRNA ...............................................................................................................................................................8 Anion-Exchange ESI/MS of Oligonucleotides—Analysis of Impurities in the Target Oligonucleotide Product .......................12 Anion-Exchange ESI/MS of Oligonucleotides—Analysis of Oxidation Products of Purified Biotinylated Oligonucleotides ..................................................................................................................................15 Analysis of 2´, 5´-Linkage Isomers in RNA and DNA, and Elucidation of Aberrant Linkage Position ......................................17 Analysis of Nucleoside Mono-, Di-, and Triphosphates ................................................................................................................22 Method Development Using Anion-Exchange Chromatography for Nucleic Acids ....................................................................23 Application Notes ..........................................................................................................................................................................29 High-Resolution Analysis and Purification of Oligonucleotides with the DNAPac PA100 Column ...........................................30 Product Focus: Systems ...............................................................................................................................................................36 Biocompatible LC Systems .............................................................................................................................................................37 Orbitrap MS Instruments .................................................................................................................................................................38 Product Focus: Consumables ......................................................................................................................................................39 Nucleic Acid Columns ...................................................................................................................................................................40 Column Selection Guide for Oligonucleotide Separations ...........................................................................................................41 Product Focus: Software .............................................................................................................................................................42 Chromeleon 7 Software ...................................................................................................................................................................43 References .......................................................................................................................................................................................44 Select Peer-Reviewed Publications .................................................................................................................................................45 Introduction THERAPEUTIC NUCLEIC ACIDS MARKETPLACE There are various models in which the active pharma- Over the last 15–20 years, development of nucleic acid ceutical ingredient (API) is mainly RNA. The first, short in- therapeutics has exploded from a modest but dedicated terfering RNA (siRNA) employs specific short (19–23 base) effort by specialty research and development organizations RNA sequences that are incorporated into a cellular complex into a widespread enterprise fully embraced by all major RNA-induced silencing complex (RISC) that functions to pharmaceutical firms. This explosion is supported by degrade RNA complementary to the siRNA. This results in the discovery of Ribonuclease H (RNase H) function for diminution of a very specific subset of cytosolic mRNA that antisense oligonucleotides, and the more recent realization is targeted by the administered sequence, severely limiting that cellular mechanisms for control of gene expression and the expression of the target gene product. transcriptome regulation can be manipulated to produce Immunostimulatory RNA (isRNA) is a mode that therapeutics for ailments that were previously considered employs sequence specific motifs, some of which are very unresponsive to drugs. Recently, options for therapeutics simple (e.g., CpG where the C is not methylated). Introduc- with exquisite selectivity also provide the opportunity for tion of the specific RNA sequence motif induces a cellular new therapeutic modes and intellectual properties. For immune system directing degradation of foreign nucleic example, Macugen successfully passed clinical trials and acids. This is mediated by the cellular toll-like receptors release protocols to become the first approved aptamer (TLRs), TLR7, 8, and 9. therapeutic. This success encourages further efforts and miRNA is another RNA model that employs a series opportunities for therapeutic nucleic acid developers. of cellular complexes to degrade mRNA in a sequence- specific fashion. This is almost certainly the pathway that THERAPEUTIC NUCLEIC ACID MODELS is conscripted by the siRNA approach, but represents a Several models of nucleic acid therapeutics have been mechanism whereby the cell controls expression and turn- described, including antisense oligodeoxynucleotides; over of specific mRNA. Many miRNA families described ribonucleic acid (RNA) interference; immunostimulatory regulate different functions in plants and animals, including nucleic acids; microRNA (miRNA) (along with other neogenesis and malignant transformations. The sources of noncoding RNA modes); and aptamers. the endogenous miRNA in humans include highly conserved Antisense oligonucleotides are nucleic acid therapeu- noncoding regions on chromosomes (initially thought to be tics that are injected into a person for distribution to target junk DNA) and sequences inserted in specific genes. There organs by various means. From there, these therapeutics are other noncoding RNA (ncRNA) models, with important recruit RNase H to degrade the resulting deoxyribonucleic ncRNA motifs being reported regularly. These may arise acid (DNA)/RNA hybrids, thus minimizing translation of the from endogenous variations on the miRNA process de- target messenger RNA (mRNA). scribed above. 3 Introduction Aptamers are synthetic nucleic acids selected to inter- 3) anion exchange-HPLC-electrospray ionization mass act with specific structures—such as cell surface receptors, spectrometry (ESI-MS) of derivatized oligonucleotides; proteins, and other chemical signatures—in a highly specific 4) preparative and analytical separations of nucleic acid manner. They are typically modified with protecting groups diastereoisomers; 5) coupling of anion-exchange HPLC to (2´-O-methyl phosphorothioate linkages, DNA bases, ESI-MS using automated desalting with a Titanium-based sometimes inverted 3´-3´ linkages, and conjugation to LC system; 6) separation and analysis of RNA contain- polyethylene glycol). Most therapeutic targets for aptamers ing aberrant linkage isomers (not resolved by single-stage have been extracellular, so they operate in the circulatory ESI-MS alone, including identification of the position of the system where these protecting groups prolong their circula- aberrant linkages); 7) analysis of nucleoside mono-, di-, and tory half-life. triphosphates; and 8) useful information for new method development. BIOSEPARATION SOLUTIONS FOR NUCLEIC ACID THERAPEUTICS THERMO SCIENTIFIC AND DIONEX INTEGRATED SYSTEMS Thermo Scientific™ offers solutions for the purification Dionex™ Products are now a part of the Thermo Scien- and analysis of essentially all classes of oligonucleotides and tific brand, creating exciting new possibilities for scientific their modifications. Purification efforts are supported by both analysis. Now, leading capabilities in LC, ion chroma- high-capacity Thermo Scientific DNASwift™ SAX-1S Mono- tography (IC), and sample preparation are together in one lith Columns (5 × 150 mm) and DNAPac™ PA-100 Semi-Pre- portfolio with those in MS. Combining Dionex’s innovation parative Columns (9 × 250 mm and 22 × 250 mm). These are in chromatography with Thermo Scientific’s leadership housed in bioinert column bodies to prevent column fouling position in MS, a new range of powerful and simplified by corrosion of stainless steel frits and tubing, and typically workflow solutions is now possible. employ either PEEK™ or Titanium high-performance liquid These Thermo Scientific integrated solutions can expand chromatography (HPLC) systems. The solutions described your capabilities and provide tools for new possibilities: in this notebook include: 1) analysis of phosphorothioate • IC and MS oligonucleotides; 2) analysis of single-stranded (ss) and • LC and MS double-stranded
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