Solid support for synthesizing nucleic acid sequences and methods for making and using
Inventors
Beaucage, Serge L. • Grajkowski, Andrzej M.
Assignees
US Department of Health and Human Services
Publication Number
US-11987599-B2
Publication Date
2024-05-21
Expiration Date
2041-06-28
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Abstract
Disclosed herein are embodiments of a solid support suitable for synthesizing nucleic acid sequences. The solid support may have a structure according to Formula I, where CPG is controlled pore glass, and m, n, x, y, R1 and R2 are as defined herein.Also disclosed are methods for making and using the solid support, kits including solid support, and a universal linker phosphoramidite suitable for use in the solid support.
Core Innovation
The invention relates to a solid support suitable for synthesizing nucleic acid sequences. The solid support has a specific chemical structure incorporating controlled pore glass (CPG) with various defined repeating units and substituents including hexaethylene glycol phosphate spacers. Embodiments of the solid support exhibit improved nucleic acid synthesis performance, particularly yielding nucleic acid compositions with a reduced amount of process-related impurities compared to those produced using current commercially available solid supports.
The disclosed solid support structures enable the reduction of shorter than full-length nucleic acid sequence impurities and contamination from partially protected sequences or side-products resulting from standard phosphoramidite synthesis. The solid support can be functionalized with multiple hexaethylene glycol spacers and possess defined loading densities, backbone lengths, and protecting groups tailored to improve synthesis efficiency and product purity.
The problem being solved addresses the challenges inherent in synthetic nucleic acid production, including instabilities, poor delivery, dose-related toxicities, and particularly the presence of process-related impurities such as shorter nucleic acid sequences and partially modified bases. These impurities are difficult to remove and may trigger immune responses or adverse therapeutic effects. The disclosed solid support aims to minimize the formation of such impurities to improve the safety and efficacy of nucleic acid-based drugs.
Claims Coverage
The patent includes multiple independent claims covering the solid support structure, methods for synthesizing nucleic acid sequences using the solid support, and kits comprising the solid support. The inventive features focus on the chemical structure of the solid support, specific substituents, and applications in nucleic acid synthesis.
Solid support comprising controlled pore glass and defined hexaethylene glycol phosphate spacers
A solid support defined by Formula I, where controlled pore glass (CPG) is functionalized with multiple hexaethylene glycol phosphate repeating units having parameters m, n, x, y, and substituents R1 and R2 within specified ranges. The backbone length from the silicon atom to the R2 moiety is from 50 to 400 atoms, improving synthesis efficiency.
Selection of specific protecting groups and base modifications
Incorporation of protecting groups suitable for nucleic acid synthesis including 4,4′-dimethoxytrityl (DMTr) as a protecting group, and exocyclic amine-protected nucleic acid bases such as benzoyl (Bz), isobutyryl (iBu), and phenoxyacetyl (Pac). The support accommodates protected DNA or RNA nucleosides to enhance synthesis fidelity.
Solid support functionalized with nucleic acid sequences
The support may have R5 as a nucleic acid sequence comprising DNA or RNA sequences, including antisense DNA, antisense RNA, microRNA (miRNA), small interfering RNA (siRNA), or repeat-associated small interfering RNA (rasiRNA), enabling direct synthesis and attachment on the support.
Method for synthesizing nucleic acid sequences using the disclosed solid support
A method comprising loading the solid support into a DNA/RNA synthesizer and operating the synthesizer to produce desired nucleic acid sequences, with preferred embodiments where R5 is a protecting group (e.g., DMTr) and R6 is tert-butyldimethylsilyl (TBDMS).
Kits comprising the solid support and protected nucleosides
Kits containing the solid support along with protected 2′-deoxynucleosides, ribonucleosides, or chemically modified nucleosides with protected exocyclic amines, facilitating efficient nucleic acid synthesis.
The claims collectively cover the specific chemical structure of the solid support with hexaethylene glycol phosphate units, protecting group selections, nucleic acid sequence attachments, methods of nucleic acid sequence synthesis using the support, and kits including the support and protected nucleosides, focusing on minimizing process-related impurities and improving nucleic acid synthesis purity.
Stated Advantages
Significant reduction in process-related impurities during solid-phase synthesis of nucleic acid sequences compared to commercial LCAA-CPG supports.
Improved purity of synthesized DNA and RNA sequences leading to safer and more efficacious nucleic acid-based drugs.
Higher yield and efficiency in nucleic acid synthesis due to better accessibility of activated phosphoramidites and reagents to the leader nucleoside.
Compatibility with standard automated DNA/RNA synthesizers and adaptability with protected nucleosides and universal linker phosphoramidites.
Documented Applications
Production of synthetic nucleic acid sequences, including DNA and RNA sequences, for use in nucleic acid-based drugs such as antisense DNA, small interfering RNA (siRNA), microRNAs (miRNAs), and repeat-associated small interfering RNA (rasiRNA).
Manufacture of synthetic nucleic acids for therapeutic applications including antisense therapy, RNA interference, mRNA, and genome editing applications.
Solid-phase synthesis of nucleic acid sequences using automated DNA/RNA synthesizers with improved purity and reduced process-related impurities.
Use of kits comprising the solid support and protected nucleosides to facilitate synthesis of modified nucleic acid sequences with reduced impurities.
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