Abstract
Oligonucleotide (ON) conjugates are increasingly important tools for various molecular diagnostics, nanotechnological applications, and for the development of nucleic acid-based therapies. Multiple labeling of ONs can further equip ON-conjugates and provide improved or additional tailored properties. Typically, the preparation of ON multiconjugates involves additional synthetic steps and/or manipulations in post-ON assembly. This report describes the simplified methodology allowing for multiple labeling of ONs on a solid support and is compatible with phosphodiester as well as phosphorothioate (PS) ONs. The current approach utilizes two novel alkyne- and amino-functionalized linker phosphoramidites that can be readily synthesized from a common aminodiol intermediate in three steps. The combination of new linkers provides orthogonal functionalities, which allow for multiple attachments of similar or varied moieties. The linkers are incorporated into ONs during automated solid-phase ON synthesis, and the conjugation with functional entities is achieved by either amide bond formation or by copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The versatility of the approach is demonstrated by the synthesis of 5′-site ON multiconjugates with small molecules, peptides, and fatty acids as well as in the preparation of an internal peptide–ON conjugate.
Highlights
Synthetic oligonucleotides (ONs) are widely used for various applications in life sciences, biotechnology, diagnostics, and represent a major part of currently used nucleic acid therapeutics.[1,2] The conjugation of ONs with various ligands, reporter groups, peptides, or proteins can provide the ON conjugate with new valuable properties for various nanotechnological applications and pharmaceutical development.[3]
ON conjugates are emerging as an important subgroup of this class of macromolecules and are considered promising candidates for clinical use.[3−5] Despite many ongoing clinical trials and several recent FDA approvals,[6] the ON delivery to the site of action remains one of the main challenges of ON-based therapeutics.[7−9] ON cellular uptake and the ability to target specific tissues can be significantly enhanced by the conjugation of uptake promoting molecules, such as N-acetylgalactosamine (GalNAc) for targeting the liver hepatocytes,[10−14] or glucagon-like peptide[1] (GLP-1) for the delivery to pancreatic β-cells.[15]
Several approaches have been established for the preparation of ON multiconjugates using copper(I)-catalyzed “click” chemistry, including preparation of DNA constructs with different fluorescent labels by sequential conjugation/deprotection reactions,[37] in combination with thiol-Michael-type addition,[38] using a “click/reverse-click” procedure,[39] or by solid-phase CuAAC approach for the preparation of ONGalNAc dendrimer conjugates.[27]
Summary
Synthetic oligonucleotides (ONs) are widely used for various applications in life sciences, biotechnology, diagnostics, and represent a major part of currently used nucleic acid therapeutics.[1,2] The conjugation of ONs with various ligands, reporter groups, peptides, or proteins can provide the ON conjugate with new valuable properties for various nanotechnological applications and pharmaceutical development.[3].
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