Recent developments in oligonucleotide conjugation

Abstract

Synthetic oligonucleotides (ONs) are being investigated for various therapeutic and diagnostic applications. The interest in ONs arises because of their capability to cause selective inhibition of gene expression by binding to the target DNA/RNA sequences through mechanisms such as antigene, antisense, and RNA interference. ONs with catalytic activity (ribozymes and DNAzymes) against the target sequences, and ability to bind to the target molecules (aptamers), ranging from small molecules to proteins, are also known. Therefore ONs are considered potentially useful for the treatment of viral diseases and cancer. ONs also find use in the design of DNA microchips (a powerful bio-analytical tool) and novel materials in nanotechnology. However, the clinical success achieved so far with ONs has not been satisfactory, and the major impediments have been recognised as their instability against nucleases, lack of target specificity, and poor uptake and targeted delivery. Tremendous efforts have been made to improve the ON properties by either incorporating chemical modifications in the ON structure or covalently linking (conjugation) reporter groups, with biologically relevant properties, to ONs. Conjugation is of great interest because it can be used not only to improve the existing ON properties but also to impart entirely new properties. This tutorial review focuses on the recent developments in ON conjugation, and describes the key challenges in efficient ON conjugation and major synthetic approaches available for successful ON conjugate syntheses. In addition, an overview on major classes of ON conjugates along with their use in therapeutics, diagnostics and nanotechnology is provided.

Efficient Synthesis of DNA Conjugates by Strain‐Promoted Azide–Cyclooctyne Cycloaddition in the Solid Phase

“…Following removal of particulate matter by filtration, the filtrate was evaporated to yield the crude product as a white solid, which was washed with CH 2 Cl 2 (5 ϫ 10 mL) to yield pure 7 (745 mg, 86 %). 1 …”

“…[1] The high chemical stability and orthogonal reactivity of azides and alkynes has made the copper-catalysed 1,3-dipolar cycloaddition a highly attractive conjugation strategy in materials science and biotechnology. [2] However, in spite of its vast successes, this reaction it is not ideal for oligonucleotide applications; Cu I -mediated oxidative DNA degradation can be problematic, and copper ion contamination of the final product can culminate in issues of cytotoxicity and nucleic acid hydrolysis.…”

A Highly Convenient Procedure for Oligodeoxynucleotide Purification

“…Synthetic oligodeoxynucleotides (ODNs) have found wide applications in areas such as molecular biology, synthetic biology and antisense drug development [1]. Their syntheses have been automated.…”

Oligonucleotide Tagging for Copper-Free Click Conjugation

“…Generally, such applications require the introduction of a suitable handle in an oligonucleotide to enable selective conjugation to a functionality of interest [6][7][8]. For example, attachment of a cell-penetrating ligand is the most commonly applied strategy to tackle the low internalization rate of ONs into target cells [2], currently the main bottleneck in oligonucleotide-based therapeutics (antisense, siRNA).…”

“…Similarly, the preparation of oligonucleotide-based microarrays requires the selective immobilization of ONs on a suitable solid surface, e.g., glass [1]. Conventional post-synthetic labeling protocols, based on amide bond formation or sulfide-based chemistry [8][9][10] typically suffer from low yield and long reaction times and often require a high concentration of the biomolecule in combination with a large excess of coupling partner. One promising alternative to the traditional conjugation technologies involves the copper-catalyzed cycloaddition of alkynes and azides, a procedure commonly referred to as "click reaction" [11,12].…”