Two site-specific oligonucleotide conjugations per antibody allowing better signal-to-noise and a lower detection limit.


Aptamers are short RNA or DNA oligonucleotides, selected from a large random library, that bind to macromolecular (e.g., proteins) or small molecule targets with high affinity and specificity. DNA and RNA aptamers are also known as chemical antibodies due to their ability to specifically bind targets with affinities similar to that of their immunological counterparts. Aptamers are obtained by a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). In this process, a chemically derived random DNA or RNA library is challenged with a target, usually a protein or small molecule. Those members of the library that bind to the target are recovered and amplified, yielding an enriched library available for another round of selection and amplification. After several rounds of selection, the best binders compose the library and are typically cloned and characterized. The full potential of SELEX, however, has not been realized due to the limited ability of polymerases to recognize modified bases. A recent method termed SELection with Modified Aptamers (SELMA) was introduced for obtaining heavily chemically modified aptamers. In SELMA, an unmodified, double-stranded copy of the aptamer remains covalently bound to the single stranded, chemically modified aptamer.

Several technologies for the ultra-sensitive measurement and detection of proteins (immuno-PCR, proximity ligation, proximity extension, microfluidics-based single-cell proteomics) rely on the use of oligonucleotide-antibody conjugates. The most popular method for conjugation is via surface amine modification. The drawback to this method is limited or no specificity resulting in a broad range in the number of conjugates per antibody, general instability and interference with epitope binding, leading to a decrease in positive signal and increase in background signal in protein measurement or detection methods where antibody-DNA conjugates are used. Additionally, the generation of these conjugates is time-consuming, involving multiple steps and optimization.

Researchers at the University of Hawai‘i John A. Burns School of Medicine have developed novel antibody-oligonucleotide conjugates (AOCs) and a method for acquiring them. Incorporating activated esters (e.g., N-hydroxysuccinimidyl esters) into DNA or RNA aptamer libraries enables the selection of members of these libraries that can proficiently react with nucleophiles (e.g., primary amines) on a target protein to form a specific covalent bond with the target. Incorporation of NHS ester groups into nucleic acid libraries can be accomplished through a variety of available chemistries such as the highly efficient copper-catalyzed azide-alkyne cycloaddition (CuAAC, aka click chemistry). To mitigate the effect of chemical modification on the enzymatic amplification of nucleic acids, SELMA may be used, where an unmodified double stranded DNA copy of the aptamer is physically bound to the modified copy. Library generation involves incorporation of an alkyne-containing nucleotide analog (5-ethynyldeoxyuridine or 5-ethynyluridine) and addition of azido-NHS ester with click chemistry. NHS ester-modified libraries are incubated with the antibody and conjugates are isolated. The recovered library is amplified by polymerase chain reaction, the NHS ester-modified library is regenerated, and the selection procedure is repeated as necessary until the library is enriched for conjugate-forming sequences.

A major benefit of this technology is that the conjugation reaction is specific and limited to two conjugations per antibody, which will likely result in better signal-to-noise and a lower detection limit. Furthermore, the preparation of conjugates can be vastly simplified for the end user. Oligonucleotide synthesis companies can produce the NHS ester-modified oligonucleotide in house and the end user can simply move their antibody of interest to a compatible buffer and add the NHS ester-modified oligonucleotide, incubate for 1-2 hours and purify the conjugates if necessary.

Practical to this invention is the modularity of aptamers, where exogenous sequence can be attached at permissible points on the covalent aptamer enabling the conjugation of user-defined sequences with antibodies. The simplest attachment method is incorporation of exogenous sequence to either end of the aptamer sequence during oligonucleotide synthesis.

Site-specific antibody conjugation reactions are also relevant to therapeutics. Antibody-drug conjugates are becoming critical tools in treatment of disease. The state-of-the art in antibody-drug conjugation is either 1) introduction of a stable cysteine or non-natural reactive residue via site-directed mutagenesis and subsequent reaction with them or 2) the enzymatic processing and chemical modification of sugar moieties which are located on the Fc region of antibodies. However, these types of modifications can result in antibody instability or immunogenicity; therefore DNA or RNA aptamer-mediated site-specific modification of proteins may have a use in the production of antibody-drug conjugates. Additionally, conjugation of antibodies to drugs via nucleic acid aptamers allows for optimized drug release kinetics, where the nuclease susceptibility of the nucleic acid can be fine-tuned for detachment of the drug from the antibody in a specific timeframe.

Furthermore, the technology covers novel phosphoramidites for the chemical synthesis of NHS-ester modified aptamers. Although click-chemistry modification with N-hydroxysulfosuccinimidyl azidobenzoate after solid phase synthesis of the ethynyluracil-containing oligonucleotide is a viable method for NHS ester incorporation, the use of copper-catalyzed methods is sensitive to oxygen and exceedingly high copper concentrations are required in large scale reactions due to sequestration of the copper by DNA. Therefore, the CuAAC modification has been incorporated into a phosphoramidite, allowing for the chemical synthesis of triazolyl benzoate-modified oligonucleotides. The benzoate group is then activated with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for the addition of NHS or sulfo-NHS to yield the covalent aptamer.

Key Benefits

Two site-specific oligonucleotide conjugations per antibody (Fc region)
Fc conjugation prevents interference with epitope binding
Simplified antibody-oligonucleotide conjugation process
Conjugation reaction can be performed by the end user
Cost and time savings over current antibody conjugation methods


Site specific antibody-oligonucleotide conjugation
Antibody-Drug conjugation

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