Antibody-oligonucleotide conjugates (AOCs) are used in techniques such as immuno-PCR, proximity ligation assay (PLA) and proximity extension assay (PEA), and are also being examined as potential therapeutic agents for gene silencing. There are many approaches to create AOCs, ranging from stochastic lysine-based conjugations to site-directed methods, which involve genetic engineering to introduce reactive handles at predetermined positions on the antibody.
The random approaches utilize heterobifunctional linkers which have two different reactive groups, one a highly reactive amine-directed succinimidyl ester (also called NHS ester), which is first reacted with the antibody, and a second more stable functional group (e.g. maleimide, DBCO), which is subsequently reacted with a chemical handle (e.g., thiol, azide, respectively) added during oligonucleotide synthesis to the 5’ or 3’ end.
In an unusual approach, Li & Moellering (Ref 1) used instead a homobifunctional crosslinking reagent, disuccinimidyl suberate (DSS), which has two identical reactive groups, i.e., two succinimidyl esters, separated by an 8-atom spacer. DSS was reacted in 250-fold molar excess over an aminated oligo (to reduce the probability of oligo-to-oligo crosslinking) and resulted in oligo derivatives with the potentially unstable terminal succinimidyl ester. The reaction mixture was purified by hplc in acidic buffers and the sample lyophilized in aliquots from a 0.1% trifluoroacetic acid solution. The reactive groups under these acidic conditions were stable for many months, as assessed by MALDI-TOF.
The DSS-activated oligos could be reacted directly with native antibodies. By avoiding the antibody processing steps required with heterobifunctional reagents, conjugation could be carried out at 10ug scale, thus avoiding wastage of valuable antibody material. For labs equipped with hplc and lyophilizers, an attractive feature of this approach is that DSS linker is about one-tenth the price of typical heterobifunctional reagents. Moreover, amination is one of the cheapest oligo modifications.
Antibody conjugations were carried out overnight with a 4-fold molar excess of DSS-modified oligos. Three commercial antibodies were conjugated and assessed by SDS-PAGE followed by Sybr-gold staining. Conjugates with one or two attached oligonucleotides dominated the mixture, with some antibodies carrying three, or possibly four oligos, with the antibody heavy chains the main sites of attachment (Ref 1 Fig 2b). Some free antibody was also evident on non-reducing gels (Ref 1, Fig 2c).
At higher scales of production, chromatographic separation of the small amount of free antibody would be feasible, though an alternative approach would be to increase slightly the input of DSS-modified oligo, which would avoid adding extra steps to the process.
Oligonucleotide conjugates prepared with polyclonal anti-GAPDH enzyme performed as well as reagents prepared by DBCO-based chemistry in PLA assays with pure protein. Using DSS-linked AOCs the authors showed quantitation of GAPDH in whole cell lysates with a limit of detection (LOD) in the sub-picogram range of whole proteome.
Conclusions
Although homobifunctional reagents are generally seen as being inferior to heterbifunctionals, the use of a DSS linker combined with hplc purification and lyophilization from 0.1% TFA allowed the production of a stable activated oligonucleotide derivative. Direct conjugation to native antibodies at 10ug scale was possible and the resulting AOCs were functional in PLA.
Reference