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. 2024 Dec 20;5(4):103329.
doi: 10.1016/j.xpro.2024.103329. Epub 2024 Sep 28.

Protocol to generate, purify, and analyze antibody-oligonucleotide conjugates from off-the-shelf antibodies

Tony Rady  1 , Victor Lehot  2 , Julien Most  2 , Stephane Erb  3 , Sarah Cianferani  3 , Guilhem Chaubet  2 , Nicolas Basse  4 , Alain Wagner  2
Affiliations

Protocol to generate, purify, and analyze antibody-oligonucleotide conjugates from off-the-shelf antibodies

Tony Rady et al. STAR Protoc. .

Abstract

Antibody-oligonucleotide conjugates (AOCs) are a fast-expanding modality for targeted delivery of therapeutic oligonucleotides to tissues. Here, we present a protocol to generate, purify, and analyze AOCs from off-the-shelf antibodies. We describe steps to conjugate single/double-stranded oligonucleotides bearing amine handles to linkers and, then, to antibodies using well-established chemistry. In addition, we provide details regarding the purification techniques and analytical methods suitable for AOC. This protocol can be applied for several purposes where AOC is a modality of interest. For complete details on the use and execution of this protocol, please refer to Rady et al.1.

Keywords: Antibody; Chemistry; Molecular/Chemical Probes.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Conjugation of amine-modified oligonucleotide using BCN-PEG6-PFP linker and hybridization with complementary sequence
Figure 2
Figure 2
Mechanism of pH-mediated cleavage of RNA molecules
Figure 3
Figure 3
Lysine conjugation of antibody using ABF
Figure 4
Figure 4
Conjugation of BCN-siRNA to antibody-azide to generate antibody-siRNA conjugates
Figure 5
Figure 5
Representative purification SEC profile obtained after conjugation of siRNA to antibody
Figure 6
Figure 6
Comparison of SEC-MS profile obtained when hybridization is performed before or after antibody conjugation (A) Representative mass spectrum of antibody-siRNA conjugates. (B) Representative mass spectrum of antibody-siRNA conjugates if the hybridization step is performed after the conjugation to the antibody.
See this image and copyright information in PMC

References

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