Sergey L. Andreev1, Valentina N. Buneva1,2, Georgy A. Nevinsky1,2
- Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
- Novosibirsk State University, Novosibirsk, Russia
In the literature, there are no available data on how anti-DNA antibodies recognize DNA. In the present work, to study the molecular mechanism of DNA recognition by antibodies, we have used anti-DNA IgGs from blood sera of patients with multiple sclerosis. A stepwise increase in ligand complexity approach was used to estimate the relative contributions of virtually every nucleotide unit of different single- (ss) and double-stranded (ds) oligonucleotides to their affinity for IgG fraction having high affinity to DNA-cellulose. DNA-binding site disposed on the heavy chain demonstrates higher affinity to different dNMPs (Kd = 0.63μM-3.8μM) than the site located on the light chain (28μM-170μM). The heavy and light chains interact independently forming relatively strong contacts with 2 to 4 nucleotides of short homo- and hetero-d(pN)2-9. Then the increase in the affinity of different d(pN)n became minimal, and at n ≥ 8 to 9, all dependencies reached plateaus: approximately 3.2nM to 20nM and approximately 200nM to 460nM for the heavy and light chains, respectively. A similar situation was observed for different ribooligonucleotides, in which their affinity is 6-fold to 100-fold lower than that for d(pN)n. Transition from ss to ds d(pN)n leads to a moderate increase in affinity of ligands to DNA-binding site of heavy chains, while light chains demonstrate the same affinity for ss and ds d(pN)n. Long supercoiled DNA interacts with both heavy and light chains with affinity of approximately 10-fold higher than that for short oligonucleotides. The thermodynamic models were constructed to describe the interactions of IgGs light and heavy chains with DNA.
CovalX Technology Used (Click each option to learn more)
To analyze IgG light and heavy chain interactions with ODNs, crosslinking was performed using 0.5 mM glutaric dialdehyde 25 mM HEPES-NaOH and modification of Abs was performed using 0.27 mM of a CovalX cross linking reagent (ethylene glycol bis- succinimidylsuccinate, iodoacetic acid N-hydroxysuccinimide ester and octaneodic acid di-N-hydroxysuccinimide ester) HEPES-NaOH.