Selective recognition of a DNA G-quadruplex by an engineered antibody

Abstract

Particular guanine rich nucleic acid sequences can fold into stable secondary structures called G-quadruplexes. These structures have been identified in various regions of the genome that include the telomeres, gene promoters and UTR regions, raising the possibility that they may be associated with biological function(s). Computational analysis has predicted that intramolecular G-quadruplex forming sequences are prevalent in the human genome, thus raising the desire to differentially recognize genomic G-quadruplexes. We have employed antibody phage display and competitive selection techniques to generate a single-chain antibody that shows >1000-fold discrimination between G-quadruplex and duplex DNA, and furthermore >100-fold discrimination between two related intramolecular parallel DNA G-quadruplexes. The amino acid sequence composition at the antigen binding site shows conservation within the light and heavy chains of the selected scFvs, suggesting sequence requirements for G-quadruplex recognition. Circular dichroism (CD) spectroscopic data showed that the scFv binds to the prefolded G-quadruplex and does not induce G-quadruplex structure formation. This study demonstrates the strongest discrimination that we are aware of between two intramolecular genomic G-quadruplexes.

Figure 1

(A) Target DNA sequences and their binding interactions with hf2 scFv. DNA sequences of c-kit1, c-kit2, bcl2Mid, MYC22-G14T/G23T, and duplex DNA. Guanines involved in the formation of a G-tetrad are shown in bold. (B) ELISA binding curves of hf2-DNA interactions. (Top) hf2 binding c-kit2 G-quadruplex, K_d of 1.6 ± 0.4 nM (blue); c-kit1 G-quadruplex, K_d of 486 ± 80 nM (green); and duplex DNA, K_d of 4.6 ± 0.3 μM (black). (Bottom) hf2 binding bcl2Mid G-quadruplex, K_d of 21 ± 2 nM (red) and MYC22-G14T/G23T G-quadruplex, K_d of 26 ± 3 nM (cerise). (C) Sensorgram overlays of hf2 scFv at 6.1 nM (red), 12.2 nM (green), 25 nM (cyan), and 50 nM (blue) concentrations interacting with the c-kit2 G-quadruplex. The black lines indicate the Biaevaluation 3.0 global fitting of the binding kinetics.

Figure 2

CD spectra of 1 μM c-kit1 (yellow), 1 μM c-kit2 (blue), and 1 μM c-kit2 with 1 μM hf2 (red) in 50 mM Tris-HCl containing 100 mM KCl.