The Functional Role of Loops and Flanking Sequences of G-Quadruplex Aptamer to the Hemagglutinin of Influenza a Virus

Abstract

Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5'- and 3'-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385-hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer.

Keywords: DNA aptamer; G-quadruplex; affinity; hemagglutinin; influenza virus; structure−activity relationship.

Figure 1

The proposed structure of RHA0385 as a parallel G-quadruplex with three propeller loops; one of them is 7 nucleotides long (a major loop), whereas two others are single-nucleotide (minor loops).

Figure 2

BLI binding assays of aptamer to HA of influenza A virus: (a) two strategies of the experiment with immobilization of recombinant HA and immobilization of the aptamer. Influenza virus picture was taken from Kisscc0 website [24]; sensorgrams for the complex of RHA0385 aptamer with (b) rHA and (c) vHA.

Figure 3

Aptamer binding vHA measured by BLI (concentration of vHA was set as to 14 nM): (a) RHA0385 and aptamers with flanking sequences truncated; (b) G7nt and aptamers with single-nucleotide loops; (c) RHA0385 and aptamers with major loop altered. (d) Hemagglutination inhibition assay of 3.6 nM vHA with RHA0385, G7-TTA_A_C and G7n5t at different concentrations.

Figure 4

Circular dichroism spectroscopy of aptamers to HA: (a) CD spectra of RHA0385 and its variants obtained at the same conditions (20 °C, 2 µM solution in sodium-potassium phosphate buffer pH 7.4 in the presence of 10 mM K⁺); (b) normalized curves of the CD melting of RHA0385 and its variants.

Figure 5

The plots of rate constants (a) association rate constants, (b) dissociation rate constants versus the aptamer’s loop length for complexes aptamer–rHA.