Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin-biotin recognition

Abstract

We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin–biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics.

Comparative Electrostatic Force Microscopy of Tetra‐ and Intra‐Molecular G4‐DNA

“…[6] When using the same number of tetrads for the construction of the tetra-molecular G4-DNA and the 2 intra-molecular G4-DNA, the former are thicker and shorter than the latter molecules. [5] This suggests that the folding orientation of the strands, which form the backbone, affects the molecular structure, i.e. the tetrad unit and the tetrad-tetrad stacking.…”

“…This type of intra-molecular G4-DNA possessed distinct polarizability, in contrast to native dsDNA, which gave no discernible signal. [4] That study was followed by the synthesis and EFM measurements of tetra-molecular G4-DNA [5] (Figure 1b, right), which is stable in the absence of metal cations, and was shown to possess a clear signal of polarizability as well.For intra-molecular and tetra-molecular G4-DNA that are composed of the same number of tetrads, AFM imaging [5] showed that tetra-molecular G4-DNA, adsorbed on a mica surface, resembles a tadpole with a large avidin "head" and a straight elongated "tail" (Figure 1c), while intra-molecular G4-DNA appears longer and thinner and is more flexible on the surface, forming curved lines. In this study, we synthesized two pairs of corresponding lengths of tetramolecular G4-DNA and intra-molecular G4-DNA (see Figure 1c and Figure S1).…”

“…This type of intra-molecular G4-DNA possessed distinct polarizability, in contrast to native dsDNA, which gave no discernible signal. [4] That study was followed by the synthesis and EFM measurements of tetra-molecular G4-DNA [5] (Figure 1b, right), which is stable in the absence of metal cations, and was shown to possess a clear signal of polarizability as well.…”

“…[5] This suggests that the folding orientation of the strands, which form the backbone, affects the molecular structure, i.e. the tetrad unit and the tetrad-tetrad stacking.…”

“…For intra-molecular and tetra-molecular G4-DNA that are composed of the same number of tetrads, AFM imaging [5] showed that tetra-molecular G4-DNA, adsorbed on a mica surface, resembles a tadpole with a large avidin "head" and a straight elongated "tail" (Figure 1c), while intra-molecular G4-DNA appears longer and thinner and is more flexible on the surface, forming curved lines. In this study, we synthesized two pairs of corresponding lengths of tetramolecular G4-DNA and intra-molecular G4-DNA (see Figure 1c and Figure S1).…”