Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy

Abstract

Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy silane (APTES), enabling one to obtain a smooth surface termed AP-mica. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations and in a broad range of pH. Another method utilizes aminopropyl silatrane (APS) to yield an APS-mica surface. The advantage of APS-mica compared with AP-mica is the ability to obtain reliable and reproducible time-lapse images in aqueous solutions. The chapter describes the methodologies for the preparation of AP-mica and APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The applications are illustrated with a number of examples.

Fig. 1

Scheme for the reaction of APTES with mica. APTES reacts with hydroxyl groups on the mica surface formed spontaneously after the mica is cleaved.

Fig. 2

APS-mica preparation. (a) Scheme for synthesis of 1-(3-aminopropyl)silatrane (APS, III) using APTES (I) and triethanolamine (II). APS: Molecular weight 232.36; molecular formula C₉H₂₀N₂O₃Si. (b) Scheme for reaction of APS (III) with mica surface. Similarly to APTES, APS reacts with hydroxyl groups on mica surface formed spontaneously after the cleavage. Three different stages of the reaction with the formation of adducts IV and V are shown. Note the last stage, exposure of APS-functionalized mica to water; it leads to dissociation of triethanolamine yielding product VI.

Fig. 3

The scheme for the vacuum distillation apparatus of APTES. The distillation flask is immersed into the heated oil bath. A regular faucet aspirator (Nalgene) creates a necessary vacuum for the distillation process.

Fig. 4

Photo of the setup used for the preparation of AP-mica. The mica sheet (3), clipped with the paper clip, is mounted at the top on a plastic rod (4); the two plastic caps with APTES (1) and DIPEA (2) reagents are placed at the bottom of the desiccator.

Fig. 5

AFM images of supercoiled 5.6 Kb plasmid DNA deposited onto AP-mica. Images were acquired with the MultiMode AFM (Nanoscope III controller) operating in Tapping Mode.

Fig. 6

AFM images of complexes of single stranded DNA binding protein (SSB) with DNA. The specially designed DNA substrate has a single stranded region (69 nucleotide) that binds the protein. The proteins appear as bright spherical features at one of the ends of the DNA substrate.

Fig. 7

Time-lapse AFM imaging of DNA cruciform dynamics captured in TE buffer with the use of AP-mica. The cruciform’s arms are indicated with arrows; the angle is ~60° in plate (a), one arm was in motion and is barely seen in plate (b), and both arms are almost parallel in plate (c). Images were acquired by the MultiMode AFM (Nanoscope IV) operating in Tapping Mode.

Fig. 8

Time-lapse AFM imaging of the nucleosome unwrapping, with the use of APS-mica. Frames 1, 2, and 3 correspond to different times of the unwrapping process. Schematically, the structure of the nucleosome at each stage is indicated with insets. Images were acquired by the MultiMode AFM (Nanoscope IV) operating in Tapping mode.