Single-molecule pH sensors have been developed by utilizing molecular imaging of pH-responsive shape transition of nanomechanical DNA origami devices with atomic force microscopy (AFM). Short DNA fragments that can form i-motifs were introduced to nanomechanical DNA origami devices with pliers-like shape (DNA Origami Pliers), which consist of two levers of 170-nm long and 20-nm wide connected at a Holliday-junction fulcrum. DNA Origami Pliers can be observed as in three distinct forms; cross, antiparallel and parallel forms, and cross form is the dominant species when no additional interaction is introduced to DNA Origami Pliers. Introduction of nine pairs of 12-mer sequence (5′-AACCCCAACCCC-3′), which dimerize into i-motif quadruplexes upon protonation of cytosine, drives transition of DNA Origami Pliers from open cross form into closed parallel form under acidic conditions. Such pH-dependent transition was clearly imaged on mica in molecular resolution by AFM, showing potential application of the system to single-molecular pH sensors.
Materials. Staple DNA strands were purchased from Sigma Genosys (Japan) or Hokkaido System Science Co., Ltd. (Japan) then used without further purification. Synthesis of bis-PNA. The bis-PNA was synthesized by Boc chemistry, purified by reversed-phase HPLC, and characterized by MALDI-TOF mass spectrometry (Bruker, AutoFLEX). The detailed protocols were described elsewhere 1. Preparation of nanomechanical DNA origami devices. Formation of nanomechanical DNA origami was performed with M13mp18 ssDNA (4 nM, Takara, Japan), staples and zipper elements (16 nM for each strand) in a solution containing Tris (40 mM), acetic acid (20 mM), EDTA (10 mM), and magnesium acetate (12.5 mM, 1 X TAE/Mg buffer, 100 µL). This mixture was cooled from 90˚C to 25˚C at a rate of-1.0˚C/min using a PCR thermal cycler to anneal the strands.
Significant enhancement of single-molecular binding to a miRNA target and bidentate and asymmetric conjugation of two distinct thiolated DNA strands to single gold nanoparticles (AuNPs) were visibly demonstrated, by introducing two groups of ligands into our nanomechanical DNA origami devices (DNA pliers) to construct allosterically controllable systems.
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