A DNA nanomachine that maps spatial and temporal pH changes inside living cells

Abstract

DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upon stimulation by external triggers. Previously, the performance of DNA devices has been limited to in vitro applications. Here we report the construction of a DNA nanomachine called the I-switch, which is triggered by protons and functions as a pH sensor based on fluorescence resonance energy transfer (FRET) inside living cells. It is an efficient reporter of pH from pH 5.5 to 6.8, with a high dynamic range between pH 5.8 and 7. To demonstrate its ability to function inside living cells we use the I-switch to map spatial and temporal pH changes associated with endosome maturation. The performance of our DNA nanodevices inside living systems illustrates the potential of DNA scaffolds responsive to more complex triggers in sensing, diagnostics and targeted therapies in living systems.

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

“…1a ). Th e pHresponsive elements in this assembly are single-stranded cytosinerich tracts present as overhangs on the 5 ′ and 3 ′ ends of O1 and O2, respectively 10 . At acidic pH, these cytosines form CH + · C nonWatson Crick base pairs, forming an intramolecular I-motif 11 that yields the ' closed state ' of the I-switch.…”

“…Donor (Alexa 488) and acceptor (Alexa 647) fl uorophores, that undergo FRET, are attached to O1 and O2, respectively, resulting in the assembly showing high FRET at pH 5 and low FRET at pH 7. Th us, when a solution of the doubly labelled I-switch (I A488 / A647 ) is excited at the donor excitation wavelength (488 nm), the donor / acceptor (D / A) ratio shows a characteristic sigmoidal profi le as a function of pH 10 .…”

“…Previous work from our laboratory has outlined the construction and working of an autonomous DNA nanomachine that undergoes a conformational switch in response to pH changes 10 . Th is DNA nanomachine, called the I-switch, consists of two DNA duplexes connected to each other by a fl exible hinge and bearing cytosinerich single-stranded overhangs at the duplex termini ( Fig.…”

“…Th is forms the molecular basis of a fl uorescence resonance energy transfer (FRET)-based pH sensor using the I-switch. Previous work has shown that this DNA nanomachine could function autonomously inside living cells in culture by using it to map spatiotemporal pH changes associated with endosome maturation 10 . Th us far, this represents rare evidence of quantitative functionality of a rationally designed DNA nanodevice inside cultured cells.…”

The unnatural order of things

“…It has been suggested that certain devices have potential uses in medicine, but artificial DNA devices have until recently been limited to in vitro applications. Now, Yamuna Krishnan and colleagues have shown that a DNA nanomachine can function inside living cells 9 . The device, called an I-switch, is triggered by protons and acts as a pH sensor inside the cell.…”