Souvik Modi

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

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.

Two DNA nanomachines map pH changes along intersecting endocytic pathways inside the same cell

DNA is a versatile scaffold for molecular sensing in living cells, and various cellular applications of DNA nanodevices have been demonstrated. However, the simultaneous use of different DNA nanodevices within the same living cell remains a challenge. Here, we show that two distinct DNA nanomachines can be used simultaneously to map pH gradients along two different but intersecting cellular entry pathways. The two nanomachines, which are molecularly programmed to enter cells via different pathways, can map pH changes within well-defined subcellular environments along both pathways inside the same cell. We applied these nanomachines to probe the pH of early endosomes and the trans-Golgi network, in real time. When delivered either sequentially or simultaneously, both nanomachines localized into and independently captured the pH of the organelles for which they were designed. The successful functioning of DNA nanodevices within living systems has important implications for sensing and therapies in a diverse range of contexts.

The PNA–DNA hybrid I-motif: implications for sugar–sugar contacts in i-motif tetramerization

We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands from an equimolar mixture of a C-rich DNA and analogous PNA sequence. Nano-electrospray ionization mass spectrometry confirmed the formation of a tetrameric species, composed of PNA–DNA heteroduplexes. Thermal denaturation and CD experiments revealed that the structure was held together by C-H+-C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H+-C base pairs per heteroduplex. The imino protons are protected from D2O exchange suggesting intercalation of the heteroduplexes as seen in DNA4 i-motifs. FRET established the relative DNA and PNA strand polarities in the hybrid. The DNA strands were arranged antiparallel with respect to one another. The same topology was observed for PNA strands. Fluorescence quenching revealed that both PNA–DNA parallel heteroduplexes are intercalated, such that both DNA strands occupy one of the narrow grooves. H1′–H1′ NOEs show that both heteroduplexes are fully intercalated and that both DNA strands are disposed towards a narrow groove, invoking sugar–sugar interactions as seen in DNA4 i-motifs. The hybrid i-motif shows enhanced thermal stability, intermediate pH dependence and forms at relatively low concentrations making it an ideal nanoscale structural element for pH-based molecular switches. It also serves as a good model system to assess the contribution of sugar–sugar contacts in i-motif tetramerization.

The RNA2–PNA2 hybrid i-motif—a novel RNA-based building block

We report the formation of a hybrid RNA2-PNA2 i-motif comprised of two RNA and two PNA strands based on the sequence specific self assembly of RNA, with potential as a building block for structural RNA nanotechnology.

A method to map spatiotemporal pH changes inside living cells using a pH-triggered DNA nanoswitch.

A few cellular compartments maintain acidic environments in their interiors that are crucial for their proper function. Alteration in steady state organelle pH is closely linked to several diseases. Although a few probes exist to measure pH of cell compartments, each has several associated limitations. We present a high-performance pH sensor, a DNA nanoswitch, a convenient method to map spatiotemporal pH changes in endocytic pathways. DNA has been used to make a variety of nanoswitches in vitro . However, the present DNA nanoswitch functions as a pH sensing device equally efficiently intracellularly as it does in vitro. This DNA nanoswitch functions as a FRET-based pH sensor in the pH regime of 5.5-7, with high dynamic range between pH 5.8 and 7. It is efficiently engulfed by Drosophila hemocytes through endocytosis and can be used to measure the acidity of the endocytic vesicles that it marks during their maturation till their lysosomal stage.

An autonomous DNA nanodevice captures pH maps of living cells in culture and in vivo

DNA nanomachines are assemblies that rely on molecular inputs that are processed or transduced into measurable outputs. Though DNA nanotechnology has created a gamut of molecular devices, an outstanding challenge has been the demonstration of functionality and relevance of these devices in living systems. The I-switch is a DNA nanodevice that, in response to protons, changes its conformation to produce a fluorescence resonance energy transfer (FRET) signal. We show that this rationally designed molecular device is capable of measuring spatiotemporal pH changes associated with endosomes as they undergo maturation in living cells in culture. Furthermore, we show that the nanomachine retains its autonomous functionality as it maps the same biological process in cells of a living organism like C. elegans. This demonstration of the quantitative functionality of an artificially designed scaffold positions DNA nanodevices as powerful tools to interrogate biological phenomena.