Yamuna Krishnan

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.

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

Structural DNA nanotechnology seeks to build synthetic molecular machinery from DNA. DNA nanomachines are artificially designed assemblies that switch between defined conformations in response to an external cue. Though it has proved possible to create DNA machines and rudimentary walkers, the function of such autonomous DNA-based molecular devices has not yet been achieved inside living organisms. Here we demonstrate the operation of a pH-triggered DNA nanomachine inside the nematode Caenorhabditis elegans. The nanomachine uses fluorescence resonance energy transfer to effectively map spatiotemporal pH changes associated with endocytosis in wild type as well as mutant worms, demonstrating autonomous function within the organismal milieu in a variety of genetic backgrounds. From this first demonstration of the independent functionality of a DNA nanomachine in vivo, we observe that rationally designed DNA-based molecular devices retain their in vitro functionality with quantitative precision. This positions DNA nanodevices as exciting and powerful tools to interrogate complex biological phenomena.

A synthetic icosahedral DNA-based host-cargo complex for functional in vivo imaging.

The encapsulation of molecular cargo within well-defined supramolecular architectures is highly challenging. Synthetic hosts are desirable because of their well-defined nature and addressability. Encapsulation of biomacromolecules within synthetic hosts is especially challenging because of the formers large size, sensitive nature, retention of functionality post-encapsulation and demonstration of control over the cargo. Here we encapsulate a fluorescent biopolymer that functions as a pH reporter within synthetic, DNA-based icosahedral host without molecular recognition between host and cargo. Only those cells bearing receptors for the DNA casing of the host-cargo complex engulf it. We show that the encapsulated cargo is therefore uptaken cell specifically in Caenorhabditis elegans. Retention of functionality of the encapsulated cargo is quantitatively demonstrated by spatially mapping pH changes associated with endosomal maturation within the coelomocytes of C. elegans. This is the first demonstration of functionality and emergent behaviour of a synthetic host-cargo complex in vivo.

The poly dA helix: a new structural motif for high performance DNA-based molecular switches

We report a pH-dependent conformational transition in short, defined homopolymeric deoxyadenosines (dA15) from a single helical structure with stacked nucleobases at neutral pH to a double-helical, parallel-stranded duplex held together by AH+-H+A base pairs at acidic pH. Using native PAGE, 2D NMR, circular dichroism (CD) and fluorescence spectroscopy, we have characterized the two different pH dependent forms of dA15. The pH-triggered transition between the two defined helical forms of dA15 is characterized by CD and fluorescence. The kinetics of this conformational switch is found to occur on a millisecond time scale. This robust, highly reversible, pH-induced transition between the two well-defined structured states of dA15 represents a new molecular building block for the construction of quick-response, pH-switchable architectures in structural DNA nanotechnology.

ATP as a biological hydrotrope

ATP boosts protein solubility Adenosine triphosphate (ATP) has well-characterized roles in providing energy for biochemical reactions within cells. Patel et al. find that ATP may also enhance protein solubility, which could help explain why such high concentrations of ATP are maintained in cells (see the Perspective by Rice and Rosen). Protein concentrations in cells can exceed 100 mg/ml. The authors found that ATP at concentrations found in cells could act as a hydrotrope to help solubilize hydrophobic proteins. The results raise the possibility that ATP concentrations could influence processes such as protein aggregation that occur in disease or liquid-liquid phase separations that occur within cells. Science, this issue p. 753; see also p. 701 ATP at cellular concentrations can influence protein aggregation and solubility. Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells.

Controlled release of encapsulated cargo from a DNA icosahedron using a chemical trigger.

Controlled Release of Encapsulated Cargo from a DNA Icosahedron using a Chemical Trigger DNA Trojan horse : A DNA icosahedron (black, see scheme) held together with aptamers (red) was used to encapsulate molecular cargo like fluorescent dextran (green). In the presence of a molecular trigger (gray hexagons), the aptamers fold back leading to opening of the icosahedron and simultaneous release of the encapsulated cargo. Angewandte Chemie

Designing DNA nanodevices for compatibility with the immune system of higher organisms

DNA is proving to be a powerful scaffold to construct molecularly precise designer DNA devices. Recent trends reveal their ever-increasing deployment within living systems as delivery devices that not only probe but also program and re-program a cell, or even whole organisms. Given that DNA is highly immunogenic, we outline the molecular, cellular and organismal response pathways that designer nucleic acid nanodevices are likely to elicit in living systems. We address safety issues applicable when such designer DNA nanodevices interact with the immune system. In light of this, we discuss possible molecular programming strategies that could be integrated with such designer nucleic acid scaffolds to either evade or stimulate the host response with a view to optimizing and widening their applications in higher organisms.

Designer nucleic acids to probe and program the cell

Recent advances in nucleic acid sequencing, structural, and computational technologies have resulted in dramatic progress in our understanding of nucleic acid structure and function in the cell. This knowledge, together with the predictable base-pairing of nucleic acids and powerful synthesis and expression capabilities now offers the unique ability to program nucleic acids to form precise 3D architectures with diverse applications in synthetic and cell biology. The unique modularity of structural motifs that include aptamers, DNAzymes, and ribozymes, together with their well-defined construction rules, enables the synthesis of functional higher-order nucleic acid complexes from these subcomponents. As we illustrate here, these highly programmable, smart complexes are increasingly enabling researchers to probe and program the cell in a sophisticated manner that moves well beyond the use of nucleic acids for conventional genetic manipulation alone.

Combining G-Quadruplex Targeting Motifs on a Single Peptide Nucleic Acid Scaffold: A Hybrid (3+1) PNA-DNA Bimolecular Quadruplex

We describe the first G-quadruplex targeting approach that combines intercalation and hybridization strategies by investigating the interaction of a G-rich peptide nucleic acid (PNA) acridone conjugate 1 with a three-repeat fragment of the human telomere G 3 to form a hybrid PNA-DNA quadruplex that mimicks the biologically relevant (3+1) pure DNA dimeric telomeric quadruplex. Using a combination of UV and fluorescence spectroscopy, circular dichroism (CD), and mass-spectrometry, we show that PNA 1 can induce the formation of a bimolecular hybrid quadruplex even at low salt concentration upon interaction with a single-stranded three-repeat fragment of telomeric DNA. However, PNA 1 cannot invade a short fragment of B-DNA even if the latter contains a CCC motif complementary to the PNA sequence. These studies could open up new possibilities for the design of a novel generation of quadruplex ligands that target not only the external features of the quadruplex but also its central core constituted by the tetrads themselves.