Souvik Modi - Academia.edu (original) (raw)
Papers by Souvik Modi
Alzheimer's & Dementia
Density of mitochondria and their localization at specific sub-cellular regions of the neurons is... more Density of mitochondria and their localization at specific sub-cellular regions of the neurons is regulated by molecular motors, their adaptors and the cytoskeleton. However, the regulation of the mitochondrial density, the positioning of mitochondria along the neuronal process and the role of axonal mitochondria in neuronal function remain poorly understood. This study shows that the density of mitochondria in C. elegans touch receptor neuron processes remains constant through development. Simulations show that mitochondrial positioning along parts of the neuronal process that are devoid of synapses is regulated. Additionally, we also demonstrate that axonal mitochondria are necessary for maintaining touch responsiveness.
Native polyacrylamide gel electrophoresis in the case of RNA-PNA complexes were performed using N... more Native polyacrylamide gel electrophoresis in the case of RNA-PNA complexes were performed using N-terminally TMR labeled PNA (N-TMR-P). RNA and PNA were allowed to complex at total strand concentrations of 100 μM by addition of an equimolar mixture of R and N-TMR-P in 30 mM sodium acetate, pH 4.5, heated to 90oC and then cooled to room temperature at a rate of 0.33oC/min and equilibrated at 4oC for 8 hrs. 20% polyacrylamide gels were used to study the differential mobility of any complex that was performed. Robinson Britton buffer (boric acid, acetic acid and phosphoric acid at 0.04 M concentration each, pH 4.5) was used as running buffer. All gels were run at 70 V for 6 hrs at 4oC. All gels were visualized by ethidium bromide filter. Nano-electrospray Ionisation Mass spectrometry (Nano ESI-MS):
Aging Cell, 2020
The G protein‐coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes ... more The G protein‐coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. Here, we show that C. elegans longevity is regulated by a chemosensory GPCR STR‐2, expressed in AWC and ASI amphid sensory neurons. STR‐2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR‐2 regulates expression of delta‐9 desaturases, fat‐5, fat‐6 and fat‐7, and of diacylglycerol acyltransferase dgat‐2. Rescue of the STR‐2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat‐5, dgat‐2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wild‐type levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR‐2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.
SummaryAstrocytic GLT-1 is the main glutamate transporter involved in glutamate buffering in the ... more SummaryAstrocytic GLT-1 is the main glutamate transporter involved in glutamate buffering in the brain, pivotal for glutamate removal at excitatory synapses to terminate neurotransmission and for preventing excitotoxicity. We show here that the surface expression and function of GLT-1 can be rapidly modulated through the interaction of its N-terminus with the nonadrenergic imidazoline-1 receptor protein, Nischarin. The phox domain of Nischarin is critical for interaction and internalization of surface GLT-1. Using live super-resolution imaging, we found that glutamate accelerated Nischarin-GLT-1 internalization into endosomal structures. The surface GLT-1 level increased in Nischarin knockout astrocytes, and this correlated with a significant increase in transporter uptake current. Furthermore, Nischarin knockout in astrocytes is neuroprotective against glutamate excitotoxicity. These data provide new molecular insights into regulation of GLT-1 surface level and function and suggest...
Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential m... more Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts (MEF) we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and endoplasmic reticulum-mitochondria contacts sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale orga...
Nanoscale, Jan 31, 2018
Neurons communicate with each other through synapses, which show enrichment for specialized recep... more Neurons communicate with each other through synapses, which show enrichment for specialized receptors. Although many studies have explored spatial enrichment and diffusion of these receptors in dissociated neurons using single particle tracking, much less is known about their dynamic properties at synapses in complex tissue like brain slices. Here we report the use of smaller and highly specific quantum dots conjugated with a recombinant single domain antibody fragment (VHH fragment) against green fluorescent protein to provide information on diffusion of adhesion molecules at the growth cone and neurotransmitter receptors at synapses. Our data reveals that QD-nanobodies can measure neurotransmitter receptor dynamics at both excitatory and inhibitory synapses in primary neuronal cultures as well as in ex vivo rat brain slices. We also demonstrate that this approach can be applied to tagging multiple proteins to simultaneously monitor their behavior. Thus, we provide a strategy for m...
Schizophrenia Research, 2017
Ion transport during neuronal signalling utilizes the majority of the brain's energy supply. Mito... more Ion transport during neuronal signalling utilizes the majority of the brain's energy supply. Mitochondria are key sites for energy provision through ATP synthesis and play other important roles including calcium buffering. Thus, tightly regulated distribution and function of these organelles throughout the intricate architecture of the neuron is essential for normal synaptic communication. Therefore, delineating mechanisms coordinating mitochondrial transport and function is essential for understanding nervous system physiology and pathology. While aberrant mitochondrial transport and dynamics have long been associated with neurodegenerative disease, they have also more recently been linked to major mental illness including schizophrenia, autism and depression. However, the underlying mechanisms have yet to be elucidated, due to an incomplete understanding of the combinations of genetic and environmental factors contributing to these conditions. Consequently, the DISC1 gene has undergone intense study since its discovery at the site of a balanced chromosomal translocation, segregating with mental illness in a Scottish pedigree. The precise molecular functions of DISC1 remain elusive. Reported functions of DISC1 include regulation of intracellular signalling pathways, neuronal migration and dendritic development. Intriguingly, a role for DISC1 in mitochondrial homeostasis and transport is fast emerging. Therefore, a major function of DISC1 in regulating mitochondrial distribution, ATP synthesis and calcium buffering may be disrupted in psychiatric disease. In this review, we discuss the links between DISC1 and mitochondria, considering both trafficking of these organelles and their function, and how, via these processes, DISC1 may contribute to the regulation of neuronal behavior in normal and psychiatric disease states.
The Journal of biological chemistry, Jan 9, 2015
The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bi... more The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but effects in dendrites have yet to be studied. Further, the mechanisms of this regulation, and its role in neuronal development and brain function are poorly understood. Here we demonstrate that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane (OMM) GTPase proteins, Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins Mitofusins. Using live cell imaging, we show that disruption of the DISC1 Miro/TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localises to mitochondria whe...
Nature Nanotechnology, 2009
DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upo... more 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.
Molecular Genetics and Metabolism
Methods in Molecular Biology, 2011
A few cellular compartments maintain acidic environments in their interiors that are crucial for ... more 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.
Lecture Notes in Computer Science, 2011
ABSTRACT DNA nanomachines are assemblies that rely on molecular inputs that are processed or tran... more ABSTRACT 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. KeywordsI-switch–pH sensor–hemocytes–coelomocytes
Nucleic Acids Research, 2006
We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands f... more 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 1 -C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H 1 -C base pairs per heteroduplex. The imino protons are protected from D 2 O exchange suggesting intercalation of the heteroduplexes as seen in DNA 4 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 0 -H1 0 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 DNA 4 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.
Nature Nanotechnology, 2013
DNA is a versatile scaffold for molecular sensing in living cells, and various cellular applicati... more 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 Journal of Physical Chemistry Letters, 2010
... Since 2007, he has been full professor of physics at TU Munich. ... methods to cut, copy, and... more ... Since 2007, he has been full professor of physics at TU Munich. ... methods to cut, copy, and covalently link B-DNA double helices sequence-specifically, which allows manipulation of theconstruction material, (vii ... These tiles assemble into various 2D shapes using molecular logic. ...
Alzheimer's & Dementia
Density of mitochondria and their localization at specific sub-cellular regions of the neurons is... more Density of mitochondria and their localization at specific sub-cellular regions of the neurons is regulated by molecular motors, their adaptors and the cytoskeleton. However, the regulation of the mitochondrial density, the positioning of mitochondria along the neuronal process and the role of axonal mitochondria in neuronal function remain poorly understood. This study shows that the density of mitochondria in C. elegans touch receptor neuron processes remains constant through development. Simulations show that mitochondrial positioning along parts of the neuronal process that are devoid of synapses is regulated. Additionally, we also demonstrate that axonal mitochondria are necessary for maintaining touch responsiveness.
Native polyacrylamide gel electrophoresis in the case of RNA-PNA complexes were performed using N... more Native polyacrylamide gel electrophoresis in the case of RNA-PNA complexes were performed using N-terminally TMR labeled PNA (N-TMR-P). RNA and PNA were allowed to complex at total strand concentrations of 100 μM by addition of an equimolar mixture of R and N-TMR-P in 30 mM sodium acetate, pH 4.5, heated to 90oC and then cooled to room temperature at a rate of 0.33oC/min and equilibrated at 4oC for 8 hrs. 20% polyacrylamide gels were used to study the differential mobility of any complex that was performed. Robinson Britton buffer (boric acid, acetic acid and phosphoric acid at 0.04 M concentration each, pH 4.5) was used as running buffer. All gels were run at 70 V for 6 hrs at 4oC. All gels were visualized by ethidium bromide filter. Nano-electrospray Ionisation Mass spectrometry (Nano ESI-MS):
Aging Cell, 2020
The G protein‐coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes ... more The G protein‐coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. Here, we show that C. elegans longevity is regulated by a chemosensory GPCR STR‐2, expressed in AWC and ASI amphid sensory neurons. STR‐2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR‐2 regulates expression of delta‐9 desaturases, fat‐5, fat‐6 and fat‐7, and of diacylglycerol acyltransferase dgat‐2. Rescue of the STR‐2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat‐5, dgat‐2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wild‐type levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR‐2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.
SummaryAstrocytic GLT-1 is the main glutamate transporter involved in glutamate buffering in the ... more SummaryAstrocytic GLT-1 is the main glutamate transporter involved in glutamate buffering in the brain, pivotal for glutamate removal at excitatory synapses to terminate neurotransmission and for preventing excitotoxicity. We show here that the surface expression and function of GLT-1 can be rapidly modulated through the interaction of its N-terminus with the nonadrenergic imidazoline-1 receptor protein, Nischarin. The phox domain of Nischarin is critical for interaction and internalization of surface GLT-1. Using live super-resolution imaging, we found that glutamate accelerated Nischarin-GLT-1 internalization into endosomal structures. The surface GLT-1 level increased in Nischarin knockout astrocytes, and this correlated with a significant increase in transporter uptake current. Furthermore, Nischarin knockout in astrocytes is neuroprotective against glutamate excitotoxicity. These data provide new molecular insights into regulation of GLT-1 surface level and function and suggest...
Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential m... more Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts (MEF) we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and endoplasmic reticulum-mitochondria contacts sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale orga...
Nanoscale, Jan 31, 2018
Neurons communicate with each other through synapses, which show enrichment for specialized recep... more Neurons communicate with each other through synapses, which show enrichment for specialized receptors. Although many studies have explored spatial enrichment and diffusion of these receptors in dissociated neurons using single particle tracking, much less is known about their dynamic properties at synapses in complex tissue like brain slices. Here we report the use of smaller and highly specific quantum dots conjugated with a recombinant single domain antibody fragment (VHH fragment) against green fluorescent protein to provide information on diffusion of adhesion molecules at the growth cone and neurotransmitter receptors at synapses. Our data reveals that QD-nanobodies can measure neurotransmitter receptor dynamics at both excitatory and inhibitory synapses in primary neuronal cultures as well as in ex vivo rat brain slices. We also demonstrate that this approach can be applied to tagging multiple proteins to simultaneously monitor their behavior. Thus, we provide a strategy for m...
Schizophrenia Research, 2017
Ion transport during neuronal signalling utilizes the majority of the brain's energy supply. Mito... more Ion transport during neuronal signalling utilizes the majority of the brain's energy supply. Mitochondria are key sites for energy provision through ATP synthesis and play other important roles including calcium buffering. Thus, tightly regulated distribution and function of these organelles throughout the intricate architecture of the neuron is essential for normal synaptic communication. Therefore, delineating mechanisms coordinating mitochondrial transport and function is essential for understanding nervous system physiology and pathology. While aberrant mitochondrial transport and dynamics have long been associated with neurodegenerative disease, they have also more recently been linked to major mental illness including schizophrenia, autism and depression. However, the underlying mechanisms have yet to be elucidated, due to an incomplete understanding of the combinations of genetic and environmental factors contributing to these conditions. Consequently, the DISC1 gene has undergone intense study since its discovery at the site of a balanced chromosomal translocation, segregating with mental illness in a Scottish pedigree. The precise molecular functions of DISC1 remain elusive. Reported functions of DISC1 include regulation of intracellular signalling pathways, neuronal migration and dendritic development. Intriguingly, a role for DISC1 in mitochondrial homeostasis and transport is fast emerging. Therefore, a major function of DISC1 in regulating mitochondrial distribution, ATP synthesis and calcium buffering may be disrupted in psychiatric disease. In this review, we discuss the links between DISC1 and mitochondria, considering both trafficking of these organelles and their function, and how, via these processes, DISC1 may contribute to the regulation of neuronal behavior in normal and psychiatric disease states.
The Journal of biological chemistry, Jan 9, 2015
The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bi... more The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but effects in dendrites have yet to be studied. Further, the mechanisms of this regulation, and its role in neuronal development and brain function are poorly understood. Here we demonstrate that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane (OMM) GTPase proteins, Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins Mitofusins. Using live cell imaging, we show that disruption of the DISC1 Miro/TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localises to mitochondria whe...
Nature Nanotechnology, 2009
DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upo... more 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.
Molecular Genetics and Metabolism
Methods in Molecular Biology, 2011
A few cellular compartments maintain acidic environments in their interiors that are crucial for ... more 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.
Lecture Notes in Computer Science, 2011
ABSTRACT DNA nanomachines are assemblies that rely on molecular inputs that are processed or tran... more ABSTRACT 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. KeywordsI-switch–pH sensor–hemocytes–coelomocytes
Nucleic Acids Research, 2006
We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands f... more 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 1 -C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H 1 -C base pairs per heteroduplex. The imino protons are protected from D 2 O exchange suggesting intercalation of the heteroduplexes as seen in DNA 4 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 0 -H1 0 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 DNA 4 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.
Nature Nanotechnology, 2013
DNA is a versatile scaffold for molecular sensing in living cells, and various cellular applicati... more 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 Journal of Physical Chemistry Letters, 2010
... Since 2007, he has been full professor of physics at TU Munich. ... methods to cut, copy, and... more ... Since 2007, he has been full professor of physics at TU Munich. ... methods to cut, copy, and covalently link B-DNA double helices sequence-specifically, which allows manipulation of theconstruction material, (vii ... These tiles assemble into various 2D shapes using molecular logic. ...