Adam Marblestone

Based on a survey of the literature, we attempt to answer Frequently Asked Questions on issues of cortical uniformity vs. non-uniformity, the neural mechanisms of symbolic variable binding, and other issues highlighted in (Marcus, Marblestone and Dean. "The Atoms of Neural Computation". Science. 31 October 2014. Vol 346. Issue 6209).

The human cerebral cortex is central to a wide array of cognitive functions, from vision to language, reasoning, decision-making, and motor control. Yet, nearly a century after the neuroanatomical organization of the cortex was first defined, its basic logic remains unknown. One hypothesis is that cortical neurons form a single, massively repeated “canonical” circuit, characterized as a kind of a “nonlinear spatiotemporal filter with adaptive properties” (1). In this classic view, it was… Show more

The human cerebral cortex is central to a wide array of cognitive functions, from vision to language, reasoning, decision-making, and motor control. Yet, nearly a century after the neuroanatomical organization of the cortex was first defined, its basic logic remains unknown. One hypothesis is that cortical neurons form a single, massively repeated “canonical” circuit, characterized as a kind of a “nonlinear spatiotemporal filter with adaptive properties” (1). In this classic view, it was “assumed that these…properties are identical for all neocortical areas.” Nearly four decades later, there is still no consensus about whether such a canonical circuit exists, either in terms of its anatomical basis or its function. Likewise, there is little evidence that such uniform architectures can capture the diversity of cortical function in simple mammals, let alone characteristically human processes such as language and abstract thinking (2). Analogous software implementations in artificial intelligence (e.g., deep learning networks) have proven effective in certain pattern classification tasks, such as speech and image recognition, but likewise have made little inroads in areas such as reasoning and natural language understanding. Is the search for a single canonical cortical circuit misguided? Show less

We propose a neural connectomics strategy called Fluorescent In-Situ Sequencing of Barcoded Individual Neuronal Connections (FISSEQ-BOINC), leveraging fluorescent in situ nucleic acid sequencing in fixed tissue (FISSEQ). FISSEQ-BOINC exhibits different properties from BOINC, which relies on bulk nucleic acid sequencing. FISSEQ-BOINC could become a scalable approach for mapping whole-mammalian-brain connectomes with rich molecular annotations.

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8742 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is… Show more

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8742 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ. Show less

We analyze the scaling and cost-performance characteristics of current and projected connectomics approaches, with reference to the potential implications of recent advances in diverse contributing fields. Three generalized strategies for dense connectivity mapping at the scale of whole mammalian brains are considered: electron microscopic axon tracing, optical imaging of combinatorial molecular markers at synapses, and bulk DNA sequencing of trans-synaptically exchanged nucleic acid barcode… Show more

We analyze the scaling and cost-performance characteristics of current and projected connectomics approaches, with reference to the potential implications of recent advances in diverse contributing fields. Three generalized strategies for dense connectivity mapping at the scale of whole mammalian brains are considered: electron microscopic axon tracing, optical imaging of combinatorial molecular markers at synapses, and bulk DNA sequencing of trans-synaptically exchanged nucleic acid barcode pairs. Due to advances in parallel-beam instrumentation, whole mouse brain electron microscopic image acquisition could cost less than $100 million, with total costs presently limited by image analysis to trace axons through large image stacks. Optical microscopy at 50 to 100 nm isotropic resolution could potentially read combinatorially multiplexed molecular information from individual synapses, which could indicate the identifies of the pre-synaptic and post-synaptic cells without relying on axon tracing. An optical approach to whole mouse brain connectomics may be achievable for less than $10 million and could be enabled by emerging technologies to sequence nucleic acids in-situ in fixed tissue via fluorescent microscopy. Novel strategies relying on bulk DNA sequencing, which would extract the connectome without direct imaging of the tissue, could produce a whole mouse brain connectome for $100k to $1 million or a mouse cortical connectome for $10k to $100k. Anticipated further reductions in the cost of DNA sequencing could lead to a $1000 mouse cortical connectome. Show less

Recording of physiological signals from inaccessible microenvironments is often hampered by the macroscopic sizes of current recording devices. A signal-recording device constructed on a molecular scale could advance biology by enabling the simultaneous recording from millions or billions of cells. We recently proposed a molecular device for recording time-varying ion concentration signals: DNA polymerases (DNAPs) copy known template DNA strands with an error rate dependent on the local ion… Show more

Recording of physiological signals from inaccessible microenvironments is often hampered by the macroscopic sizes of current recording devices. A signal-recording device constructed on a molecular scale could advance biology by enabling the simultaneous recording from millions or billions of cells. We recently proposed a molecular device for recording time-varying ion concentration signals: DNA polymerases (DNAPs) copy known template DNA strands with an error rate dependent on the local ion concentration. The resulting DNA polymers could then be sequenced, and with the help of statistical techniques, used to estimate the time-varying ion concentration signal experienced by the polymerase. We develop a statistical framework to treat this inverse problem and describe a technique to decode the ion concentration signals from DNA sequencing data. We also provide a novel method for estimating properties of DNAP dynamics, such as polymerization rate and pause frequency, directly from sequencing data. We use this framework to explore potential application scenarios for molecular recording devices, achievable via molecular engineering within the biochemical parameter ranges of known polymerases. We find that accurate recording of neural firing rate responses across several experimental conditions would likely be feasible using molecular recording devices with kinetic properties similar to those of known polymerases. Show less

Adam H. Marblestone*, Bradley M. Zamft*, Yael G. Maguire, Mikhail G. Shapiro, Thaddeus R. Cybulski, Joshua I. Glaser, Ben Stranges, Reza Kalhor, David A. Dalrymple, Dongjin Seo, Elad Alon, Michel M. Maharbiz, Jose Carmena, Jan Rabaey, Edward S. Boyden**, George M. Church**, Konrad P. Kording**

Simultaneously measuring the activities of all neurons in a mammalian brain at millisecond resolution is a challenge beyond the limits of existing techniques in neuroscience. Entirely new… Show more

Adam H. Marblestone*, Bradley M. Zamft*, Yael G. Maguire, Mikhail G. Shapiro, Thaddeus R. Cybulski, Joshua I. Glaser, Ben Stranges, Reza Kalhor, David A. Dalrymple, Dongjin Seo, Elad Alon, Michel M. Maharbiz, Jose Carmena, Jan Rabaey, Edward S. Boyden**, George M. Church**, Konrad P. Kording**

Simultaneously measuring the activities of all neurons in a mammalian brain at millisecond resolution is a challenge beyond the limits of existing techniques in neuroscience. Entirely new approaches may be required, motivating an analysis of the fundamental physical constraints on the problem. We outline the physical principles governing brain activity mapping using optical, electrical,magnetic resonance, and molecular modalities of neural recording. Focusing on the mouse brain, we analyze the scalability of each method, concentrating on the limitations imposed by spatiotemporal resolution, energy dissipation, and volume displacement. We also study the physics of powering and communicating with microscale devices embedded in brain tissue.
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High-throughput recording of signals embedded within inaccessible micro-environments is a technological challenge. The ideal recording device would be a nanoscale machine capable of quantitatively transducing a wide range of variables into a molecular recording medium suitable for long-term storage and facile readout in the form of digital data. We have recently proposed such a device, in which cation concentrations modulate the misincorporation rate of a DNA polymerase (DNAP) on a known… Show more

High-throughput recording of signals embedded within inaccessible micro-environments is a technological challenge. The ideal recording device would be a nanoscale machine capable of quantitatively transducing a wide range of variables into a molecular recording medium suitable for long-term storage and facile readout in the form of digital data. We have recently proposed such a device, in which cation concentrations modulate the misincorporation rate of a DNA polymerase (DNAP) on a known template, allowing DNA sequences to encode information about the local cation concentration. In this work we quantify the cation sensitivity of DNAP misincorporation rates, making possible the indirect readout of cation concentration by DNA sequencing. Using multiplexed deep sequencing, we quantify the misincorporation properties of two DNA polymerases – Dpo4 and Klenow exo− – obtaining the probability and base selectivity of misincorporation at all positions within the template. We find that Dpo4 acts as a DNA recording device for Mn2+ with a misincorporation rate gain of ~2%/mM. This modulation of misincorporation rate is selective to the template base: the probability of misincorporation on template T by Dpo4 increases >50-fold over the range tested, while the other template bases are affected less strongly. Furthermore, cation concentrations act as scaling factors for misincorporation: on a given template base, Mn2+ and Mg2+ change the overall misincorporation rate but do not alter the relative frequencies of incoming misincorporated nucleotides. Characterization of the ion dependence of DNAP misincorporation serves as the first step towards repurposing it as a molecular recording device. Show less

We consider classical and entanglement-assisted versions of a distributed computation scheme that computes nonlinear Boolean functions of a set of input bits supplied by separated parties. Communication between the parties is restricted to take place through a specific apparatus which enforces the constraints that all nonlinear, nonlocal classical logic is performed by a single receiver, and that all communication occurs through a limited number of one-bit channels. In the entanglement-assisted… Show more

We consider classical and entanglement-assisted versions of a distributed computation scheme that computes nonlinear Boolean functions of a set of input bits supplied by separated parties. Communication between the parties is restricted to take place through a specific apparatus which enforces the constraints that all nonlinear, nonlocal classical logic is performed by a single receiver, and that all communication occurs through a limited number of one-bit channels. In the entanglement-assisted version, the number of channels required to compute a Boolean function of fixed nonlinearity can become exponentially smaller than in the classical version. We demonstrate this exponential enhancement for the problem of distributed integer addition. Show less

DNA nanotechnology exploits the programmable specificity afforded by base-pairing to produce self-assembling macromolecular objects of custom shape. For building megadalton-scale DNA nanostructures, a long ‘scaffold’ strand can be employed to template the assembly of hundreds of oligonucleotide ‘staple’ strands into a planar antiparallel array of cross-linked helices. We recently adapted this ‘scaffolded DNA origami’ method to producing 3D shapes formed as pleated layers of double helices… Show more

DNA nanotechnology exploits the programmable specificity afforded by base-pairing to produce self-assembling macromolecular objects of custom shape. For building megadalton-scale DNA nanostructures, a long ‘scaffold’ strand can be employed to template the assembly of hundreds of oligonucleotide ‘staple’ strands into a planar antiparallel array of cross-linked helices. We recently adapted this ‘scaffolded DNA origami’ method to producing 3D shapes formed as pleated layers of double helices constrained to a honeycomb lattice. However, completing the required design steps can be cumbersome and time-consuming. Here we present caDNAno, an open-source software package with a graphical user interface that aids in the design of DNA sequences for folding 3D honeycomb-pleated shapes A series of rectangular-block motifs were designed, assembled, and analyzed to identify a well-behaved motif that could serve as a building block for future studies. The use of caDNAno significantly reduces the effort required to design 3D DNA-origami structures. The software is available at http://cadnano.org/, along with example designs and video tutorials demonstrating their construction. The source code is released under the MIT license. Show less

De novo synthesis of long double-stranded DNA constructs has a myriad of applications in
biology and biological engineering. However, its widespread adoption has been hindered
by high costs. Cost can be significantly reduced by using oligonucleotides synthesized
on high-density DNA chips. However, most methods for using off-chip DNA for gene
synthesis have failed to scale due to the high error rates, low yields, and high chemical
complexity of the chip-synthesized… Show more

De novo synthesis of long double-stranded DNA constructs has a myriad of applications in
biology and biological engineering. However, its widespread adoption has been hindered
by high costs. Cost can be significantly reduced by using oligonucleotides synthesized
on high-density DNA chips. However, most methods for using off-chip DNA for gene
synthesis have failed to scale due to the high error rates, low yields, and high chemical
complexity of the chip-synthesized oligonucleotides. We have recently demonstrated
that some commercial DNA chip manufacturers have improved error rates, and that the
issues of chemical complexity and low yields can be solved by using barcoded primers
to accurately and efficiently amplify subpools of oligonucleotides. This unit includes
protocols for computationally designing the DNA chip, amplifying the oligonucleotide
subpools, and assembling 500- to 800-bp constructs. Show less

We present “molecular threading”, a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA… Show more

We present “molecular threading”, a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA deposited in arrays onto elastomeric surfaces and thin membranes. Molecular threading demonstrates high straightness and uniformity over length scales from nanometers to micrometers, and represents an alternative to existing DNA deposition and linearization methods. These results point towards scalable and high-throughput precision manipulation of single-molecule polymers.
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Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced in vitro reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein… Show more

Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced in vitro reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein complexes and pathways from the model organism Escherichia coli to reconstitute functional synthetic proteomes in vitro. By application of over 110 MAGE cycles, we successfully inserted hexa-histidine sequences into 38 essential genes in vivo that encode for the entire translation machinery. Streamlined co-purification and reconstitution of the translation protein complex enabled protein synthesis in vitro. Our approach can be applied to a growing area of applications in in vitro one-pot multienzyme catalysis (MEC) to manipulate or enhance in vitro pathways such as natural product or carbohydrate biosynthesis. Show less

We present the theory of a Josephson parametric amplifier employing two-pump sources. Our calculations are based on input-output theory, and can easily be generalized to any coupled system involving parametric interactions. We analyze the operation of the device, taking into account the feedback introduced by the reaction of the signal and noise on the pump power, and in this framework, compute the response functions of interest—signal and idler gains, internal gain of the amplifier, and… Show more

We present the theory of a Josephson parametric amplifier employing two-pump sources. Our calculations are based on input-output theory, and can easily be generalized to any coupled system involving parametric interactions. We analyze the operation of the device, taking into account the feedback introduced by the reaction of the signal and noise on the pump power, and in this framework, compute the response functions of interest—signal and idler gains, internal gain of the amplifier, and self-oscillation signal amplitude. To account for this back action between signal and pump, we adopt a mean-field approach and self-consistently explore the boundary between amplification and self-oscillation. The coincidence of bifurcation and self-oscillation thresholds reveals that the origin of coherent emission of the amplifier lies in the multiwave mixing of the noise components. Incorporation of the back action leads the system to exhibit hysteresis, dependent on parameters such as temperature and detuning from resonance. Our analysis also shows that the resonance condition itself changes in the presence of back action and this can be understood in terms of the change in plasma frequency of the junction. The potential of the double-pump amplifier for quantum-limited measurements and as a squeezer is also discussed. Show less