Search

CRISPR-Cas9 nucleases, and particularly Streptococcus pyogenes Cas9, are widespread tools for genome editing. However, many aspects of intracellular Cas9 activity and the ensuing DNA damage response remain incompletely characterized. In order to address these issues, we developed a multiplexed CRISPR approach, where a single, degenerate multi-target gRNA (mgRNA) directs the Cas9 enzyme to target hundred endogenous sites at once. When combined with next-generation sequencing readouts, this system enables interrogation of Cas9 activity and DNA double-strand break (DSB) repair response in high-throughput. Here, we present a step-by-step protocol to deliver a Cas9:mgRNA ribonucleoprotein complex into cultured cells and measure key processes related to Cas9 activity and DSB repair.

The release of anticoagulant drugs such as warfarin from human serum albumin (HSA) has been important not only mechanistically but also clinically for patients who take multiple drugs simultaneously. In this study, the role of some commonly used drugs, including s-ibuprofen, ascorbic acid, and salicylic acid, was investigated in the release of warfarin bound to HSA in silico. The effects of the aforementioned drugs on the HSA-warfarin complex were investigated with molecular dynamics (MD) simulations using two approaches; in the first perspective, molecular docking was used to model the interaction of each drug with the HSA-warfarin complex, and in the second approach, drugs were positioned randomly and distant from the binary complex (HSA-warfarin) in a physiologically relevant concentration. The results obtained from both approaches indicated that s-ibuprofen and ascorbic acid both displayed allosteric effects on the release of warfarin from HSA. Although ascorbic acid aided in warfarin release, leading to destabilization of HSA, ibuprofen demonstrated a stabilizing effect on releasing the anticoagulant drug through several noncovalent interactions, including hydrophobic, electrostatic, and hydrogen-bonding interactions with the protein. The calculated binding free energy and energy contribution of involved residues using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method, along with root mean square deviation (RMSD) values, protein gyration, and free energy surface (FES) mapping of the protein, provided valuable details on the nature of the interactions of each drug on the release of warfarin from HSA. These results can provide important information on the mechanisms of anticoagulant release that has not been revealed in molecular details previously.

Proteins possess a variety of nucleophiles, which can carry out different reactions in the functioning cells. Proteins endogenously and synthetically can be modified through their nucleophilic sites. The roles of these chemical modifications have not been completely revealed. These modifications can alter the protein folding process. Protein folding directly affects the function of proteins. If an error in protein folding occurs, it may cause protein malfunction leading to several neurodegenerative disorders such as Alzheimer's and Parkinson's. In this study, Hen Egg White Lysozyme (HEWL) and bovine insulin, as model proteins for studying the amyloid formation, were covalently attached with 5(6)-thiophenolfluorescein. The amyloid formation of the covalently labeled lysozyme and insulin were compared with the native proteins. Interestingly, the results indicated that the covalent attachment of fluorescein slowed down the amyloid formation of HEWL and insulin significantly. The amyloid formation was examined using Thioflavin T (ThT) fluorescence assay, circular dichroism, FTIR, and gel electrophoresis. Tandem mass spectrometry was employed to identify the sites of covalent modifications in HEWL. It turned out that two surface lysine residues (K97 and K 116) in HEWL were modified. Computational studies, including docking and molecular simulations, revealed that 5(6)-thiophenolfluorescein makes several non-covalent interactions with HEWL residues, including Lys 97, leading to the reduction of the β-sheet in the protein. Additionally, AFM analysis confirmed the amyloid fibril reduction of lysine-modified bovine insulin and HEWL. Altogether, our results expand mechanistic insights into preventing amyloid formation by providing an approach for reducing amyloid formation by modifying specific lysine residues in the proteins.

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; however, whether these lineage barcodes can capture the dynamics of complex progenitor fields remains unclear. Here, we introduce quantitative fate mapping, an approach to simultaneously map the fate and quantify the commitment time, commitment bias, and population size of multiple progenitor groups during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a generalizable barcoding mutagenesis model. We validate these approaches using realistically-simulated barcoding results as well as experimental results from a barcoding stem cell line. We further establish criteria for the minimum number of cells that must be analyzed for robust quantitative fate mapping. Overall, this work demonstrates how lineage barcodes, natural or synthetic, can be used to obtain quantitative fate maps, thus enabling analysis of progenitor dynamics long after embryonic development in any organism.

Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

We present an approach that combines a Cas9 that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing technologies to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of novel multi-target gRNAs (mgRNAs), degenerate gRNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, discovering rapid post-cleavage Cas9 departure and repair factor loading at PAM-proximal genomic DNA. Moreover, by bypassing confounding effects from gRNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and Cas9 cleavage is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double strand breaks with high temporal resolution, discovering the presence, extent (under 2 kb), and kinetics (~ 0.5 hr) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

A number of conformational diseases in humans have been associated with protein/peptide fibrillation known as amyloid. Although extensive studies have been conducted in understanding the molecular basis of amyloid formation, a detailed mechanism is still missing. Experimentally, HEWL (hen egg white lysozyme) has been exploited ubiquitously as a model protein for amyloid fibrillation and drug inhibition. However, computational studies investigating fibril formation of HEWL have been a difficult task to perform mainly due to high stability of lysozymes and the absence of crystal structures of HEWL fibril oligomers. In this study, we have examined various conditions of HEWL amyloid formation computationally; the results indicated that, at high concentration of ethanol (90%), significant unfolding of the protein was apparent. Higher values for RMSD, solvent accessibility, and solvent diffusion into the core, as well as conversion of native α-helical structures to random coils, were detected in the ethanol solution. REMD (replica exchange molecular dynamics) analysis demonstrated that the presence of ethanol significantly altered the minimum structure of HEWL into partially unfolded states. It has been observed that unfolding of the protein was initiated from the C-terminal region, exposing the protein to the solvent. The interaction of previously known anti amyloid drug (RS-0406) with HEWL was analyzed in high concentration of ethanol both

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.

Employing DNA as a high-density data storage medium has paved the way for next-generation digital storage and biosensing technologies. However, the multipart architecture of current DNA-based recording techniques renders them inherently slow and incapable of recording fluctuating signals with sub-hour frequencies. To address this limitation, we developed a simplified system employing a single enzyme, terminal deoxynucleotidyl transferase (TdT), to transduce environmental signals into DNA. TdT adds nucleotides to the 3’ ends of single-stranded DNA (ssDNA) in a template-independent manner, selecting bases according to inherent preferences and environmental conditions. By characterizing TdT nucleotide selectivity under different conditions, we show that TdT can encode various physiologically relevant signals like Co(2+), Ca(2+), Zn(2+) concentrations and temperature changes in vitro. Further, by considering the average rate of nucleotide incorporation, we show that the resulting ssDNA functions as a molecular ticker tape. With this method we accurately encode a temporal record of fluctuations in Co(2+) concentration to within 1 minute over a 60-minute period. Finally, we engineer TdT to allosterically turn off in the presence of physiologically relevant concentration of calcium. We use this engineered TdT in concert with a reference TdT to develop a two-polymerase system capable of recording a single step change in Ca(2+) signal to within 1 minute over a 60-minute period. This work expands the repertoire of DNA-based recording techniques by developing a novel DNA synthesis-based system that can record temporal environmental signals into DNA with minutes resolution.

Employing DNA as a high-density data storage medium has paved the way for next-generation digital storage and biosensing technologies. However, the multipart architecture of current DNA-based recording techniques renders them inherently slow and incapable of recording fluctuating signals with sub-hour frequencies. To address this limitation, we developed a simplified system employing a single enzyme, terminal deoxynucleotidyl transferase (TdT), to transduce environmental signals into DNA. TdT adds nucleotides to the 3’ ends of single-stranded DNA (ssDNA) in a template-independent manner, selecting bases according to inherent preferences and environmental conditions. By characterizing TdT nucleotide selectivity under different conditions, we show that TdT can encode various physiologically relevant signals like Co2+, Ca2+, Zn2+ concentrations and temperature changes in vitro. Further, by considering the average rate of nucleotide incorporation, we show that the resulting ssDNA functions as a molecular ticker tape. With this method we accurately encode a temporal record of fluctuations in Co2+ concentration to within 1 minute over a 60-minute period. Finally, we engineer TdT to allosterically turn off in the presence of physiologically relevant concentration of calcium. We use this engineered TdT in concert with a reference TdT to develop a two-polymerase system capable of recording a single step change in Ca2+ signal to within 1 minute over a 60-minute period. This work expands the repertoire of DNA-based recording techniques by developing a novel DNA synthesis-based system that can record temporal environmental signals into DNA with minutes resolution.

DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA., Adoption of DNA as a data storage medium could be accelerated with specialized synthesis processes and codecs. The authors describe TdT-mediated DNA synthesis in which data is stored in transitions between non-identical nucleotides and the use of synchronization markers to provide error tolerance.

Conformational diseases, constituting a large number of diseases, have been connected with protein misfolding, leading to aggregation known as amyloid fibrils. Mainly due to the lack of detailed molecular mechanisms, there has not been an effective drug to combat amyloid-associated diseases. Recently, a small organic pyridazine-based molecule (RS-0406) has shown significant reductions in amyloid fibrils in both in vitro and in vivo animal studies. However, no information on molecular details of inhibition for the small molecule has been reported. In this work, we have decided to explore structure-activity relationship of pyridazine-based compounds to investigate structural parameters for amyloid inhibition. A number of closely related derivatives of RS-0406 were designed and synthesized to delineate the roles of structural properties, including bulkiness and halogen bonding, hydrogen-bonding ability, and the position of substituents on the flanking aromatic rings of the synthetic molecules. To examine the effectiveness of the synthesized compounds, amyloid fibril formation of hen egg white lysozyme was measured in the presence of each synthetic molecule. Our results indicated that in addition to the type of the aryl substituent, their positions on the ring were also important for their inhibitory roles in amyloid fibrils formation. Moreover, a fluorinated compound turned out to be a more effective kinetic inhibitor, displaying a delayed fibril nucleation than the original lead compound. Furthermore, biochemical structural analyses and molecular dynamics simulation revealed that the pyridazine-based compounds may mediate the inhibition of amyloid fibrils through stabilization of the protein monomer during partially unfolded state. The cytotoxicity assay revealed that the amounts of amyloid intermediates were reduced in the presence of the synthetic compounds. Eventually, IC50 values were obtained for the synthetic compounds, and quantitative structure-activity relationship method was employed to suggest more effective amyloid inhibitors.

Keratinocyte Growth Factor (KGF) is a paracrine-acting, epithelial mitogen that plays a prominent role in the regeneration of damaged epithelial tissues. In spite of different attempts to produce recombinant human KGF in many organisms, including bacteria, mammalian cells, plant cells and insect cells; production of recombinant form suffers from lower yields and recovery relative to other recombinant proteins of similar size and properties. Due to many advantages of Pichia pastoris expression systems for producing industrial enzymes and pharmaceutical proteins, in this study P. pastoris was chosen as a host for KGF expression. For preparing human KGF coding sequence, MCF-7 cell line was treated with 1,25-Dihydroxy vitamin D3 for inducing the expression of KGF. The coding sequence of 23N-terminal truncated KGF form was amplified using RT-PCR technique and then cloned into the yeast expression vector in frame with the yeast α-factor secretion signal. The recombinant plasmid was integrated into Pichia pastoris strain X-33 genome. Western blotting and Mass Spectrometry demonstrated that recombinant human KGF (rhKGF) was correctly expressed after methanol induction and secreted into the media. The recombinant protein was purified from the media by heparin affinity chromatography. MTT assay showed that the purified rhKGF had a proliferative effect on NIH3T3 and A549 cell lines. In addition, protective effect of recombinant KGF was assessed in A549 cell line after irradiation. The results showed that the recombinant protein was biologically active. Finally, the effect of recombinant KGF was investigated on proliferation of MCF-7 cell line and its response to radiation. The results showed that pre-treatment of KGF have a protective effect on MCF-7 cell line after irradiation.

A number of human diseases, including Alzheimer's and Parkinson's have been linked to amyloid formation. To search for an anti-amyloidogenic product, alkaloid enriched extract from borage leaves was examined for anti-amyloidogenic activity using Hen Egg White Lysozyme (HEWL) as a model protein. After isolation of the plant extract using rHPLC, only one fraction indicated a significant bioactivity. TEM analysis confirmed a remarkable reduction of amyloid fibrils in the presence of the bioactive fraction. To identify the effective substance in the fraction, mass spectrometry, FTIR, and NMR were performed. Our analyses determined that the bioactive compound as 1-acetyl-19,21-epoxy-15,16-dimethoxyaspidospermidine-17-ol, a derivative of aspidospermine. To investigate the mechanism of the inhibition, ANS binding, intrinsic fluorescence, and amide I content were performed in the presence of the bioactive compound. All the results confirmed the role of the compound in assisting the proper folding of the protein. In addition, molecular docking indicated the aspidospermine derivative binds the amyloidogenic region of the protein. Our results show that the alkaloid extracted from borage leaves reduces protein aggregation mediating through structural elements of the protein, promoting the correct folding of lysozyme. Since a number of aspidospermine compounds have been shown to possess potent antimalarial activities, the action of compound identified in the present study suggests a possible link between protein aggregation and aspidospermine drugs.

Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.

We present here an approach for engineering evolving DNA barcodes in living cells. The methodology entails using a homing guide RNA (hgRNA) scaffold that directs the Cas9-hgRNA complex to target the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cultured cell populations and show that they can record lineage history and and that their RNA can be assayed as single moleculesin situ. This integrated approach will have wide ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.

Recording biological signals can be difficult in three-dimensional matrices, such as tissue. We present a DNA polymerase-based strategy that records temporal biosignals locally onto DNA to be read out later, which could obviate the need to extract information from tissue on the fly. We use a template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT) that probabilistically adds dNTPs to single-stranded DNA (ssDNA) substrates without a template. We show thatin vitro, the dNTP-incorporation preference of TdT changes with the presence of Co2+, Ca2+, Zn2+and temperature. Extracting the signal profile over time is possible by examining the dNTP incorporation preference along the length of synthesized ssDNA strands like a molecular ticker tape. We call this TdT-based untemplated recording of temporal local environmental signals (TURTLES). We show that we can determine the time of Co2+addition to within two minutes over a 60-minute period. Further, TURTLES has the capability to record multiple fluctuations. We can estimate the rise and fall of an input Co2+pulse to within three minutes. TURTLES has at least 200-fold better temporal resolution than all previous DNA-based recording techniques.

Keratinocyte Growth Factor (KGF) is a paracrine-acting and epithelium-specific growth factor produced by cells of mesenchymal origin. Based on preclinical data, recombinant KGF plays a critical role in protecting and repairing of damaged epithelial tissues. Despite great efforts to express recombinant human KGF(rhKGF) in different organisms, attempts for finding appropriate protein expression system with the ability of producing a properly folded and processed KGF needs further investigation. Pichia pastoris has been used successfully and extensively for production of industrial enzymes and pharmaceutical proteins. Herein, we investigated the affect of pro-region-α-factor early deletion on production and secretion of rhKGF in Pichia pastoris. Initially, expression of human KGF induced in MCF-7 cell line treated with 1, 25-Dihydroxy vitamin D3. The coding sequence of full-length rhKGF194 was then cloned into the yeast integrative expression vector, downstream of α-factor and was integrated into P. pastoris genome. KGF protein was expressed in P. pastoris x33 cells, usingα-factor signal peptide for translocation of KGF to ER. An internal human signal peptide was also arranged after α-factorfor early removal of the pro-region in ER. RT-PCR results demonstrated that KGF mRNA was expressed successfully after induction by methanol. Recombinant KGF protein expression was detected by Western blotting in cell lysats, but not in conditioned media. A molecular weight of 17 kD for rhKGF194 indicates that the α-factor and internal human signal peptideshad been removed in x33 cells. The results indicate that in the absence of pro-region-α-factor, the recombinant KGF protein was not efficiently processed and transported within the biosynthesis-secretory pathway. As KGF protein is an unstable growth factor and tend to aggregate because of some native properties, It seems that presence of a chaperon molecule fusion with KGF is necessary for efficient secretion of the recombinant protein.

A simple platform based on a hairpin oligonucleotide switch and multi-walled carbon nanotubes (MWCNTs) for the ultrasensitive detection of specific DNA sequences has been developed. In this approach, the π-stacking interaction of single-strand DNA–MWCNT was employed to construct an electrochemical DNA biosensor. Changes to the surface conductivity, based on the MWCNT replacement, were monitored by using the electrochemical species [Fe(CN)6]3−/4− as a redox probe. Morphological and voltammetric characterizations of the electrode surface were performed using atomic force microscopy (AFM), energy dispersive spectroscopy (EDS), cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. This biosensor has a good linear correlation range, from 0.1 μM to 10 pM, for the model target DNA. This approach provides a precise method for the sensitive detection of sequence-specific, non-complementary and mismatched DNA with significant advantages in terms of simplicity, low cost, and stability.

INTRODUCTION The remarkable development of a single cell, the zygote, into the full organism occurs through a complex series of division and differentiation events that resemble a tree, with the zygote at the base branching through lineages that end in the terminal cell types at the top. Characterizing this tree of development has long been a subject of interest, and the combination of modern genome engineering and sequencing technologies promises a powerful strategy in its service: in vivo barcoding. For in vivo barcoding, heritable random mutations are induced to accumulate during development and sequenced post hoc to reconstruct the lineage tree. Demonstrations thus far have largely focused on lower vertebrates and have used a barcoding element with a constrained window of activity for clonal tracing of individual cells or cell types. Implementation in mammalian model systems, such as the mouse, incurs unique challenges that require major enhancements. RATIONALE To address the complexity of mammalian development, we reasoned that multiple independent in vivo barcoding elements could be deployed in parallel to exponentially expand their recording power. Independence requires both an absence of cross-talk between the elements and an absence of interference between their mutation outcomes. A system with the potential to deliver on these requirements is homing CRISPR, a modified version of canonical CRISPR wherein the homing guide RNA (hgRNA) combines with CRISPR-Cas9 nuclease for repeated targeting of its own locus, leading to diverse mutational outcomes. Therefore, in mouse embryonic stem cells, we scattered multiple hgRNA loci with distinct spacers in the genome to serve as barcoding elements. With this arrangement, each hgRNA acts independently as a result of its unique spacer sequence, and undesirable deletion events between multiple adjacent cut sites are less likely. Using these cells, we generated a chimeric mouse with 60 hgRNAs as the founder of the MARC1 (Mouse for Actively Recording Cells 1) line that enables barcoding and recording of cell lineages. RESULTS In the absence of Cas9, hgRNAs are stable and dormant; to initiate barcoding, we crossed MARC1 mice with Cas9 knock-in mice. In the resulting offspring, hgRNAs were activated, creating diverse mutations such that an estimated 10 23 distinct barcode combinations can be generated with only 10 hgRNAs. Furthermore, hgRNAs showed a range of activity profiles, with some mutating soon after conception while others exhibited lower activity through most of the gestation period. This range resulted in sustained barcoding throughout gestation and recording of developmental lineages: Each cell inherits a set of unique mutations that are passed on to its daughter cells, where further unique mutations can be added. Consequently, at any stage in such developmentally barcoded mice, closely related cells have a more similar mutation profile, or barcode, than the more distant ones. These recordings remain embedded in the genomes of the cells and can be extracted by sequencing. We used these recordings to carry out bottom-up reconstruction of the mouse lineage tree, starting with the first branches that emerged after the zygote, and observed robust reconstruction of the correct tree. We also investigated axis development in the brain by sequencing barcodes from the left and right side of the forebrain, midbrain, and hindbrain regions. We found that barcodes from the left and right sides of the same region were more closely related than those from different regions; this result suggests that in the precursor of the brain, commitment to the anterior-posterior axis is established prior to the lateral axis. CONCLUSION This system provides an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system. It can straightforwardly create developmentally barcoded mice in which lineage information is prerecorded in cell genomes. Combining multiple independently acting molecular recording devices greatly enhances their capacity and allows for reliable information recovery and reconstruction of deep lineage trees.

Surface functionality of nanoparticles has been pivotal in defining interactions of nanoparticles and biomolecules. To explore various functionalities on the surface of nanoparticle through a facile procedure, various carbon-based nanoparticles, modified with a specific natural amino acid, were synthesized; the amino acids were chosen in order that almost all classes of amino acids were included. After characterizations of the nanoparticles using several spectroscopic methods, the effects of surface modification of nanoparticles were examined against amyloid formation, exploiting insulin as a model amyloidogenic polypeptide. Although most amino acids afforded carbon nanoparticles, only glycine, glutamine, and asparagine containing nanoparticles demonstrated significant inhibition of amyloid formation. These bioactive nanoparticles displayed low toxicity, and they allowed higher cell viability when mixed with the amyloid fibrils. The mechanism of the inhibition was investigated by monitoring conformational changes, and intermediates in the presence of bioactive nanoparticles. All in all, the results indicated that flexibility and the amide functionality can be considered as two main factors that enhance inhibitory roles of the modified nanoparticles likely through making networks of hydrogen bonding via functionalized surface of nanoparticles.

DNA is an emerging storage medium for digital data but its adoption is hampered by limitations of phosphoramidite chemistry, which was developed for single-base accuracy required for biological functionality. Here, we establish ade novoenzymatic DNA synthesis strategy designed from the bottom-up for information storage. We harness a template-independent DNA polymerase for controlled synthesis of sequences with user-defined information content. We demonstrate retrieval of 144-bits, including addressing, from perfectly synthesized DNA strands using batch-processed Illumina and real-time Oxford Nanopore sequencing. We then develop a codec for data retrieval from populations of diverse but imperfectly synthesized DNA strands, each with a ~30% error tolerance. With this codec, we experimentally validate a kilobyte-scale design which stores 1 bit per nucleotide. Simulations of the codec support reliable and robust storage of information for large-scale systems. This work paves the way for alternative synthesis and sequencing strategies to advance information storage in DNA.

Cellular barcoding using nuclease-induced genetic mutations is an effective approach that is emerging for recording biological information, including developmental lineages. We have previously introduced the homing CRISPR system as a promising methodology for generating such barcodes with scalable diversity and without crosstalk. Here, we present a mouse line (MARC1) with multiple genomically-integrated and heritable homing guide RNAs (hgRNAs). We determine the genomic locations of these hgRNAs, their activity profiles during gestation, and the diversity of their mutants. We apply the line for unique barcoding of mouse embryos and differential barcoding of embryonic tissues. We conclude that this mouse line can address the unique challenges associated with in vivo barcoding in mammalian model organisms and is thus an enabling platform for recording and lineage tracing applications in a mammalian model system.

RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

Conformational diseases have been investigated extensively in recent years; as a result, a number of drug candidates have been introduced as amyloid inhibitors; however, no effective therapies have been put forward. RS-0406 with pyridazine as its core chemical structure has so far shown promising results in inhibiting amyloid formation. In the present work, using molecular dynamics, we undertook the investigation of RS-0406 interactions with U-shaped Aβ1–42 and Aβ1–40 pentamers, Aβ1–42 monomers, and double-horseshoe-like Aβ1–42. To set better parameters for the small molecule, experimental and computational log P values were obtained. In addition, an analogue of RS-0406 was also simulated for comparison. The results indicate that RS-0406 may inhibit amyloid formation exploiting two different mechanisms. One mechanism includes stabilizing the α helix, in the monomer peptide, by binding to the flanking sites of the amyloidogenic region. The second mechanism mediates through interaction of the small molecule...

Viruses have evolved a variety of mechanisms to interact with host cells for their adaptive benefits, including subverting host immune responses and hijacking host DNA replication/transcription machineries [1–3]. Although interactions between viral and host proteins have been studied extensively, little is known about how the vial genome may interact with the host genome and how such interactions could affect the activities of both the virus and the host cell. Since the three-dimensional organization of a genome can have significant impact on genomic activities such as transcription and replication, we hypothesize that such structure-based regulation of genomic functions also applies to viral genomes depending on their association with host genomic regions and their spatial locations inside the nucleus. Here, we used Tethered Chromosome Conformation Capture (TCC) to investigate viral-host genome interactions between the adenovirus and human lung fibroblast cells. We found viral-host genome interactions were enriched in certain active chromatin regions and chromatin domains marked by H3K27me3. The contacts by viral DNA seems to impact the structure and function of the host genome, leading to remodeling of the fibroblast epigenome. Our study represents the first comprehensive analysis of viral-host interactions at the genome structure level, revealing unexpectedly specific virus-host genome interactions. The non-random nature of such interactions indicates a deliberate but poorly understood mechanism for targeting of host DNA by foreign genomes.

In recent years, applications of dielectrophoresis-based platforms have been recognized as effective and dependable approach to separate cells and bioparticles, suspended in different carrier fluids, based on particle size and electrical properties. In this study, a microfluidic device was fabricated by an unprecedented electrode pattern, and several experiments were performed to enrich samples including either of yeast, Escherichia coli, or latex particles. A chemical deposition-based method was employed for fabrication of microelectrodes, inducing nonuniform electric field required for dielectrophoresis-based separation. One major advantage of our employed method is low fabrication cost, in addition to its accuracy and operation at low voltages. The performance of the microfluidic device in enriching either of injected samples was studied using spectrophotometric techniques. The effects of experimentally controllable parameters (applied-voltage amplitude and frequency, and flow rate) were studied by changing a parameter while keeping the others constant. It became evident that all the aforementioned parameters had modulating impact on the performance of the microfluidic device. Furthermore, to investigate binary interactions among the parameters, response surface methodology was exploited, resulting in a second-order polynomial model for the performance of the device as a function of the parameters. The model was employed for finding the optimum values of the parameters at which the performance of the device is the highest. At optimum values for the experimentally controllable parameters, enrichment efficiencies of 87 ± 2, 82 ± 4, and 86 ± 3% for, respectively, yeast, E. coli, and latex particles were obtained experimentally, confirming the ability of the proposed method for biological and polymeric particles enrichment.

Transcripts Visualized in Situ Despite advances, current methods for single-cell sequencing are unable to resolve transcript location within the cell, so Lee et al. (p. 1360 , published online 27 February) developed a method of fluorescent in situ RNA sequencing (FISSEQ) that works in vivo to show messenger RNA localization within cells. The method amplifies complementary DNA targets by rolling circle amplification, and then in situ cross-linking locks amplicons to produce ample, highly localized templates for three-dimensional sequencing. The technique was tested in fibroblasts to reveal the differences between individual cells during wound repair.

Hepcidin is a peptide hormone that plays an important role in iron metabolism. We have produced a recombinant mouse hepcidin-1 by using baculovirus expression system. Its expression yield was 25 μg/ml when cell culture media were supplemented with a protease inhibitor cocktail. The recombinant mouse hepcidin-1 and synthetic human hepcidin-25 had similar effects on reducing ferroportin expression in J774A cell line and in peritoneal macrophages. However, synthetic human hepcidin-25 was more efficient than recombinant mouse hepcidin-1 in reducing iron concentration in blood circulation (p < 0.01).

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

ApolipoproteinD (ApoD) is a human glycoprotein from the lipocalin family. ApoD contains a conserved central motif of an 8-stranded antiparallel β-sheet, which forms a beta-barrel that can be used for transport and storage of diverse hydrophobic ligands. Due to hydrophobic nature of ApoD, it has been difficult to generate a recombinant version of this protein. In the present work, we aimed at the production of ApoD in the robust Pichia pastoris expression system. To this end, the ApoD gene sequence was synthesized and subcloned for expression in the yeast host cells. Following integration of the ApoD gene into the yeast genomic region using homologous recombination, the ApoD recombinant protein was induced using methanol, reaching its maximum induction at 96 h. Having purified the ApoD recombinant protein by affinity chromatography, we measured the dissociation constant (KD) using its natural ligands: progesterone and arachidonic acid. Our results provide a viable solution to the production of recombinant ApoD protein in lieu of previous obstacles in generating soluble and functional ApoD protein.

We describe tethered conformation capture (TCC), a method for genome-wide mapping of chromatin interactions. By performing ligations on solid substrates rather than in solution, TCC substantially enhances the signal-to-noise ratio, thereby facilitating a detailed analysis of interactions within and between chromosomes. We identified a group of regions in each chromosome in human cells that account for the majority of interchromosomal interactions. These regions are marked by high transcriptional activity, suggesting that their interactions are mediated by transcriptional machinery. Each of these regions interacts with numerous other such regions throughout the genome in an indiscriminate fashion, partly driven by the accessibility of the partners. As a different combination of interactions is likely present in different cells, we developed a computational method to translate the TCC data into physical chromatin contacts in a population of three-dimensional genome structures. Statistical analysis of the resulting population demonstrates that the indiscriminate properties of interchromosomal interactions are consistent with the well-known architectural features of the human genome.

Density functional theory calculations were performed to investigate the properties of purine-functionalised carbon nanotube (CNT), including adenine and guanine functionalised models (CNT-A and CNT-G). The results indicated that the CNT-G model could be better dispersed in the hydrated systems, whereas the CNT-A model could be expected as a more favourably stable structure. The results also indicated that the quadrupole coupling constant (C(Q)) for the nitrogen atoms of the CNT-G model detects more significant changes than the CNT-A model with respect to the individual guanine and adenine structures.

We have investigated the electronic and structural properties of covalent functionalization of the tip of (5,0) carbon nanotube (CNT) by di-keto and keto-enol forms of thymine (T) and uracil (U) nucleobases. Density functional theory (DFT) calculations have been performed to optimize the investigated structures and to calculate the properties such as dipole moment, bond length, band gap, total energy, binding energy and quadrupole coupling constant. The results indicated that, due to the functionalization of CNT by T and U, the hybrids exhibit new properties in which they are similar in both types of CNT-T and CNT-U hybrids.

Gold nanoparticles were synthesized in a single step and a facile procedure by γ-irradiation using bovine serum albumin protein as a stabilizer. The results of UV–vis absorption spectroscopy, transmission electron microscopy and X-ray diffraction analyses indicated that spherical well-dispersed gold nanoparticles ranging from 2 to 7 nm were generated, depending on the irradiation doses. The conjugation of BSA to the gold nanoparticles was indicated using FTIR spectroscopy and dynamic light scattering. Moreover, the effect of irradiation on the BSA structure in the presence and absence of Au3+ was studied by SDS-PAGE analysis. It became evident that gold nanoparticles formation partially protected against the degradation and aggregation of BSA. Methyl tetrazolium (MTT) assay demonstrated that the generated gold nanoparticles are not cytotoxic to mammalian cells in cultures.

In searching for alternative ways to reduce or inhibit amyloid formation, we have studied this process using hen egg white lysozyme (HEWL) in the presence of a low concentration of protic ionic liquids. The ionic liquids were synthesized in a combinatorial fashion maintaining the cationic part (tetramethylguanidinium) with alteration of the anionic component of each compound tested. It was observed that one of these compounds (tetramethylguanidinium acetate) inhibited amyloid formation of HEWL in vitro by nearly 50%. Examination under transmission electron microscopy confirmed the fibril inhibition, and fibrils were observed to be morphologically thinner. To investigate the mechanism of inhibition, intrinsic fluorescence, ANS binding, and circular dichroism analyses were performed. These analyses indicated that the native structure of HEWL was maintained in the presence of the ionic liquid. Performing native PAGE and nondenaturing agarose electrophoresis, it became evident that some of the intermediate oligomers were not converted to protofibrils and that the oligomers were trapped in more stable conformations. Additionally, it was observed that this inhibitory effect was related to the ionic liquid itself and not the solvated ions. It also became evident that the carboxyl functional group was important in the inhibition. The size of the anions and kosmotropicity did not play significant roles in the fibril inhibition.

The host factor, nuclear factor of activated T-cells (NFAT), regulates the transcription and replication of HIV-1. Here, we have determined the crystal structure of the DNA binding domain of NFAT bound to the HIV-1 long terminal repeat (LTR) tandem kappaB enhancer element at 3.05 A resolution. NFAT binds as a dimer to the upstream kappaB site (Core II), but as a monomer to the 3' end of the downstream kappaB site (Core I). The DNA shows a significant bend near the 5' end of Core I, where a lysine residue from NFAT bound to the 3' end of Core II inserts into the minor groove and seems to cause DNA bases to flip out. Consistent with this structural feature, the 5' end of Core I become hypersensitive to dimethylsulfate in the in vivo footprinting upon transcriptional activation of the HIV-1 LTR. Our studies provide a basis for further investigating the functional mechanisms of NFAT in HIV-1 transcription and replication.

RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

We demonstrate that the product of the yeast open reading frame YML005w is required for wybutosine (yW) formation in the phenylalanine-accepting tRNA of the yeast Saccharomyces cerevisiae. tRNA isolated from a deletion mutant of the YML005w gene accumulates 4-demethylwyosine (ImG-14), a precursor lacking three of the methyl groups of the yW hypermodified base. Since the amino acid sequence of the YML005w gene contains the signature motifs of the seven β-strand methyltransferases, we now designate the gene TRM12 for tRNA methyltransferase. Using pulse-chase labeling of intact yeast cells with S-adenosyl- l -[methyl-3H]methionine, we show that the methylesterified form of yW is metabolically stable.

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.