Biology subjects

Strauss, K.

Publications and source records attributed to Strauss, K..

14 recordsLinked to original sources

bioRxiv2024

Control of alveolar bone development, homeostasis, and socket healing by salt inducible kinases

Alveolar bone supports and anchors teeth. The parathyroid hormone-related protein (PTHrP) pathway plays a key role in alveolar bone biology. Salt inducible kinases (SIKs) are important downstream regulators of PTH/PTHrP signaling in the appendicular skeleton where SIK inhibition increases bone formation and trabecular bone mass. However, the function of these kinases in alveolar bone remains unknown. Here, we report a critical role for SIK2/SIK3 in alveolar bone development, homeostasis, and socket healing after tooth extraction. Inducible SIK2/SIK3 deletion led to dramatic alveolar bone defects without changes in tooth eruption. Ablating these kinases impairs alveolar bone formation due to disrupted osteoblast maturation, a finding associated with ectopic periostin expression by fibrous cells in regions of absent alveolar bone at steady state and following molar extraction. Distinct phenotypic consequences of SIK2/SIK3 deletion in appendicular versus craniofacial bones prompted us to identify a specific transcriptomic signature in alveolar versus long bone osteoblasts. Thus, SIK2/SIK3 deletion illuminates a key role for these kinases in alveolar bone biology and highlights the emerging concept that different osteoblast subsets utilize unique genetic programs. Summary statementSIK2/SIK3 deletion in alveolar bone reduces bone formation and mass by impairing osteoblast maturation, unlike in long bones, where it increases bone formation and mass.

developmental biology↗

bioRxiv2023

DNA storage in thermoresponsive microcapsules for repeated random multiplexed data access

Owing to its longevity and extremely high information density, DNA has emerged as an attractive medium for archival data storage. Scalable parallel random access of information is a desirable property of any storage system. For DNA-based storage systems, however, this yet has to be robustly established. Here we develop thermoconfined PCR, a novel method that enables multiplexed, repeated random access of compartmentalized DNA files. Our strategy is based on stable localization of biotin-functionalized oligonucleotides inside microcapsules with temperature-dependent membrane permeability. At low temperatures, microcapsules are permeable to enzymes, primers, and amplified products, while at high temperatures membrane collapse prevents molecular crosstalk during amplification. We demonstrate that our platform outperforms non-compartmentalized DNA storage with respect to repeated random access and reducing amplification bias during multiplex PCR. Using fluorescent sorting, we additionally demonstrate sample pooling and data retrieval by barcoding of microcapsules. Our thermoresponsive microcapsule technology offers a scalable, sequence-agnostic approach for repeated random access of archival DNA files.

synthetic biology↗

bioRxiv2022

Information Decay and Enzymatic Information Recovery for DNA Data Storage

Synthetic DNA has been proposed as a storage medium for digital information due to its high theoretical storage density and anticipated long storage horizons. However, under all ambient storage conditions, DNA undergoes a slow chemical decay process resulting in nicked (broken) DNA strands, and the information stored in these strands is no longer readable. In this work we design an enzymatic repair procedure, which is applicable to the DNA pool prior to readout and can partially reverse the damage. Through a chemical understanding of the decay process, an overhang at the 3 end of the damaged site is identified as obstructive to repair via the base excision-repair (BER) mechanism. The obstruction can be removed via the enzyme apurinic/apyrimidinic endonuclease I (APE1), thereby enabling repair of hydrolytically damaged DNA via Bst polymerase and Taq ligase. Simulations of damage and repair reveal the benefit of the enzymatic repair step for DNA data storage, especially when data is stored in DNA at high storage densities (= low physical redundancy) and for long time durations.

synthetic biology↗

bioRxiv2021

A nanopore interface for high bandwidth DNA computing

DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies MinION device). We show that engineered reporter probes can be detected and classified with high accuracy at the single-molecule level directly from raw nanopore signals using deep learning. We then demonstrate this methods utility in multiplexed detection of clinically relevant microRNA sequences. These results increase DSD output bandwidth by an order of magnitude over what is possible with fluorescence spectroscopy, laying the foundations for a new paradigm in DNA circuit readout and programmable multiplexed molecular diagnostics using portable nanopore devices.

bioengineering↗

bioRxiv2021

A Combinatorial PCR Method for Efficient, Selective Oligo Retrieval from Complex Oligo Pools

With the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications, including gene synthesis, CRISPR-based gene editing, and DNA data storage. Selectively retrieving specific oligos from these complex pools traditionally uses Polymerase Chain Reaction (PCR), in which any selected oligos are exponentially amplified to quickly outnumber non-selected ones. In this case, the number of orthogonal PCR primers is limited due to interactions between them. This lack of specificity presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool (i.e., a DNA database) is orders of magnitude larger than it is for other applications. Although a nested file address system was recently developed to increase the number of accessible files for DNA storage, it requires a more complicated lab protocol and more expensive reagents to achieve high specificity. Instead, we developed a new combinatorial PCR method that outperforms prior work without compromising the fidelity of retrieved material or complicating wet lab processes. Our method quadratically increases the number of accessible oligos while maintaining high specificity. In experiments, we accessed three arbitrarily chosen files from a DNA prototype database that contained 81 different files. Initially comprising only 1% of the original database, the selected files were enriched to over 99.9% using our combinatorial primer method. Our method thus provides a viable path for scaling up DNA data storage systems and has broader utility whenever scientists need access to a specific target oligo and can design their own primer regions.

synthetic biology↗

bioRxiv2020

A Comprehensive Study of Synthetic DNA Preservation for DNA Data Storage

Synthetic DNA has recently risen as a viable alternative for long-term digital data storage. To ensure that information is safely recovered after storage, it is essential to appropriately preserve the physical DNA molecules encoding the data. While preservation of biological DNA has been studied previously, synthetic DNA differs in that it is typically much shorter in length, it has different sequence profiles with fewer, if any, repeats (or homopolymers), and it has different contaminants. In this paper we evaluate nine different methods used to preserve data files encoded in synthetic DNA by accelerated aging of nearly 29,000 DNA sequences. In addition to a molecular count comparison, we also sequence and analyze the DNA after aging. Our findings show that errors and erasures are stochastic and show no practical distribution difference between preservation methods. Finally, we compare the physical density of these methods and provide a stability versus density trade-offs discussion.

synthetic biology↗

bioRxiv2020

Content-Based Similarity Search in Large-Scale DNA Data Storage Systems

Synthetic DNA has the potential to store the worlds continuously growing amount of data in an extremely dense and durable medium. Current proposals for DNA-based digital storage systems include the ability to retrieve individual files by their unique identifier, but not by their content. Here, we demonstrate content-based retrieval from a DNA database by learning a mapping from images to DNA sequences such that an encoded query image will retrieve visually similar images from the database via DNA hybridization. We encoded and synthesized a database of 1.6 million images and queried it with a variety of images, showing that each query retrieves a sample of the database containing visually similar images are retrieved at a rate much greater than chance. We compare our results with several algorithms for similarity search in electronic systems, and demonstrate that our molecular approach is competitive with state-of-the-art electronics. One Sentence SummaryLearned encodings enable content-based image similarity search from a database of 1.6 million images encoded in synthetic DNA.

bioengineering↗

bioRxiv2020

Porcupine: Rapid and robust tagging of physical objects using nanopore-orthogonal DNA strands

Molecular tagging is an approach to labeling physical objects using DNA or other molecules that can be used in cases where methods like RFID tags and QR codes are not suitable. No molecular tagging method exists that is inexpensive, fast and reliable to decode, and usable outside a lab setting to create or read tags. To address this, we present Porcupine, an end-user molecular tagging system that features DNA-based tags readable within seconds using a portable nanopore device. Porcupines digital bits are represented by the presence or absence of distinct, nanopore-orthogonal DNA strands, which we call molecular bits (molbits). We classify molbits directly from the raw nanopore signal, avoiding basecalling. To extend the tags shelf life, decrease readout time, and make tags robust to environmental contamination, molbits are prepared for readout during tag assembly and can be stabilized by dehydration. The result is an extensible, real time, high accuracy tagging system that includes a novel approach to developing nanopore-orthogonal barcodes. One sentence summaryPorcupine lets end-users label physical objects with custom DNA tags, without requiring a lab to create or read tags, and offers rapid readout using nanopore sequencing.

synthetic biology↗

bioRxiv2019

Multiplexed direct detection of barcoded protein reporters on a nanopore array

Genetically encoded reporter proteins are a cornerstone of molecular biology. While they are widely used to measure many biological activities, the current number of uniquely addressable reporters that can be used together for one-pot multiplexed tracking is small due to overlapping detection channels such as fluorescence. To address this, we built an expanded library of orthogonally-barcoded Nanopore-addressable protein Tags Engineered as Reporters (NanoporeTERs), which can be read and demuxed by nanopore sensors at the single-molecule level. By adapting a commercially available nanopore sensor array platform typically used for real-time DNA and RNA sequencing (Oxford Nanopore Technologies MinION), we show direct detection of NanoporeTER expression levels from unprocessed bacterial culture with no specialized sample preparation. These results lay the foundations for a new class of reporter proteins to enable multiplexed, real-time tracking of gene expression with nascent nanopore sensor technology.

synthetic biology↗

bioRxiv2019

Cortical Overgrowth in a Preclinical Forebrain Organoid Model of CNTNAP2-Associated Autism Spectrum Disorder

Autism spectrum disorder (ASD) represents a major public health burden but translating promising treatment findings from preclinical non-human models of ASD to the clinic has remained challenging. The recent development of forebrain organoids generated from human induced pluripotent stem cells (hiPSCs) derived from subjects with brain disorders is a promising method to study human-specific neurobiology, and may facilitate the development of novel therapeutics.\n\nIn this study, we utilized forebrain organoids generated from hiPSCs derived from patients from the Old Order Amish community with a rare syndromic form of ASD, carrying a homozygous c.3709DelG mutation in CNTNAP2 and healthy controls to investigate the effects of this mutation on cortical embryonic development.\n\nPatients carrying the c.3709DelG mutation in CNTNAP2 present with an increased head circumference and brain MRI reveals an increase in gray matter volume. Patient-derived organoids displayed an increase in total volume that was driven by an increased proliferation in neural progenitor cells, leading to an increase in the generation of cortical neuronal and non-neuronal cell types. The observed phenotypes were rescued after correction of the pathogenic mutation using CRISPR-Cas9. RNA sequencing revealed 339 genes differentially expressed between patient- and control-derived organoids of which a subset are implicated in cell proliferation and neurogenesis. Notably, these differentially expressed genes included previously identified ASD-associated genes and are enriched for genes in ASD-associated weighted gene co-expression networks.\n\nThis work provides a critical step towards understanding the role of CNTNAP2 in human cortical development and has important mechanistic implications for ASD associated with brain overgrowth. This CNTNAP2 organoid model provides opportunity for further mechanistic inquiry and development of new therapeutic strategies for ASD.

neuroscience↗

bioRxiv2019

Experimental Assessment of PCR Specificity and Copy Number for Reliable Data Retrieval in DNA Storage

Synthetic DNA has been gaining momentum as a potential storage medium for archival data storage1-9. Digital information is translated into sequences of nucleotides and the resulting synthetic DNA strands are then stored for later individual file retrieval via PCR7-9 (Fig. 1a). Using a previously presented encoding scheme9 and new experiments, we demonstrate reliable file recovery when as few as 10 copies per sequence are stored, on average. This results in density of about 17 exabytes/g, nearly two orders of magnitude greater than prior work has shown6. Further, no prior work has experimentally demonstrated access to specific files in a pool more complex than approximately 106 unique DNA sequences9, leaving the issue of accurate file retrieval at high data density and complexity unexamined. Here, we demonstrate successful PCR random access using three files of varying sizes in a complex pool of over 1010 unique sequences, with no evidence that we have begun to approach complexity limits. We further investigate the role of file size on successful data recovery, the effect of increasing sequencing coverage to aid file recovery, and whether DNA strands drop out of solution in a systematic manner. These findings substantiate the robustness of PCR as a random access mechanism in complex settings, and that the number of copies needed for data retrieval does not compromise density significantly.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC=\"FIGDIR/small/565150_fig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@4b79a7org.highwire.dtl.DTLVardef@11fca45org.highwire.dtl.DTLVardef@18b830org.highwire.dtl.DTLVardef@e47101_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO (a) A high-level representation of the DNA data storage pipeline. (b) (Left) The bar chart depicts contents of the initial, undiluted pool. (Right) The illustration shows the serial nature of subsequent dilutions. Mean copy number refers to the mean number of copies of each files unique sequences as determined by qPCR (Supplemental Section 1). One serial dilution used water as the diluent in each step; the other used a solution of 150Nmers to dilute the pool to much greater complexity. (c) Details of how the samples were diluted. Note that the dilution steps were identical regardless of diluent. The smallest percent of pool accessed is calculated by dividing the size of the smallest file by the number of unique sequences in the 1 {micro}L of solution used for PCR random access. This percentage refers to the 150N diluent pool since the small file in the water diluent pool is a constant 0.13%.\n\nC_FIG

synthetic biology↗

bioRxiv2019

Quantifying Molecular Bias in DNA Data Storage

DNA has recently emerged as an attractive medium for future digital data storage because of its extremely high information density and potential longevity. Recent work has shown promising results in developing proof-of-principle prototype systems. However, very uneven (biased) sequencing coverage distributions have been reported, which indicates inefficiencies in the storage process and points to optimization opportunities. These deviations from the average coverage in oligonucleotide copy distribution result in sequence drop-out and make error-free data retrieval from DNA more challenging. The uneven copy distribution was believed to stem from the underlying molecular processes, but the interplay between these molecular processes and the copy number distribution has been poorly understood until now. In this paper, we use millions of unique sequences from a DNA-based digital data archival system to study the oligonucleotide copy unevenness problem and show that two important sources of bias are the synthesis process and the Polymerase Chain Reaction (PCR) process. By mapping the sequencing coverage of a large complex oligonucleotide pool back to its spatial distribution on the synthesis chip, we find that significant bias comes from array-based oligonucleotide synthesis. We also find that PCR stochasticity is another main driver of oligonucleotide copy variation. Based on these findings, we develop a statistical model for each molecular process as well as the overall process and compare the predicted bias with our experimental data. We further use our model to explore the trade-offs between synthesis bias, storage physical density and sequencing redundancy, providing insights for engineering efficient, robust DNA data storage systems.

synthetic biology↗

bioRxiv2018

Demonstration of End-to-End Automation of DNA Data Storage

We developed a complete end-to-end DNA data storage device. The device enables the encoding of data, which is then written to a DNA oligonucleotide using a custom DNA synthesizer, pooled for liquid storage, and read using a nanopore sequencer and a novel, minimal preparation protocol. We demonstrate an automated 5-byte write, store, and read cycle with the ability to expand as new technology is available.

synthetic biology↗

bioRxiv2017

Scaling up DNA data storage and randomaccess retrieval

Current storage technologies can no longer keep pace with exponentially growing amounts of data. 1 Synthetic DNA offers an attractive alternative due to its potential information density of ~ 1018 B/mm3, 107 times denser than magnetic tape, and potential durability of thousands of years.2 Recent advances in DNA data storage have highlighted technical challenges, in particular, coding and random access, but have stored only modest amounts of data in synthetic DNA. 3,4,5 This paper demonstrates an end-to-end approach toward the viability of DNA data storage with large-scale random access. We encoded and stored 35 distinct files, totaling 200MB of data, in more than 13 million DNA oligonucleotides (about 2 billion nucleotides in total) and fully recovered the data with no bit errors, representing an advance of almost an order of magnitude compared to prior work. 6 Our data curation focused on technologically advanced data types and historical relevance, including the Universal Declaration of Human Rights in over 100 languages,7 a high-definition music video of the band OK Go,8 and a CropTrust database of the seeds stored in the Svalbard Global Seed Vault.9 We developed a random access methodology based on selective amplification, for which we designed and validated a large library of primers, and successfully retrieved arbitrarily chosen items from a subset of our pool containing 10.3 million DNA sequences. Moreover, we developed a novel coding scheme that dramatically reduces the physical redundancy (sequencing read coverage) required for error-free decoding to a median of 5x, while maintaining levels of logical redundancy comparable to the best prior codes. We further stress-tested our coding approach by successfully decoding a file using the more error-prone nanopore-based sequencing. We provide a detailed analysis of errors in the process of writing, storing, and reading data from synthetic DNA at a large scale, which helps characterize DNA as a storage medium and justify our coding approach. Thus, we have demonstrated a significant improvement in data volume, random access, and encoding/decoding schemes that contribute to a whole-system vision for DNA data storage.

bioengineering↗