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1.
ACS Synth Biol ; 12(11): 3156-3169, 2023 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-37935025

RESUMO

Synthetic Biology has overcome many of the early challenges facing the field and is entering a systems era characterized by adoption of Design-Build-Test-Learn (DBTL) approaches. The need for automation and standardization to enable reproducible, scalable, and translatable research has become increasingly accepted in recent years, and many of the hardware and software tools needed to address these challenges are now in place or under development. However, the lack of connectivity between DBTL modules and barriers to access and adoption remain significant challenges to realizing the full potential of lab automation. In this review, we characterize and classify the state of automation in synthetic biology with a focus on the physical automation of experimental workflows. Though fully autonomous scientific discovery is likely a long way off, impressive progress has been made toward automating critical elements of experimentation by combining intelligent hardware and software tools. It is worth questioning whether total automation that removes humans entirely from the loop should be the ultimate goal, and considerations for appropriate automation versus total automation are discussed in this light while emphasizing areas where further development is needed in both contexts.


Assuntos
Automação Laboratorial , Biologia Sintética , Humanos , Automação , Software , Padrões de Referência , Projetos de Pesquisa
2.
ACS Synth Biol ; 12(6): 1716-1726, 2023 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-37192389

RESUMO

Novel enzymatic methods are poised to become the dominant processes for de novo synthesis of DNA, promising functional, economic, and environmental advantages over the longstanding approach of phosphoramidite synthesis. Before this can occur, however, enzymatic synthesis methods must be parallelized to enable production of multiple DNA sequences simultaneously. As a means to this parallelization, we report a polymerase-nucleotide conjugate that is cleaved using electrochemical oxidation on a microelectrode array. The developed conjugate maintains polymerase activity toward surface-bound substrates with single-base control and detaches from the surface at mild oxidative voltages, leaving an extendable oligonucleotide behind. Our approach readies the way for enzymatic DNA synthesis on the scale necessary for DNA-intensive applications such as DNA data storage or gene synthesis.


Assuntos
DNA Nucleotidilexotransferase , Nucleotídeos , DNA Nucleotidilexotransferase/genética , DNA , Oligonucleotídeos , Sequência de Bases
3.
Nat Nanotechnol ; 18(8): 912-921, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37142708

RESUMO

DNA has emerged as an attractive medium for archival data storage due to its durability and high information density. Scalable parallel random access to information is a desirable property of any storage system. For DNA-based storage systems, however, this still needs to be robustly established. Here we report on a thermoconfined polymerase chain reaction, which enables multiplexed, repeated random access to compartmentalized DNA files. The strategy is based on localizing biotin-functionalized oligonucleotides inside thermoresponsive, semipermeable microcapsules. At low temperatures, microcapsules are permeable to enzymes, primers and amplified products, whereas at high temperatures, membrane collapse prevents molecular crosstalk during amplification. Our data show that the platform outperforms non-compartmentalized DNA storage compared with repeated random access and reduces amplification bias tenfold during multiplex polymerase chain reaction. Using fluorescent sorting, we also demonstrate sample pooling and data retrieval by microcapsule barcoding. Therefore, the thermoresponsive microcapsule technology offers a scalable, sequence-agnostic approach for repeated random access to archival DNA files.


Assuntos
DNA , Armazenamento e Recuperação da Informação , Cápsulas , DNA/genética , Oligonucleotídeos , Sequenciamento de Nucleotídeos em Larga Escala
4.
Commun Biol ; 5(1): 1117, 2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36266439

RESUMO

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.


Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/genética , Armazenamento e Recuperação da Informação , Desoxirribonuclease I , Ligases
5.
Langmuir ; 38(37): 11191-11198, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36083165

RESUMO

A core-shell strategy was developed to protect synthetic DNA in organosilica particles encompassing dithiol linkages allowing for a DNA loading of 1.1 wt %. DNA stability tests involving bleach as an oxidant showed that following the procedure DNA was sandwiched between core particles of ca. 450 nm size and a protective outer layer, separating the DNA from the environment. Rapid aging tests at 60 °C and 50% relative humidity revealed that the DNA protected within this material was significantly more stable than nonprotected DNA, with an expected ambient temperature half-life of over 60 years. Still, and due to the presence of the dithiol linkages in the backbone of the organosilica material, the particles degraded in the presence of reducing agents (TCEP and glutathione) and disintegrated within several days in a simulated compost environment, which was employed to test the biodegradability of the material. This is in contrast to DNA encapsulated following state of the art procedures in pure SiO2 particles, which do not biodegrade in the investigated timeframes and conditions. The results show that synthetic DNA protected within dithiol comprising organosilica particles presents a strategy to store digital data at a high storage capacity for long time frames in a fully biodegradable format.


Assuntos
Nanopartículas , Dióxido de Silício , DNA/genética , Glutationa , Oxidantes , Substâncias Redutoras , Tolueno/análogos & derivados
6.
Nat Commun ; 13(1): 4904, 2022 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-35987925

RESUMO

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). These results increase DSD output bandwidth by an order of magnitude over what is currently feasible with fluorescence spectroscopy.


Assuntos
Nanoporos , DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Recombinação Genética , Análise de Sequência de DNA/métodos
7.
Small ; 18(15): e2107381, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35218608

RESUMO

Using DNA as a durable, high-density storage medium with eternal format relevance can address a future data storage deficiency. The proposed storage format incorporates dehydrated particle spots on glass, at a theoretical capacity of more than 20 TB per spot, which can be efficiently retrieved without significant loss of DNA. The authors measure the rapid decay of dried DNA at room temperature and present the synthesis of encapsulated DNA in silica nanoparticles as a possible solution. In this form, the protected DNA can be readily applied to digital microfluidics (DMF) used to handle retrieval operations amenable to full automation. A storage architecture is demonstrated, which can increase the storage capacity of today's archival storage systems by more than three orders of magnitude: A DNA library containing 7373 unique sequences is encapsulated and stored under accelerated aging conditions (4 days at 70 °C, 50% RH) corresponding to 116 years at room temperature and the stored information is successfully recovered.


Assuntos
DNA , Microfluídica , Vidro , Armazenamento e Recuperação da Informação , Temperatura
8.
Chem Commun (Camb) ; 58(19): 3174-3177, 2022 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-35171192

RESUMO

The resilience of ancient DNA (aDNA) in bone gives rise to the preservation of synthetic DNA with bioinorganic materials such as calcium phosphate (CaP). Accelerated aging experiments at elevated temperature and humidity displayed a positive effect of co-precipitated, crystalline dicalcium phosphate on the stability of synthetic DNA in contrast to amorphous CaP. Quantitative PXRD in combination with SEM and EDX measurements revealed distinct CaP phase transformations of calcium phosphate dihydrate (brushite) to anhydrous dicalcium phosphate (monetite) influencing DNA stability.


Assuntos
Materiais Biocompatíveis/química , Fosfatos de Cálcio/química , DNA/química , Teste de Materiais
9.
ACS Synth Biol ; 11(5): 1727-1734, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35191684

RESUMO

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. The selective retrieval of specific oligos from these complex pools traditionally uses polymerase chain reaction (PCR). Designing a large number of primers to use in PCR presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool is orders of magnitude larger than other applications. Although a nested primer address system was recently developed to increase the number of accessible files for DNA storage, it requires more complicated lab protocols and more expensive reagents to achieve high specificity, as well as more DNA address space. Here, we present a new combinatorial PCR method that has none of those drawbacks and outperforms in retrieval specificity. In experiments, we accessed three files that each comprised 1% of a DNA prototype database that contained 81 different files and enriched them to over 99.9% using our combinatorial primer method. Our method provides a viable path for scaling up DNA data storage systems and has broader utility whenever one must access a specific target oligo and can design their own primer regions.


Assuntos
DNA , Armazenamento e Recuperação da Informação , DNA/genética , Primers do DNA/genética , Bases de Dados de Ácidos Nucleicos , Reação em Cadeia da Polimerase/métodos
10.
Nat Commun ; 13(1): 352, 2022 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-35039502

RESUMO

Synthetic DNA is a growing alternative to electronic-based technologies in fields such as data storage, product tagging, or signal processing. Its value lies in its characteristic attributes, namely Watson-Crick base pairing, array synthesis, sequencing, toehold displacement and polymerase chain reaction (PCR) capabilities. In this review, we provide an overview of the most prevalent applications of synthetic DNA that could shape the future of information technology. We emphasize the reasons why the biomolecule can be a valuable alternative for conventional electronic-based media, and give insights on where the DNA-analog technology stands with respect to its electronic counterparts.


Assuntos
DNA/química , Tecnologia da Informação , Redes Neurais de Computação
11.
Nat Biotechnol ; 40(1): 42-46, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34385692

RESUMO

Detection of specific proteins using nanopores is currently challenging. To address this challenge, we developed a collection of over twenty nanopore-addressable protein tags engineered as reporters (NanoporeTERs, or NTERs). NTERs are constructed with a secretion tag, folded domain and a nanopore-targeting C-terminal tail in which arbitrary peptide barcodes can be encoded. We demonstrate simultaneous detection of up to nine NTERs expressed in bacterial or human cells using MinION nanopore sensor arrays.


Assuntos
Nanoporos , Bactérias , Humanos
12.
Small Methods ; 5(5): e2001094, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34928102

RESUMO

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, nine different methods used to preserve data files encoded in synthetic DNA are evaluated by accelerated aging of nearly 29 000 DNA sequences. In addition to a molecular count comparison, the DNA is also sequenced and analyzed after aging. These findings show that errors and erasures are stochastic and show no practical distribution difference between preservation methods. Finally, the physical density of these methods is compared and a stability versus density trade-offs discussion provided.


Assuntos
DNA/química , Sequência de Bases , DNA/metabolismo , Meia-Vida , Sequenciamento de Nucleotídeos em Larga Escala , Nanopartículas de Magnetita/química , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Temperatura , Fatores de Tempo , Trealose/química
13.
Sci Adv ; 7(48): eabi6714, 2021 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-34818035

RESUMO

Synthetic DNA is an attractive medium for long-term data storage because of its density, ease of copying, sustainability, and longevity. Recent advances have focused on the development of new encoding algorithms, automation, preservation, and sequencing technologies. Despite progress in these areas, the most challenging hurdle in deployment of DNA data storage remains the write throughput, which limits data storage capacity. We have developed the first nanoscale DNA storage writer, which we expect to scale DNA write density to 25 × 106 sequences per square centimeter, three orders of magnitude improvement over existing DNA synthesis arrays. We show confinement of DNA synthesis to an area under 1 square micrometer, parallelized over millions of nanoelectrode wells and then successfully write and decode a message in DNA. DNA synthesis on this scale will enable write throughputs to reach megabytes per second and is a key enabler to a practical DNA data storage system.

14.
Nat Commun ; 12(1): 4764, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362913

RESUMO

As global demand for digital storage capacity grows, storage technologies based on synthetic DNA have emerged as a dense and durable alternative to traditional media. Existing approaches leverage robust error correcting codes and precise molecular mechanisms to reliably retrieve specific files from large databases. Typically, files are retrieved using a pre-specified key, analogous to a filename. However, these approaches lack the ability to perform more complex computations over the stored data, such as similarity search: e.g., finding images that look similar to an image of interest without prior knowledge of their file names. Here we demonstrate a technique for executing similarity search over a DNA-based database of 1.6 million images. Queries are implemented as hybridization probes, and a key step in our approach was to learn an image-to-sequence encoding ensuring that queries preferentially bind to targets representing visually similar images. Experimental results show that our molecular implementation performs comparably to state-of-the-art in silico algorithms for similarity search.


Assuntos
Biologia Computacional/métodos , DNA/química , Bases de Dados Genéticas , Armazenamento e Recuperação da Informação , Algoritmos , Sequência de Bases , Simulação por Computador , DNA/genética , Sondas de DNA , Bases de Dados Factuais , Redes Neurais de Computação
15.
Nat Commun ; 11(1): 5454, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144581

RESUMO

Molecular tagging is an approach to labeling physical objects using DNA or other molecules that can be used when methods such as RFID tags and QR codes are unsuitable. No molecular tagging method exists that is inexpensive, fast and reliable to decode, and usable in minimal resource environments to create or read tags. To address this, we present Porcupine, an end-user molecular tagging system featuring DNA-based tags readable within seconds using a portable nanopore device. Porcupine's digital bits are represented by the presence or absence of distinct DNA strands, called molecular bits (molbits). We classify molbits directly from raw nanopore signal, avoiding basecalling. To extend 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 an approach to developing highly separable barcodes.


Assuntos
DNA/genética , Nanoporos , Biologia Sintética/métodos , Algoritmos , Biologia Computacional , Sistemas Computacionais , Processamento Eletrônico de Dados , Aprendizado de Máquina , Análise de Sequência de DNA
16.
Nat Commun ; 11(1): 3264, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601272

RESUMO

DNA has recently emerged as an attractive medium for archival data storage. Recent work has demonstrated proof-of-principle prototype systems; however, very uneven (biased) sequencing coverage has been reported, which indicates inefficiencies in the storage process. Deviations from the average coverage in the sequence copy distribution can either cause wasteful provisioning in sequencing or excessive number of missing sequences. Here, we use millions of unique sequences from a DNA-based digital data archival system to study the oligonucleotide copy unevenness problem and show that the two paramount sources of bias are the synthesis and amplification (PCR) processes. Based on these findings, we develop a statistical model for each molecular process as well as the overall process. We further use our model to explore the trade-offs between synthesis bias, storage physical density, logical redundancy, and sequencing redundancy, providing insights for engineering efficient, robust DNA data storage systems.


Assuntos
Armazenamento e Recuperação da Informação , Análise de Sequência de DNA , Viés , Modelos Teóricos , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/estatística & dados numéricos
17.
J Am Chem Soc ; 142(21): 9587-9593, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32364723

RESUMO

Chemical reaction networks (CRNs) provide a powerful abstraction to formally represent complex biochemical processes. DNA provides a promising substrate to implement the abstract representation (or programming language) of CRNs due to its programmable nature. Prior works that used DNA to implement CRNs either used DNA-only systems or multienzyme DNA circuits. Architectures with DNA-only components had the rationale of being biologically simple systems. Multienzyme systems, on the other hand, aimed at using natural enzymes to improve circuit performance, although, at the cost of increased complexity. In this work, we explore an alternative architecture that lies along the spectrum in between DNA-only systems and multienzyme DNA systems. Our architecture relies on only a strand displacing polymerase enzyme and DNA hybridization reactions for implementing CRNs. First, we briefly introduce the theory and DNA design of simple CRNs and then explore the fundamental properties of polymerase-based strand displacement systems. Finally, we engineer a catalytic amplifier in vitro as a use-case of our framework since such amplifiers require the intricate design of DNA sequences and reaction conditions.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Redes e Vias Metabólicas , DNA/química , DNA Polimerase Dirigida por DNA/química , Hibridização de Ácido Nucleico
18.
Nat Commun ; 11(1): 1080, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081865

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

19.
Chem Commun (Camb) ; 56(25): 3613-3616, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-32107514

RESUMO

Rapid aging tests (70 °C, 50% RH) of solid state DNA dried in the presence of various salt formulations, showed the strong stabilizing effect of calcium phosphate, calcium chloride and magnesium chloride, even at high DNA loadings (>20 wt%). A DNA-based digital information storage system utilizing the stabilizing effect of MgCl2 was tested by storing a DNA file, encoding 115 kB of digital data, and the successful readout of the file by sequencing after accelerated aging.


Assuntos
Cloreto de Cálcio/química , Fosfatos de Cálcio/química , DNA/química , Armazenamento e Recuperação da Informação , Cloreto de Magnésio/química , DNA/síntese química , Tamanho da Partícula , Sais/química , Propriedades de Superfície
20.
Nat Commun ; 11(1): 616, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001691

RESUMO

Synthetic DNA is gaining momentum as a potential storage medium for archival data storage. In this process, digital information is translated into sequences of nucleotides and the resulting synthetic DNA strands are then stored for later retrieval. Here, we demonstrate reliable file recovery with PCR-based random access when as few as ten copies per sequence are stored, on average. This results in density of about 17 exabytes/gram, nearly two orders of magnitude greater than prior work has shown. We successfully retrieve the same data in a complex pool of over 1010 unique sequences per microliter with no evidence that we have begun to approach complexity limits. Finally, we also investigate the effects of file size and sequencing coverage on successful file retrieval and look for systematic DNA strand drop out. These findings substantiate the robustness and high data density of the process examined here.


Assuntos
DNA/química , Armazenamento e Recuperação da Informação/métodos , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Dosagem de Genes , Engenharia Genética/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Ciência da Informação
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