Building in vitro transcriptional regulatory networks by successively integrating multiple functional circuit modules

Schaffter, Samuel W.; Schulman, Rebecca

doi:10.1038/s41557-019-0292-z

Cited by 94 publications

(107 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The interface of biosensing with TMSD creates a potentially powerful molecular computation paradigm for engineering molecular devices that can perform programmed tasks in response to specific chemical triggers. This is especially true since TMSD circuits are much easier to program than protein-based circuits as a result of their simpler design rules [47], computational models that accurately predict their behavior [34, 35] and the emerging suite of design tools to build TMSD circuits [26, 48]. We, therefore, sought to leverage these features of TMSD circuits to create an information processing layer for cell-free biosensors that could be used to expand their function.…”

Section: Resultsmentioning

confidence: 99%

“…While simple in concept, we found that the combination of TMSD with cell-free biosensing reactions did not work immediately. This was due to the incompatibility of 3' toehold overhangs in DNA gates with T7 RNAP-driven IVT reactions [48]. A careful analysis of the issues determined that this incompatibility is due to undesired transcription of these 3' toehold overhangs by T7 RNAP, which can be solved by changing toehold overhangs to be on the 5' ends ( Fig.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Programming Cell-Free Biosensors with DNA Strand Displacement Circuits

Jung¹,

Kk²,

Lucks³

2021

Preprint

View full text Add to dashboard Cite

Cell-free biosensors are emerging as powerful platforms for monitoring human and environmental health. Here, we expand the capabilities of biosensors by interfacing their outputs with toehold- mediated strand displacement circuits, a dynamic DNA nanotechnology that enables molecular computation through programmable interactions between nucleic acid strands. We develop design rules for interfacing biosensors with strand displacement circuits, show that these circuits allow fine-tuning of reaction kinetics and faster response times, and demonstrate a circuit that acts like an analog-to-digital converter to create a series of binary outputs that encode the concentration range of the target molecule being detected. We believe this work establishes a pathway to create 'smart' diagnostics that use molecular computations to enhance the speed, robustness and utility of biosensors.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Programming Cell-Free Biosensors with DNA Strand Displacement Circuits

Jung¹,

Kk²,

Lucks³

2021

Preprint

View full text Add to dashboard Cite

show abstract

“…To demonstrate how transcriptional valves might be built, we attempted to construct proof-of-concept designs for T7 RNAP. T7 RNAP was selected due to its broad use in synthetic biology, which stems from the fact that it is a single-subunit RNAP with high processivity, making it ideal for both in vitro use 20 as well as an orthogonal transcription system in vivo 21,22 . Whilst diverse terminators are available for native Escherichia coli RNAP 6,11 , for T7 RNAP only a single terminator exists in the T7 phage genome 23 and only a few alternatives have been characterized 17,24,25 .…”

Section: Resultsmentioning

confidence: 99%

Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing

Tarnowski

Gorochowski

2021

Preprint

View full text Add to dashboard Cite

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as ‘transcriptional valves’ which can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly we construct 1183 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to simultaneously characterize the entire pool at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate their function using nearby sequence context. This work provides new avenues for controlling transcription and demonstrates the value of long-read sequencing for exploring complex sequence-function landscapes.

show abstract

Section: Design Strategies Of Sdr For Biocomputingmentioning

confidence: 99%

Biocomputing Based on DNA Strand Displacement Reactions

Shi

et al. 2021

ChemPhysChem

View full text Add to dashboard Cite

The high sequence specificity and precise base complementary pairing principle of DNA provides a rich orthogonal molecular library for molecular programming, making it one of the most promising materials for developing bio‐compatible intelligence. In recent years, DNA has been extensively studied and applied in the field of biological computing. Among them, the toehold‐mediated strand displacement reaction (SDR) with properties including enzyme free, flexible design and precise control, have been extensively used to construct biological computing circuits. This review provides a systemic overview of SDR design principles and the applications. Strategies for designing DNA‐only, enzymes‐assisted, other molecules‐involved and external stimuli‐controlled SDRs are described. The recently realized computing functions and the application of DNA computing in other fields are introduced. Finally, the advantages and challenges of SDR‐based computing are discussed.

show abstract

Building in vitro transcriptional regulatory networks by successively integrating multiple functional circuit modules

Cited by 94 publications

References 68 publications

Programming Cell-Free Biosensors with DNA Strand Displacement Circuits

Programming Cell-Free Biosensors with DNA Strand Displacement Circuits

Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing

Biocomputing Based on DNA Strand Displacement Reactions

Contact Info

Product

Resources

About