Branched DNA Architectures Produced by PCR‐Based Assembly as Gene Compartments for Cell‐Free Gene‐Expression Reactions

Guo, Xiaocui; Bai, Lihui; Feng, Li; Huck, Wilhelm T. S.; Yang, Dayong

doi:10.1002/cbic.201900094

Cited by 25 publications

(20 citation statements)

References 49 publications

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“…The eGFP yields of the four gene circuits that separately anchored on AuNP were all comparable with that of the free gene circuit system (Figure S7). For the underlying mechanism, it was speculated that the pure gene compartment effect enhanced the gene expression as we reported previously; whereas, the synthesized T7 RNAP was mainly localized in pure T7 RNAP gene compartment, which was unfavorable to the diffusion of T7 RNAP to pure eGFP gene compartment, thus downregulating the gene expression. The synergy effect of the two mentioned factors led to comparable gene expression in the separately anchoring system and free gene circuit system.…”

Section: Resultsmentioning

confidence: 77%

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Gene Circuit Compartment on Nanointerface Facilitatating Cascade Gene Expression

Guo

Bai

et al. 2019

J. Am. Chem. Soc.

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Cellular genes that are functionally related to each other are usually confined in specialized subcellular compartments for efficient biochemical reactions. Construction of spatially controlled biosynthetic systems will facilitate the study of biological design principles. Herein, we fabricated a gene circuit compartment by coanchoring two functionrelated genes on surface of gold nanoparticles and investigated the compartment effect on cascade gene expression in a cell-free system. The gene circuit consisted of a T7 RNA polymerase (T7 RNAP) expression cassette as regulatory gene and a fluorescent protein expression cassette as regulated reporter gene. Both the expression cassettes were attached on a Y-shaped DNA nanostructure whose other two branches were mercapto-modified in order to steadily anchor the gene expression cassettes on the surface of gold nanoparticles. Experimental results demonstrated that both the yield and initial expression rate of the fluorescent reporter protein in the gene circuit compartment system were enhanced compared with those in free gene circuit system. Mechanism investigation revealed that the gene circuit compartment on nanoparticle made the regulatory gene and regulated reporter gene spatially proximal at nanoscale, thus effectively improving the transfer efficiency of the regulatory proteins (T7 RNAP) from regulatory genes to the regulated reporter genes in the compartments, and consequently, the biochemical reaction efficiency was significantly increased. This work not only provided a simplified model for rational molecular programming of genes circuit compartments on nanointerface but also presented implications for the cellular structure−function relationship.

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Section: Resultsmentioning

confidence: 77%

“…The protein expressions were conducted in 96-well plates at 30 °C and monitored in real time for 12 h by ELISA (BioTek synergy/H1 microplate reader). The concentration of protein was determined as in our previous method …”

Section: Methodsmentioning

confidence: 99%

Gene Circuit Compartment on Nanointerface Facilitatating Cascade Gene Expression

Guo

Bai

et al. 2019

J. Am. Chem. Soc.

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“…Taking the molecular design and synthesis method of four-branched DNA (also termed as X-shaped DNA, Figure 2a) as an example, each constituent ssDNA is separated into two segments, where the domain marked in black is designed to be pairwise complementary sequences to each other, and the expansion domain Reproduced with permission. [17] Copyright 2019, Elsevier Publishing Group. marked in red contributes to functional modification or structural extension.…”

Section: Construction Of Branched Dna Nanostructure 21 Branched Dna M...mentioning

confidence: 99%

Construction of Branched DNA‐based Nanostructures for Diagnosis, Therapeutics and Protein Engineering

Dong

Yao

et al. 2022

Chemistry An Asian Journal

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Branched DNA with multibranch-like anisotropic topology serves as a promising and powerful building block in constructing multifunctional-integrated nanomaterials in a programmable and controllable manner. Recently, a series of branched DNA-based functional nanomaterials were developed by elaborate molecular design. In this review, we focused on the construction of branched DNA-based nanostructures for biological and biomedical applications. First, the molecular design and synthesis method of branched DNA monomer were briefly described. Then, the construction strategies of branched DNA-based nanostructures were categorially discussed, including target-triggered polymerization, enzymatic extension and hybrid assembly. Finally, the biological and biomedical applications including diagnosis, therapeutics and protein engineering were summarized. We envision that the review will contribute to the further development of branched DNA-based nanomaterials with great application potential in the field of biomedicine, thus building a new bridge between material chemistry and biomedicine.

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“…Reproduced with permission. [ 82 ] Copyright 2019, Wiley‐VCH. C) Schematic illustration of gene circuit compartment on the nanointerface of gold nanoparticles, and the expression kinetics (lower left) and eGFP yield (lower right) with altered stoichiometric ratios.…”

Section: Compartmentmentioning

confidence: 99%

“…Our group recently made use of Y‐shaped DNA nanostructure as the scaffold to probe the gene proximity effects. [ 82 ] The “graft‐from” design enabled the linkage of gene‐length DNA with up to 1000 bp (Figure 4B). Since the regulatory gene cassette and reporter gene cassette were spatially correlated, expressed RNA polymerase could shuttle between neighboring transcription units, which resulted in enhanced gene expression efficiency.…”

Section: Compartmentmentioning

confidence: 99%

Emerging Advances of Cell‐Free Systems toward Artificial Cells

et al. 2020

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models have attracted considerable attentions in order to unravel the inherent complexity. Although engineering modern cells has achieved some progress, [2] the arbitrary elimination of genomic information could probably remove some distinguished characteristics (top-down strategy). Building chemical systems that resemble the structure and behaviors of living cells by stepwise integration of pivotal components is also conceivable (bottom-up strategy). [3] These synthetic systems with biomimetic cellularity provide conceptual pathway from inanimate matter to artificial life, which are often known as artificial cells. [4] Due to the lack of full comprehension about the molecular mechanism of living cells, laborious reconstitution of complicated networks would be difficult and the disparities of protein behaviors in the artificial cells have been observed. Herein, we prefer to construct artificial cells by preserving certain endogenous machinery already existing in living cells. Such principle is conducible for maintaining essential features of living cells while leaving design space for synthetic biologists to study specific cellular behavior in bottom-up manner. [5] Cell-free system, also referred to as in vitro transcription and translation system is a universal toolbox to study the organisms of cell biology. [6] Cell-free extracts inherit most of the endogenous machinery including functional macromolecules as well as metabolic networks and the addition of desired resources could initiate coupled protein expressions. Indeed, the first appearance of cell-free systems could date back to 1961 when Nirenberg and Matthaei deciphered the genetic codes. [7] Featured by the accessible utilization of molecular machinery, cellfree systems could circumvent the frangibility and membrane barrier of biological cells. The transformative development of protein synthesis using recombinant elements (PURE) system with well-defined components greatly enhances the systemic manipulation and controllability. [8] In principle, the quintessence behind cell-free system is the central dogma that reflects the flow of genetic information, from plasmid DNA to messenger RNA (mRNA), to eventually make functional proteins. As such, artificial cells encapsulated with cell-free system could serve as the valuable candidate to construct numerous biological processes. [9] The powerful ability of protein expressions, which is deprived in other synthetic systems, offers advantageous scenario for enzymatic processes that are also guided by flexible and programmable regulations of gene circuit. [10] The execution of larger genetic programs could further permit the construction of complex biochemical systems. [11] More importantly, Artificial cells that mimic the architectural and functional characteristics of living cells not only shed light on the physical principle of life but also facilitate development in the areas such as cell engineering and biomedicine. Cell-free systems carry out the central dogma that refers to the transcription and translation ...

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Branched DNA Architectures Produced by PCR‐Based Assembly as Gene Compartments for Cell‐Free Gene‐Expression Reactions

Cited by 25 publications

References 49 publications

Gene Circuit Compartment on Nanointerface Facilitatating Cascade Gene Expression

Gene Circuit Compartment on Nanointerface Facilitatating Cascade Gene Expression

Construction of Branched DNA‐based Nanostructures for Diagnosis, Therapeutics and Protein Engineering

Emerging Advances of Cell‐Free Systems toward Artificial Cells

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