DNA Nanoparticles for Improved Protein Synthesis In Vitro

Abstract

The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System

“…Consistent with this, the observed nanoparticles could be stained by DNA‐binding dye (Figure S3). Notably, previous work had shown that the nanoparticles are still accessible to transcription and translation machineries …”

“…In a bacterial cell, DNA is condensed both by supercoiling and by several DNA‐binding proteins. Although in vitro reconstitution of these activities might in principle be possible, the inorganic DNA precipitates produced as by‐products of in vitro DNA synthesis provide a promising alternative. As these DNA nanoparticles are accessible for protein binding and functional as templates for transcription, they might indeed mimic the condensed state of cellular DNA and serve as templates for successive replication in minimal systems.…”

“…Although in vitro reconstitution of these activities might in principle be possible, the inorganic DNA precipitates produced as by‐products of in vitro DNA synthesis provide a promising alternative. As these DNA nanoparticles are accessible for protein binding and functional as templates for transcription, they might indeed mimic the condensed state of cellular DNA and serve as templates for successive replication in minimal systems. Moreover, DNA nanoparticles might be superior to individual plasmids as segregation substrates, as the presence of multiple parC sites and slow diffusion facilitate nucleation of multifilamented bipolar spindles.…”

Microfluidics‐Based Biomaterials and Biodevices

“…The herringbone structures of microfluidic channel can induce chaotic advection and increase mass transfer, providing fine‐tuned features for lipid nanoparticles. More recently, the integration of microfluidics and DNA nanotechnology has made great progress for constructing a living cell in vitro . Overall, as a precision technology, has remarkably expanded the scope for complexity in synthetic biology.…”

Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

“…Compartmentalization of reagents into microscopic droplets can shift reaction equilibrium towards product formation and as a result improve the efficacy of chemical reactions [ 21 ]. As was shown recently, in the case of the DNA isothermal amplification reaction, the compartmentalization enhances amplified nucleic acid condensation into crystalline-like structures [ 22 ]. It was shown that during the multiple displacement amplification (MDA) reaction, the magnesium ions from the buffer and DNA replication reaction byproduct inorganic pyrophosphate trigger amplified DNA condensation into DNA-magnesium-inorganic pyrophosphate (DNA-Mg-PP i ) particles.…”