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Abstract
The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Here, we show the ability to express ssDNAs in Escherichia coli (32–205 nt), which can form structures in vivo or be purified for in vitro assembly. Each ssDNA is encoded by a gene that is transcribed into non-coding RNA containing a 3′-hairpin (HTBS). HTBS recruits HIV reverse transcriptase, which nucleates DNA synthesis and is aided in elongation by murine leukemia reverse transcriptase. Purified ssDNA that is produced in vivo is used to assemble large 1D wires (300 nm) and 2D sheets (5.8 μm2) in vitro. Intracellular assembly is demonstrated using a four-ssDNA crossover nanostructure that recruits split YFP when properly assembled. Genetically encoding DNA nanostructures provides a route for their production as well as applications in living cells.
Highlights
The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs to assemble into desired 3D structures
We present a method that enables the short single-stranded DNA (ssDNA) to be encoded as a gene (r_oligo) that is expressed as a non-coding RNA that is enzymatically converted to ssDNA
The possibility to functionally reverse transcribe RNA to DNA in bacteria using these eukaryotic retroviral reverse transcriptase (RT) has not yet been shown. This may be due to the lack of eukaryotic t-RNALYS, which is required for binding to the RT at the protein-binding site (PBS) and recruiting it to viral RNA to initiate polymerization (Fig. 1b)[45,46]
Summary
The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Different nucleotide sequences yield complementary strands that direct short single-stranded DNA (ssDNA) to hybridize with high specificity into a set of branched junctions, including the crossover[2] and paranemic crossover[3] motifs. These are the architectural elements that enable the self-assembly of larger 2D and three-dimensional (3D) nanostructures[4,5,6]. We demonstrate the ability to express and assemble DNA nanostructure within living cells This is shown by building a four-ssDNA ‘crossover motif’ that can act as a scaffold for proteins. This work offers both a route by which these structures could be made in bulk via biotechnology and to be induced in cells for in vivo applications
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