T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

Abstract

The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

Dynamic self-assembly of compartmentalized DNA nanotubes

“…These experiments indicate that more complex genetic programs relying on RNA production and degradation may be encapsulated to control nanotube assembly 35 . A notable challenge toward sustained transcription and degradation dynamics is the accumulation of abortive and elongated transcripts, which can cause side reactions and crosstalk 70 . These undesired transcripts may bind to inactive tiles producing “waste” complexes that can no longer be activated by the correct RNA trigger 36 .…”

Growth and site-specific organization of micron-scale biomolecular devices on living mammalian cells

“…These DNA nanotubes can grow from DNA origami templates, seeds (Figure 1c) 22 , and can reach 100 µm in length [24][25][26] . DNA nanotube growth kinetics 25,[27][28][29][30] , hierarchical assembly pathways 31 and diffusion rates 22 have also been extensively characterized, allowing kinetic control over their growth and interactions with cells. Nanotubes can be functionalized with polymers, gold nanoparticles 32 , proteins 33 and peptides 34 , and thus could be templates for constructing diverse functional devices.…”

Programming Cell-Free Biosensors with DNA Strand Displacement Circuits

“…2d). Previous literature reported the ability of T7 RNAP to initiate promoter-independent transcription from exposed, linear ssDNA regions [30][31][32][33]. Based on these observations, we hypothesized that T7 RNAP was initiating transcription from the 3' end toehold region of the DNA signal gate, causing strand displacement and signal generation ( Fig.…”