Strategies to Reduce Promoter-Independent Transcription of DNA Nanostructures and Strand Displacement Complexes

Bacteriophage RNA polymerases, in particular T7 RNA polymerase
(RNAP), are well-characterized and popular enzymes for many RNA applications
in biotechnology both in vitro and in cellular settings.
These monomeric polymerases are relatively inexpensive and have high
transcription rates and processivity to quickly produce large quantities
of RNA. T7 RNAP also has high promoter-specificity on double-stranded
DNA (dsDNA) such that it only initiates transcription downstream of
its 17-base promoter site on dsDNA templates. However, there are many
promoter-independent T7 RNAP transcription reactions involving transcription
initiation in regions of single-stranded DNA (ssDNA) that have been
reported and characterized. These promoter-independent transcription
reactions are important to consider when using T7 RNAP transcriptional
systems for DNA nanotechnology and DNA computing applications, in
which ssDNA domains often stabilize, organize, and functionalize DNA
nanostructures and facilitate strand displacement reactions. Here
we review the existing literature on promoter-independent transcription
by bacteriophage RNA polymerases with a specific focus on T7 RNAP,
and provide examples of how promoter-independent reactions can disrupt
the functionality of DNA strand displacement circuit components and
alter the stability and functionality of DNA-based materials. We then
highlight design strategies for DNA nanotechnology applications that
can mitigate the effects of promoter-independent T7 RNAP transcription.
The design strategies we present should have an immediate impact by
increasing the rate of success of using T7 RNAP for applications in
DNA nanotechnology and DNA computing.