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Title: 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.

Authors:
 [1];  [2];  [1];  [2];  [3]; ORCiD logo [2]
  1. Department of Chemical and Biomolecular Engineering – Johns Hopkins University
  2. Department of Mechanical Engineering – University of California - Riverside
  3. Department of Chemical and Biomolecular Engineering – Johns Hopkins University, Department of Computer Science – Johns Hopkins University
Publication Date:
Research Org.:
Johns Hopkins Univ., Baltimore, MD (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1435363
Alternate Identifier(s):
OSTI ID: 1502443
Grant/Contract Number:  
SC0010426
Resource Type:
Published Article
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Name: Nucleic Acids Research Journal Volume: 46 Journal Issue: 10; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Schaffter, Samuel W., Green, Leopold N., Schneider, Joanna, Subramanian, Hari K. K., Schulman, Rebecca, and Franco, Elisa. T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures. United Kingdom: N. p., 2018. Web. doi:10.1093/nar/gky283.
Schaffter, Samuel W., Green, Leopold N., Schneider, Joanna, Subramanian, Hari K. K., Schulman, Rebecca, & Franco, Elisa. T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures. United Kingdom. doi:10.1093/nar/gky283.
Schaffter, Samuel W., Green, Leopold N., Schneider, Joanna, Subramanian, Hari K. K., Schulman, Rebecca, and Franco, Elisa. Mon . "T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures". United Kingdom. doi:10.1093/nar/gky283.
@article{osti_1435363,
title = {T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures},
author = {Schaffter, Samuel W. and Green, Leopold N. and Schneider, Joanna and Subramanian, Hari K. K. and Schulman, Rebecca and Franco, Elisa},
abstractNote = {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.},
doi = {10.1093/nar/gky283},
journal = {Nucleic Acids Research},
number = 10,
volume = 46,
place = {United Kingdom},
year = {2018},
month = {4}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1093/nar/gky283

Citation Metrics:
Cited by: 3 works
Citation information provided by
Web of Science

Figures / Tables:

Figure 1 Figure 1: DNA nanotube design and disassembly in the presence of 8.6 U/μl T7 RNAP in transcription conditions. (A) DNA nanotube structure and assembly. Left: DNA tiles are DAE-E double crossover molecules (71) that assemble from 5 strands of synthetic DNA. Tiles consist of two DNA helices that are rigidlymore » bound by two crossover motifs with four single-stranded sticky end (se) regions on the two helix ends. Sticky ends are complementary across the diagonal of the tile (complementary sequences denoted by *) (1). Tiles also contain a single-stranded overhang domain on strand 2 (orange) and a Cy3 fluorophore (yellow) at the 5′ end of strand 3. Numbers next to domains indicate domain lengths in number of bases. Middle: The complementary sticky ends specifically program the DNA tiles to form the lattice shown as an abstract line representation (top) and a 3D rendering (bottom). Right: The lattice cyclizes to form nanotubes (1). (B) Fluorescence micrographs of DNA nanotubes (var1_7––SI Section 1) before and after incubation with (T7 (+)) or without (T7 (–)) T7 RNAP in transcription conditions. (C) Nanotube stability (var1_7) in the presence of T7 RNAP with ATP (ATP (+)) as the only NTP or without any NTPs (NTPs (–)). Scale bars: 10 μm.« less

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