Construction and characterization of metal ion-containing DNA nanowires for synthetic biology and nanotechnology

Austin Ray,Jesica Urbina,Le Nguyen,Alissa Greenberg,Mark C Capece,Gary Wessel,Joseph Shih,Ivan G Paulino-Lima,Vitor Pinheiro,Shalom J Wind,Lynn Rothschild,Kosuke Fujishima,Emily Toomey,Simon Vecchioni

doi:10.1038/s41598-019-43316-1

Abstract

DNA is an attractive candidate for integration into nanoelectronics as a biological nanowire due to its linear geometry, definable base sequence, easy, inexpensive and non-toxic replication and self-assembling properties. Recently we discovered that by intercalating Ag+ in polycytosine-mismatch oligonucleotides, the resulting C-Ag+-C duplexes are able to conduct charge efficiently. To map the functionality and biostability of this system, we built and characterized internally-functionalized DNA nanowires through non-canonical, Ag+-mediated base pairing in duplexes containing cytosine-cytosine mismatches. We assessed the thermal and chemical stability of ion-coordinated duplexes in aqueous solutions and conclude that the C-Ag+-C bond forms DNA duplexes with replicable geometry, predictable thermodynamics, and tunable length. We demonstrated continuous ion chain formation in oligonucleotides of 11–50 nucleotides (nt), and enzyme ligation of mixed strands up to six times that length. This construction is feasible without detectable silver nanocluster contaminants. Functional gene parts for the synthesis of DNA- and RNA-based, C-Ag+-C duplexes in a cell-free system have been constructed in an Escherichia coli expression plasmid and added to the open-source BioBrick Registry, paving the way to realizing the promise of inexpensive industrial production. With appropriate design constraints, this conductive variant of DNA demonstrates promise for use in synthetic biological constructs as a dynamic nucleic acid component and contributes molecular electronic functionality to DNA that is not already found in nature. We propose a viable route to fabricating stable DNA nanowires in cell-free and synthetic biological systems for the production of self-assembling nanoelectronic architectures.

Highlights

Nanoelectronics has become a transformative technology for both industrial and personal computing applications, yet the limitations of traditional manufacturing methods make current prototypes expensive and difficult to mass produce[1]
Thermal annealing of cytosine-mismatched DNA duplexes from cytosine-enriched DNA oligomers subjected to varying salt conditions was readily visualized by mass distribution using polyacrylamide gel electrophoresis (PAGE) (Fig. 2)
Using two complementary 32 bp ssDNA sequences with ten cytosine mismatch points (Oligo A) as a model, we found that strand annealing is directly proportional to the concentration of Ag+ ions in the reaction (Fig. 2A)

Summary

Introduction

Nanoelectronics has become a transformative technology for both industrial and personal computing applications, yet the limitations of traditional manufacturing methods make current prototypes expensive and difficult to mass produce[1]. As in any biosynthetic system, there is the potential for rapid, cheap, and environmentally-safe mass production from cellular components that can be tuned for different purposes This system does not require the heavy infrastructure utilized for inorganic production, and it has the potential for distributed manufacturing in low-resource environments, including in space, the moon or Mars. Metal-nucleobase interactions have been studied since the 1960s when an affinity was discovered between the mercury cation (Hg2+) and thymine-enriched DNA polynucleotides[52], but several decades passed before the notion of introducing a metal ion directly into a DNA duplex was first described in detail by Tanaka and coworkers in 200253 This was followed by work that demonstrated the replacement of Watson-Crick G-C base pairs with mismatched cytosine pairs capable of forming a coordinating bond with Ag+ 54,55.

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Results

Conclusion