A Novel Method for the Production of Single-Stranded DNA for Demand-Meeting Applications in DNA Nanotechnology, Biological Imaging, and Genome Editing
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CitationGuerra, Richard. 2020. A Novel Method for the Production of Single-Stranded DNA for Demand-Meeting Applications in DNA Nanotechnology, Biological Imaging, and Genome Editing. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.
AbstractSingle-stranded DNA (ssDNA) has numerous applications in the fields of DNA nanotechnology, biological imaging, and synthetic biology. While the costs of commercially available, chemically synthesized ssDNA have drastically decreased over the last decade, this synthetic process has reached its limit in terms of the length of the strands it can produce at reasonable purities. Given the increasing demand for longer, high-purity ssDNA, many enzymatic-based methods have been developed to address this unmet need. Despite the high interest for long ssDNA, almost every “in-house” method is limited either in the production scale, length scale, or purity. While commercially available, enzymatically produced, long ssDNA has reached lengths of 5000 nucleotides these vendors provide exceedingly low product yields at enormous prices, making this material prohibitively expensive to purchase at scales needed for individual experiments. In addition to linear ssDNA, many applications benefit from circular ssDNAs, however their production is limited in scale and purity and relies completely on in-house methods, as commercial options are limited. Current methods that convert ssDNA into its circular counterpart require fine-tuning of reaction conditions and often generate numerous unwanted side products, resulting in low yields upon additional purification. Proposed herein is an easy to use method for the scalable production of ssDNAs of arbitrary length and high purity. The resulting strands can then be cyclized and the capabilities of the linear and circular ssDNAs are demonstrated through several applications. The power of linear ssDNA is leveraged in DNA nanotechnology via DNA origami and crisscross cooperativity, in biological imaging through fluorescence in situ hybridization, and in CRISPR/Cas9 via homology-directed genome editing. Meanwhile, the advantage of circular ssDNA is then demonstrated through nanobioengineering of a viral-like RNA factory to aid in the production of RNAs. These methods enable the scientific exploration of novel applications of ssDNAs (linear and circular) for both therapeutic and diagnostic purposes.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37368933
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