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Networks of interacting DNA oligomers are useful for applications such as biomarker detection, targeted drug delivery, information storage, and photonic information processing. However, differences in the chemical kinetics of hybridization reactions, referred to as kinetic dispersion, can be problematic for some applications. Here, it is found that limiting unnecessary stretches of Watson-Crick base pairing, referred to as unnecessary duplexes, can yield exceptionally low kinetic dispersions. Hybridization kinetics can be affected by unnecessary intra-oligomer duplexes containing only 2 base-pairs, and such duplexes explain up to 94% of previously reported kinetic dispersion. As a general design rule, it is recommended that unnecessary intra-oligomer duplexes larger than 2 base-pairs and unnecessary inter-oligomer duplexes larger than 7 base-pairs be avoided. Unnecessary duplexes typically scale exponentially with network size, and nearly all networks contain unnecessary duplexes substantial enough to affect hybridization kinetics. A new method for generating networks which utilizes in-silico optimization to mitigate unnecessary duplexes is proposed and demonstrated to reduce in-vitro kinetic dispersions as much as 96%. The limitations of the new design rule and generation method are evaluated in-silico by creating new oligomers for several designs, including three previously programmed reactions and one previously engineered structure.

 

Deoxyribonucleic acid (DNA) is emerging as an alternative archival memory technology. Recent advancements in DNA synthesis and sequencing have both increased the capacity and decreased the cost of storing information in de novo synthesized DNA pools. In this survey, we review methods for translating digital data to and/or from DNA molecules. An emphasis is placed on methods which have been validated by storing and retrieving real-world data via in-vitro experiments.

 

DNA is a compelling alternative to non-volatile information storage technologies due to its information density, stability, and energy efficiency. Previous studies have used artificially synthesized DNA to store data and automated next-generation sequencing to read it back. Here, we report digital Nucleic Acid Memory (dNAM) for applications that require a limited amount of data to have high information density, redundancy, and copy number. In dNAM, data is encoded by selecting combinations of single-stranded DNA with (1) or without (0) docking-site domains. When self-assembled with scaffold DNA, staple strands form DNA origami breadboards. Information encoded into the breadboards is read by monitoring the binding of fluorescent imager probes using DNA-PAINT super-resolution microscopy. To enhance data retention, a multi-layer error correction scheme that combines fountain and bi-level parity codes is used. As a prototype, fifteen origami encoded with ‘Data is in our DNA!\n’ are analyzed. Each origami encodes unique data-droplet, index, orientation, and error-correction information. The error-correction algorithms fully recover the message when individual docking sites, or entire origami, are missing. Unlike other approaches to DNA-based data storage, reading dNAM does not require sequencing. As such, it offers an additional path to explore the advantages and disadvantages of DNA as an emerging memory material.

 

To bring real-world applications of DNA nanostructures to fruition, advanced microscopy techniques are needed to shed light on factors limiting the availability of addressable sites. Correlative microscopy, where two or more microscopies are combined to characterize the same sample, is an approach to overcome the limitations of individual techniques, yet it has seen limited use for DNA nanotechnology. We have developed an accessible strategy for high resolution, correlative DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution and atomic force microscopy (AFM) of DNA nanostructures, enabled by a simple and robust method to selectively bind DNA origami to cover glass. Using this technique, we examined addressable “docking” sites on DNA origami to distinguish between two defect scenarios–structurally incorporated but inactive docking sites, and unincorporated docking sites. We found that over 75% of defective docking sites were incorporated but inactive, suggesting unincorporated strands played a minor role in limiting the availability of addressable sites. We further explored the effects of strand purification, UV irradiation, and photooxidation on availability, providing insight on potential sources of defects and pathways toward improving the fidelity of DNA nanostructures.