Purification of Functionalized DNA Origami Nanostructures

Shaw, Alan; Benson, Erik; Högberg, Björn

doi:10.1021/nn507035g

Cited by 106 publications

(135 citation statements)

References 32 publications

(55 reference statements)

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“…Moreover, custom-tailored DNA scaffolds now allow the construction of DNA origami structures of different sizes and are not limited to the frequently used M13 single strand [80]. With the aid of suitable purification methods [81][82][83][84][85], these nanostructures can be prepared in pure forms that provide enhanced sensitivity. DNA, being a biomolecule, also provides an advantage of being biocompatible [86] and can be useful for biosensing in combination with biomimetic approaches.…”

Section: Discussion and Outlookmentioning

confidence: 99%

DNA Nanobiosensors: An Outlook on Signal Readout Strategies

Chandrasekaran¹

2017

Journal of Nanomaterials

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A suite of functionalities and structural versatility makes DNA an apt material for biosensing applications. DNA-based biosensors are cost-effective and sensitive and have the potential to be used as point-of-care diagnostic tools. Along with robustness and biocompatibility, these sensors also provide multiple readout strategies. Depending on the functionality of DNA-based biosensors, a variety of output strategies have been reported: fluorescence-and FRET-based readout, nanoparticle-based colorimetry, spectroscopy-based techniques, electrochemical signaling, gel electrophoresis, and atomic force microscopy.

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Section: Discussion and Outlookmentioning

confidence: 99%

DNA Nanobiosensors: An Outlook on Signal Readout Strategies

Chandrasekaran¹

2017

Journal of Nanomaterials

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“…Previously used methods based on electrophoresis, ultrafiltration and chromatography or Ni‐NTA‐affinity tag purification are often not applicable when it comes to purification of fragile DONs decorated with sensitive proteins . Högberg and co‐workers have recently evaluated a panel of purification methods for protein‐functionalized 18‐helix bundle DONs . However, these 3D structures are more stable than mechanically sensitive 2D structures and the binding of the proteins was only achieved by hybridization of previously synthesized oligonucleotide–protein conjugates.…”

Section: Figurementioning

confidence: 99%

Solid‐Phase Synthesis and Purification of Protein–DNA Origami Nanostructures

Burgahn

Garrecht

Rabe

et al. 2019

Chemistry A European J

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We present a facile method for the combined synthesis and purification of protein‐decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the “halo‐based oligonucleotide binder” (HOB), a variant of Halo‐tag, the “SpyTag/SpyCatcher” (ST/SC) system, and the enzymatic “ybbR tag” coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface‐based cell assays.

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“…Shaw et al provided a systematic study comparing seven purification methods for DNA origami nanostructures functionalized with either small molecules (Alexa 488 fluorophore), antibodies (human IgG) or large proteins (Ferritin), with the aim to provide a guideline for effectively fabricating and scalable production of modified DNA origami constructs (Fig. 4) [63]. Other than several traditional purification methods including ultrafiltration, gel filtration, glycerol gradient ultracentrifugation, PEG precipitation, gel extraction, the authors reported two new approaches for purification of DNA origami with function groups: magnetic bead capture and fast protein liquid chromatography (FPLC) using a Superose 6 column.…”

Section: Evolution Of Dna Nanotechnology For Biomedical Applicationsmentioning

confidence: 99%

“…Samples were purified using one of seven methods with violet indicating the starting solution with excess free functional groups and blue indicating purified DNA origami with functional groups. Adapted with permission from [63]. …”

Section: Figmentioning

confidence: 99%

DNA nanomaterials for preclinical imaging and drug delivery

Jiang

England

Cai

2016

Journal of Controlled Release

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Besides being the carrier of genetic information, DNA is also an excellent biological organizer to establish well-designed nanostructures in the fields of material engineering, nanotechnology, and biomedicine. DNA-based materials represent a diverse nanoscale system primarily due to their predictable base pairing and highly regulated conformations, which greatly facilitate the construction of DNA nanostructures with distinct shapes and sizes. Integrating the emerging advancements in bioconjugation techniques, DNA nanostructures can be readily functionalized with high precision for many purposes ranging from biosensors to imaging to drug delivery. Recent progress in the field of DNA nanotechnology has exhibited collective efforts to employ DNA nanostructures as smart imaging agents or delivery platforms within living organisms. Despite significant improvements in the development of DNA nanostructures, there is limited knowledge regarding the in vivo biological fate of these intriguing nanomaterials. In this review, we summarize the current strategies for designing and purifying highly-versatile DNA nanostructures for biological applications, including molecular imaging and drug delivery. Since DNA nanostructures may elicit an immune response in vivo, we also present a short discussion of their potential toxicities in biomedical applications. Lastly, we discuss future perspectives and potential challenges that may limit the effective preclinical and clinical employment of DNA nanostructures. Due to their unique properties, we predict that DNA nanomaterials will make excellent agents for effective diagnostic imaging and drug delivery, improving patient outcome in cancer and other related diseases in the near future.

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Purification of Functionalized DNA Origami Nanostructures

Cited by 106 publications

References 32 publications

DNA Nanobiosensors: An Outlook on Signal Readout Strategies

DNA Nanobiosensors: An Outlook on Signal Readout Strategies

Solid‐Phase Synthesis and Purification of Protein–DNA Origami Nanostructures

DNA nanomaterials for preclinical imaging and drug delivery

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