An Introduction to 3DNA Nanotechnology
What is 3DNA®?
3DNA® is Genisphere's core nanotechnology: a 3-dimensional structure made entirely out of DNA. The 3-dimentional structure is a highly branched molecule built from interconnected monomeric subunits of DNA.
The 3DNA monomer—the basic unit of construction
A 3DNA monomer [Figure 1] is composed of two DNA strands that share a central region of sequence complementarity. When the two strands anneal, a monomer is formed, with a central double-stranded 'waist' bordered by four single-stranded 'arms'. All of the nucleic acids in the DNA strands are natural; no modified DNA bases are used. The branched structure of a 3DNA monomer is simply due to base pairing (and lack of base pairing).
Linking monomers—the architecture of 3DNA
The single-stranded arms of each of five unique monomers base-pair with one another according to their specific sequences. Base-pairing between the arms of complementary monomers allows directed assembly of the 3DNA as a step-wise process of forming layers [Figures 2A, 2B, 2C].
The layered structure of 3DNA
Construction of a 3DNA structure begins with a single initiator monomer, to which a first layer of monomers is hybridized. The result is a one-layer 3DNA configuration with 12 single-strand arms on the outer surface. This assembly is chemically cross-linked to prevent dissociation. Next, a second layer of monomers is attached to the outer arms of the first layer by the same hybridizing and crosslinking steps. In this two-layer 3DNA scaffold the number of free single-stranded arms increases to 36. A third layer of monomers is added in an analogous fashion, creating a DNA arrangement with a total of 108 free single-stranded arms. For many applications a four-layer arrangement is desired, and the addition of the fourth set of monomers leaves 324 single-stranded arms on the surface of the molecule. As the manufacturing process is not perfect, most 4-layer structures have fewer than 324 arms, averaging about 280 +/- 20 arms per molecule.
Characteristics of the 3DNA core
Depending on the selection of DNA strands and monomers used during manufacturing, typical 2-layer assemblies have a diameter of around 90nm and consist of about 3,300 bases of DNA, while typical 4-layer assemblies have a diameter of around 170nm and consist of about 30,000 bases of DNA. The Zetapotential of 3DNA structures has been determined to be -16eV to -20eV. Typical core 3DNA reagents are 99% solvent in most aqueous media due to their dynamic DNA configuration.
Using the arms for labeling and specificity
After the 3DNA core is manufactured, its arms are functionalized with labels and targeting devices. The molecules that determine the target and labeling specificity are attached to the 3DNA nanoscaffold as oligonucleotides or as oligonucleotide conjugates. Using simple DNA labeling, hybridization and ligation reactions, the 3DNA structure may be converted into a highly labeled, application-specific probe [Figures 3A, 3B, 3C].
The architecture of the 3DNA imposes no restrictions on the type of specificity or label used, so it may be configured to meet a wide variety of detection needs. The label may be fluorescent, enzymatic (HRP, AP), nanogold, or a hapten (biotin, FITC, DIG). The targeting moiety may be an antibody, peptide, specified RNA/DNA sequence, aptamer, PNA, or a hapten (biotin, FITC, DIG). Mixing and matching a variety of labels and targets on the same 3DNA core creates a highly customized reagent.
Signal intensity is determined by the amount of label that can be localized at the reaction site. Genisphere's 3DNA products are labeled with dozens or hundreds of signal producing molecules. Since so many labels are delivered to a single 3DNA-targeted binding site, these reagents passively amplify the signal intensity in a variety of life science research applications, often improving the limit of detection and/or allowing the use of less sample.
Customization for clinical diagnostics
The typical purpose of using 3DNA in clinical diagnostic tests is to improve sensitivity without changing the label or detection reagent by simply dropping 3DNA into the assay workflow. For example, if a standard lateral flow rapid test uses a sandwich antibody approach to detect antigen using nanogold particles, the 3DNA reagent easily facilitates the collection of hundreds of nanogold particles per analyte, using the same detection antibody. Genisphere customizes all components to avoid any increase in background signal and to maintain the optimal kinetics of the assay. Specificity of the test remains unchanged. An example reagent used in clinical diagnostics is shown in Figure 4: a 4-layer 3DNA with detection antibodies and hundreds of biotin labels.
Customization for targeted drug delivery
For targeted drug delivery, 3DNA cargo can consist of small off patent drug molecules, siRNAs, microRNAs, peptides or proteins. Targeting may be achieved by specific peptides, antibodies or other devices. Dual or multiple targeting is fully enabled by the architecture of the 3DNA and may help reduce off-target effects. Since these nanocarriers are comprised of natural DNA, they are biocompatible and have no demonstrated toxicity to living cells, and facilitate the release of cargo from internal cellular compartments. Attaching labels (fluorescent, enzymatic, or radioactive) to the scaffold is useful for tracking and imaging. Example reagents used in targeted therapeutics are shown in Figures 5A and 5B: both are 2-layer 3DNA with four molecules of folic acid for targeting. Drug cargo may be attached to free arms of 3DNA (Figure 5A) or intercalated into the 3DNA (Figure 5B).
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