Dominant Analytical Techniques in DNA Nanotechnology for Various Applications

Abstract

DNA nanotechnology is rapidly gaining traction in numerous applications, each bearing varying degrees of tolerance to the quality and quantity necessary for viable nanostructure function. Despite the distinct objectives of each application, they are united in their reliance on essential analytical techniques, such as purification and characterization. This tutorial aims to guide the reader through the current state of DNA nanotechnology analytical chemistry, outlining important factors to consider when designing, assembling, purifying, and characterizing a DNA nanostructure for downstream applications.

Design and Characterization of a Gene-Encoding DNA Nanoparticle in a Cell-Free Transcription–Translation System

“…This arises since the 3D DNA NP does not migrate in the gel based solely on the mass-to-charge ratio of a simple dsDNA that is not supercoiled, but because its migration rate is now far more complex due to its origami architecture. More stringent gel methodologies, such as those based on polyacrylamide, are required when separating closely related species of DNA origami. , The band for the ssDNA promoter DNA NP (lane 7) migrates in a manner identical to that of the original Luc9 DNA NP as expected, also indicating successful formation. All the PEG-purified versions of the DNA NP (Figure A lanes 4, 5, and 7) have marginally slower migration than the crude DNA NP (lane 6) which could be attributed to the excess Na + present in the purified DNA NP samples post PEG precipitation.…”

“…By strategically routing the gene-encoded scaffold strand and leveraging staple strands for installing a Förster resonance energy transfer reporter system on the DNA NP, one could apply luminescence and fluorescence in tandem to learn the nuances of DNA NP stability and functionality. In fact, measuring protein expression efficiency from a DNA NP could serve as a much-needed analytical tool to determine nucleotide-level structural integrity of DNA NPs under physicochemical stressors . Use of DNA binding protein fusions may also allow the NPs to bring proteins attached to it into the TXTL reaction or eventually even a cell in a piggyback-like manner to complement any requirements not present or to focus activity to just a given target .…”

Purification of DNA Nanoparticles Using Photocleavable Biotin Tethers

“…When determining the efficiency of an analytical technique in purifying a DNA NP, four factors are worth considering, namely, the ease of execution, the quality of the purified DNA NP, the quantified yield of the recovered DNA NP, and the extent to which the sample suffers dilution during the purification process Table S3 summarizes our comparative analysis of representative purification techniques and the PC-biotin approach.…”

“…Collectively, the PC-biotin approach to purifying DNA NPs serves to simplify the process and caters to the growing demand for custom-scaffolded NPs. This method adds to and expands the toolset for purifying these structures, and purification is not an either–or approach where you can only pick and use one technique . Techniques can be adapted, adjusted, and combined, and our process allows for more choices and options, which will be useful, since it allows scientists to choose what suits them best.…”

“…100 μL of the crude PB origami solution was diluted 2-fold into its respective target buffer (12 mM MgCl 2 , 1× TBE, pH = 8) to obtain a starting volume of 200 μL. This was then combined in a 1:1 ratio with PEG precipitation buffer (15% PEG 8000 (w/v), 1× TBE, and 505 mM NaCl). , The sample was mixed via pipetting and was then transferred to a centrifuge to spin at 13,000 g at room temperature for 30 min. The supernatant was removed using a pipette, and the resulting pellet was resuspended in 100 μL of the target buffer.…”