This novel protocol describes the use of multiple distinct sequences of the synthetic nucleic acid mimic gamma PNA to form nanostructures in specific organic solvent mixtures. The assays described here show the need to adapt existing nucleic acid nanotechnology protocols towards organic solvents where little to no protocols have been published. Practitioners new to this technique might find it difficult to adapt existing protocols developed for aqueous-rich environments into organic solvent-rich environments because of the propensity of the nanomaterial PNA to aggregate.
Begin by preparing the gamma PNA stocks. After obtaining HPLC grade purified gamma PNA strands from a commercial manufacturer, resuspend each strand in deionized water to 300 micromolar concentrations. Store the gamma PNAs at minus 20 degrees Celsius for up to several months.
When ready to perform self-assembly, prepare anneal batch samples in 75%DMSO, 75%DMF, and 40%1, 4-dioxane. First, prepare 20 micromolar substocks of each oligomer by aliquoting 0.67 microliters from the main stock and adding deionized water for a final volume of 10 microliters. Add one microliter of each oligomer from the 20 micromolar substocks to a 200 microliter PCR tube, which will come to a total volume of nine microliters for nine oligomers.
Add 30 microliters of anhydrous DMSO or DMF with an additional one microliter of deionized water for a final volume of 40 microliters. To prepare the 40%1, 4-dioxane batch, add 16 microliters of 1, 4-dioxane and bring the volume to 40 microliters with deionized water. Anneal the samples for 22.5 hours in a thermal cycler cooling from 90 to 20 degrees Celsius.
Typically, melting temperatures obtained for two oligomer and three oligomer gamma PNA subsets lie in the range of 40 to 70 degrees Celsius for different solvent conditions. Program the thermal cycler according to manuscript directions. Once annealing is complete, samples can be stored at four degrees Celsius for 12 to 24 hours before characterization.
Make a humidity chamber from an empty pipette tips box by filling the box with approximately five milliliters of water. Prepare a 20-fold dilution of commercially available 2%collodion in amyl acetate with isoamyl acetate solvent to create the nitrocellulose solution. Use tweezers to dip a coverslip into the nitrocellulose solution and allow it to air-dry, then make a flow chamber out of a microscope slide, two double-sided tape strips, and the nitrocellulose-coated coverslip.
Prepare the biotin BSA solution by weighing one milligram of biotin BSA and dissolving it in one milliliter of PBS. Pipette 15 microliters of the biotin BSA into the flow channel at an angle and incubate it for two to four minutes at room temperature in the humidified chamber. Blow 15 microliters of the wash buffer, which consists of one milligram of BSA in one milliliter of PBS, into the channel to wash away excess biotin BSA, then passivate the surface by incubating the flow channel for two to four minutes in the humidified chamber.
Measure 0.5 milligrams of streptavidin and one milligram of BSA, then dissolve both in one milliliter of PBS. Pipette 15 microliters of the streptavidin solution into the flow channel and place it in the humidified chamber for two to four minutes and wash the channel by flowing 15 microliters of wash buffer. Next, flow 15 microliters of the annealed batch of gamma PNA oligomers in the humidified chamber, then wash the unbound nanostructures by flowing 15 microliters of one millimolar Trolox in the same solvent composition as the nanostructures.
Transfer the flow channel to the slide holder and image it with a fluorescence microscope equipped with TIRF imaging, a 60X oil immersion objective, and a 1.5X magnifier. Scan the flow channel at either 60 or 90X magnification while monitoring the Cy3 channel. Prepare 20 and 6%SDS stocks according to manuscript directions, then anneal the gamma PNA oligomers by adding one microliter of each 20 micromolar oligomer to a 200 microliter PCR tube.
Add 30 microliters of anhydrous DMSO and one microliter of 6%or 20%SDS to the PCR tubes for a final volume of 40 microliters, which will result in a final SDS concentration of 5.25 or 17.5 millimolar respectively. Anneal the gamma PNAs in the thermocycler as previously described. Characterize gamma PNA nanostructures in the presence of SDS using the TIRF or TEM protocol.
TIRF imaging of gamma PNA oligomers annealed in 75%DMSO showed well-organized architectures while annealing in 75%DMF resulted in spicule-shaped or needle-like nanostructures. The 40%1, 4-dioxane condition produced a sparse decoration of filamentous nanostructures. Furthermore, the samples of gamma PNA nanotubes formed in 75%DMSO demonstrated bundling of nanofibers at high magnification or nanoscopic resolutions during TEM imaging.
Quantitative analysis of the width of the nanostructures showed a median width of 16.3 nanometers with maximum values beyond 80 nanometers. Isosequential DNA oligomers that replaced contiguous gamma PNAs formed straight filamentous structures whereas replacement of crossover gamma PNAs result in stellate structures. The nanostructures that were replaced with contiguous DNA oligomers had median widths of around 19 nanometers.
At high SDS concentrations, gamma PNA nanofibers also adopt highly networked morphologies in comparison to its critical micelle concentrations when viewed with TIRF imaging. TEM imaging indicates that gamma PNA assembly in 5.25 millimolar SDS has the most substantial reductions in structural bundling. When attempting this protocol, it is important to remember to maintain the solvent compositions as mentioned because of the propensity of the nanostructures to aggregate.
These methods encourage practitioners to pursue other unexplored avenues of different solvent compositions, surfactants, and other PNA DNA nanostructures.
