Gling was by design (see Aptamer sub-section). A preliminary study of
Gling was by design (see Aptamer sub-section). A preliminary study of three model systems was performed:I. The model DNA/DNA system: ssDNA annealing As a test case for DNA-DNA interaction studies with the nanopore detector, a “pseudo-aptamer” was examined (Figure 10 and Figure 11). Aptamers are nucleic acids selected for their ability to bind to molecules of interest and may provide the basis for a whole new class of medicines. If the aptamer is simply a DNA molecule with an overhang (a “sticky” end) then the segment of ssDNA that complements that overhang provides a known binding target with binding strength adjustable according to length of overhang, etc II. The model DNA/Protein system: aptamers and sTF/TFBS A “reverse-engineered” TBP-binding aptamer (it has an exposed TATA box) is chosen. The first design problem, generic to all such NADIR aptamers (see Methods and Figure 12), is implementing the added functionality of toggle-blockade interactions with the nanopore. Some variants studied are shown in Figure 13. NADIR selection is constrained to the subset of aptamers that not only have desirable binding properties to their target, but that also have a common “base”, partly consisting of a blunt 10 base-pair dsDNA end that provides a sensitive “toggle” signal upon capture by the electrophoretic forces at the nanopore’s limiting aperture. This is accomplished by having the terminal base-pair perch over the limiting aperture due to a Y-branching in the DNA molecule being held at the channel’s outer (cis) mouth (performing a sim-Page 11 of(page number not for citation purposes)BMC Bioinformatics 2006, 7(Suppl 2):SPercent Occurance45.00 40.00 35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9UL Duration PlotsCA_0 CA_1 CA_2 CA_3 CA_4 CA_5 CA_1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.2.1 2.2 2.3 2.4 2.Time (ms)Figure 9 distant group very closely, the one molecule with no extra GC extent, and molecules with GC pairs (and surroundbase-pair ings), neighboring terminus (CA_2, CA_4) share this property to CA_3 has strongest interaction own characteristic curve 1 UL, the unbound variants state, has shortest life for CA_3, i.e., a lesser also separates with its with channel more than UL, the unbound terminus state, has shortest life for CA_3, i.e., CA_3 has strongest interaction with channel (and surroundings), neighboring variants (CA_2, CA_4) share this property to a lesser extent, and molecules with GC pairs more than 1 base-pair distant group very closely, the one molecule with no extra GC also separates with its own characteristic curve. This result is consistent with the increased reactivity of CA_3 to initiate complex formation [1], with weaker variants in CA_2 and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26100631 CA_4, exactly as found experimentally [1-7].ilar role to the hairpin loop in prior, DNA hairpin, studies) [7-11]. As a test case for protein-DNA interaction studies, binding of TBP to an aptamer with a TATA binding site is studied with the nanopore detector, and preliminary results are shown (Figure 14 and Figure 15).III. The model Protein/Protein system: antibody/antigen binding Antibody conformational change, and antibody-antigen binding, are examined using a nanopore detector, and provide a test case for nanopore-based MLN9708 chemical information protein-protein interaction studies. Antibody blockade results with a variety PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28854080 of “toggle” signals, are shown in Figure 16. Blockades observed before and after introduction of target antigen are shown in Figure 17.exposed to a soluti.