Abstract
BackgroundAptamers 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 dsDNA molecule with a ssDNA 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.ResultsTwo bifunctional aptamers are examined using a nanopore detector. They are chosen to provide sensitive, highly modulated, blockade signals with their captured ends, while their un-captured regions are designed to have binding moieties for complementary ssDNA targets. The bifunctional aptamers are duplex DNA on their channel-captured portion, and single-stranded DNA on their portion with binding ability. For short ssDNA, the binding is merely to the complementary strand of DNA, which is what is studied here – for 5-base and 6-base overhangs.ConclusionA preliminary statistical analysis using hidden Markov models (HMMs) indicates a clear change in the blockade pattern upon binding by the single captured aptamer. This is also consistent with the hypothesis that significant conformational changes occur during the annealing binding event. In further work the objective is to simply extend this ssDNA portion to be a well-studied ~80 base ssDNA aptamer, joined to the same bifunctional aptamer molecular platform.
Highlights
Introduction and backgroundNanopore blockade detectorOur nanopore detector is biologically based and uses a protein, the α-hemolysin (α-HL) toxin produced by the bacterium Staphylococcus aureus, to create a pore through a phospholipid bilayer by self-assembly
The overhang's binding strength can be adjusted by tailoring its length in both of these experiments, and in future work this will permit a highly precise study of DNA annealing
The α-hemolysin pore has a 2.0 nm width allowing a dsDNA molecule to be captured while a ssDNA molecule translocates
Summary
Experiments are performed with a linear molecule with a bulge in the center. To one side of the bulge is the bluntended stem sequence like that used in one of our DNA hairpin controls, where the bulge is in the position of the hairpin's loop. Besides the pseudo-aptamer, the Y-aptamer complex T6-Y10T1-GC was found to have mean dwell time values of 0.30 ms, 0.38 ms and 1.52 ms (for the first, second and third levels respectively – see Fig. 8a). When a T6 group was added to bind with the T6-Y10T1-GC to form a T6-Y10T1-GC+T6 complex, the mean dwell time statistics differed significantly, becoming dominated by fewer blockade levels, consistent with the annealed molecule "stacking" into the duplex helix, thereby having fewer accessible degrees of freedom. The statistics determined by the analysis of a single molecule are a snapshot of the conditions present at the time of data collection, as opposed to averaging numerous signals collected over time together. Targeting of cancer cells relies on the development of molecules, aptamers with toxins in tow, perhaps, that are suited to in vivo applications and that have the required affinity, specificity and favorable pharmacokinetic properties
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