Translocation Of Structurally Defined Branched Dna Through Nanopores

We use solid-state nanopores to study the translocation characteristics of different structurally defined DNA topologies. Site-specific modifications with non-natural nucleotides along the backbone of DNA fragments allow grafting of side branches at specific locations by “click” chemistry. We produce T- and pi-shaped DNA molecules with a 50bp double-stranded DNA backbone and either 25nt single-stranded or 25bp double stranded DNA branches. Nanopores ranging in size from 3 to 10nm are fabricated by controlled breakdown (CBD) in ultra thin 10-nm SiN membranes and used to electrophoretically translocate these short branched DNA molecules. We can distinguish the topologies of these DNA molecules through analysis of the ionic current blockages. Such structurally defined branched DNA molecules can be used for the development of multiplexed nanopore-based assays, and as position-controlled building blocks of much larger DNA polymers, to further our understanding of the fundamentals of molecular transport through nanopores by precisely measuring intra molecular velocity fluctuations.