Single-stranded DNA (ssDNA) is involved in many important cellular processes such as the replication, transcription and repair of our genome. It is also involved in the creation of so called telomeres, end-caps that protect chromosomes from degradation and are linked to aging. ssDNA is also used extensively in modern DNA nano-fabrication. Examples of this include DNA-origami, which can be used to create nanometer scale structures in programmable shapes and aptamers, ssDNA architectures that bind with high affinity and high specificity to a target that show great promise in use as novel therapeutics. This makes ssDNA structure an interesting topic of study. Such structural assays have been done using various techniques, including but not limited to: FRET, NMR, optical/magnetic tweezers and AFM. A downside to all these techniques is that only a couple of sequences can be measured at a time, making it difficult to sufficiently sample the vastness of sequence space available. In this thesis I demonstrate a novel technique combining single-molecule FRET combined nextgeneration high-throughput optical sequencing. I show that the two different measurements can be performed on the same chip and effectively mapped to each other. First, using traditional lowthroughput methods, the FRET efficiency was measured for a cy3-cy5 pair separated by a 8 nucleotide piece of single-stranded DNA. This was done for 12 different sequences that were selected to vary in terms of base stacking, bulkiness, hydrogen bonds and other structural factors. The experiment was repeated using the high-throughput platform. The results of the high-throughput method were compared with the results from the low-throughput method, and show a correlation
factor of 0.75. These experiments show that this technique can be used as an effective tool in performing FRET measurements on a minimum of 1500 sequences up to possibly 910,000 sequences in onemeasurement, making it an exciting new tool for structural research into ssDNA.