Aligned collagen fiber matrix as anisotropic biological substrate and fluorescent nucleotide reversible terminators with tailless linkers for four color DNA sequencing by synthesis

The first part of the thesis presents the study of aligned collagen fiber formation and its application as an anisotropic biological substrate. Straightforward techniques are developed to align collagen fibers in one direction. The first technique requires only collagen solution, surface-modified magnetic beads, a small magnet, and an incubator. The collagen gels are imaged with confocal reflectance microscopy, allowing the entire gelation procedure to be recorded. The degree of alignment is quantitatively assessed using MatLab image analysis programs that permit the identification of fiber shape, position and angular distribution. A series of control experiments show that magnetic beads coated with streptavidin lead to the most highly aligned gels. Rheology and microscopy experiments together suggest that alignment results from bead coupling to, and entrainment and entrapment in collagen fibrils during their assembly into fibers that form a sample-spanning gel. The time scales of gelation and bead motion to the poles of the external magnet must be similar to effect good alignment over large areas with this technique. The second technique, which requires only collagen solution, also achieves excellent collagen fiber alignment. The potential utility of such a system is as an anisotropic biological substrate for cancer cell metastasis, neuron development and stem cell differentiation, etc.;The third part of the thesis presents single molecule experiments with fluorescent nucleotide reversible terminators. Third generation DNA sequencing will require more sensitivity and much higher throughput. A very promising way to achieve such a goal is the use of single molecule fluorescence detection methods. Our preliminary experimental results show that single molecule fluorescence signals can be observed with three fluorescent nucleotide reversible terminators and different photo bleaching behaviors are observed. This provides new opportunities for third-generation four-color DNA sequencing by synthesis.;The second part of the thesis presents the design and synthesis of fluorescent nucleotide reversible terminators with novel "tailless" linkers for four color DNA sequencing by synthesis. Based on previous molecular frameworks, an important improvement is made to the linker structure and position which, upon cleavage, can lead to regeneration of natural nucleotides on the DNA strand. Of the four nucleotide analogues, the cytidine compound has been synthesized successfully permitting nucleotide extension with a mutant 9°N DNA polymerase. MALDI-TOF MS results show that the polymerase correctly recognizes the nucleotide and incorporates it into the growing DNA strand. What's more, after TCEP treatment, the 3'-termination group and the linker can be cleaved completely and regeneration of natural cytidine on the DNA strand is observed. Synthesis of the adenosine and guanosine compounds with a similar strategy have begun but it presented a greater challenge to attach the linker to these bases due to the lower reactivity of the amino groups on the purine rings. Continuing efforts are underway to produce the final compounds. The new tailless linker is expected to eliminate the molecular remnants on certain incorporated bases and reduce steric hindrance, which may result in longer sequencing read lengths. The use of such nucleotide analogues will very likely lead to improvements in the next-generation DNA sequencing technology.