Fluorescent dyes which are specific for duplex DNA have found a wide range of applications from staining gels to visualization of chromosomes. Porphyrin dyes have been found which are highly fluorescent in the presence of quadruplex but not duplex DNA. These dyes may offer a route to the specific detection of quadruplex DNA under biologically important conditions. There are three types of DNA quadruplex structures, and these may play important roles in telomere, centromere, triplet repeat, integration sites and other DNAs, and this first set of porphyrin dyes show some selectivity between the quadruplex types.
Multiphoton excited (MPE) photochemistry is used to fabricate model tissue engineering scaffolds directly from types I, II, and IV collagen. A modified benzophenone dimer (BPD) provides the photoactivation and becomes incorporated into the resulting collagen matrixes. Unlike xanthene photochemistries, the benzophenone dimer can be used in acidic environments, where most forms of collagen have the greatest solubility. The minimum feature sizes are investigated by using two- and three-photon excitation, where the latter provides for superior "resolution" and suggests that collagen structures can be fabricated on the size scales of focal contacts. The resulting structures display excellent retention of bioactivity as evidenced by highly specific cell adhesion as well as immunofluorescence labeling. Structural and chemical aspects of the collagen matrixes are probed through measuring the enzymatic degradation through specific and nonspecific proteases, as the resulting relative rates are consistent with the activity of these enzymes. The degradation rates can also be controlled through varying the cross-link density in the matrixes, which is achieved through tuning the exposure dose during the fabrication process. The degradation rates are also found to be consistent with swelling/shrinking measurements and thus the average mesh size of the matrixes. In all cases the enzymatic degradations are well-fit single exponentials, suggesting that the matrixes can be fabricated with a priori knowledge of their structural properties. These results coupled with the resulting bioactivity suggest that the multiphoton fabrication process may be a powerful tool for the creation of cell-sized tissue engineering scaffolds.
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