Design, synthesis, and evaluation of safety-catch photolabile linkers: Applications for gene synthesis.

摘要

This dissertation reports the synthesis and evaluation of several safety-catch photolabile linkers (SC-PLL) for use in the development of an automated gene synthesizer. These linkers allow the light-directed synthesis and spatially selective photorelease of oligonucleotides from microarrays. The linkers themselves are based upon photolabile protecting groups; photocaging groups, or photolabile linkers and made latently active by use of a safety-catch.;We have demonstrated spatial photorelease of oligonucleotides from microarrays by three methods. The first evidence of spatially selective photorelease is the loss of signal from a fluorescently tagged complement hybridized to a sequence on the array surface bound via a SC-PLL. Signal loss occurs when select regions of the array have been photoreleased after activation of the linker. We were also able to label photoreleased oligonucleotides with 32P - ATP, and confirm they were cleanly released in a spatial manner by an electrophoresis gel profile (i.e. there was no evidence of cross-contamination of the photoreleased sequences). Finally, we confirmed that the photoreleased oligonucleotides could function as templates for amplification by successfully PCR amplifying an oligonucleotide 40 base pairs in length.;We anticipate that the advantages offered by these safety-catch photolabile linkers will aid the development of a robust, automated gene synthesis platform. These advantages include a modular design, which can extend their utility to other microarray synthesis formats; complete flexibility in regards to which sequences are released from the array, enabling multiplexed PCR assembly reactions on subsets of photoreleased oligonucleotides; and improved sequence integrity (i.e. fewer errors such as deletions or insertions) of photoreleased oligonucleotides.