Nucleic acid reagents for imaging and detection.

摘要

In order to better understand how nucleic acids function as natural molecules, it is necessary to develop probes that allow the study of these molecules. The studies described herein utilize fluorescent labeled PNA and aptamers to provide new techniques and molecules for use as probes in the imaging and detection of biological molecules.; In order to site-specifically and noncovalently label mRNA transcripts, we have utilized fluorescent PNA hybridization probes. These labels deliver dyes to specific regions of the mRNA through specific Watson-Crick base pairing. In order to study mRNA splicing, two probes were used, one labeled with a thiazole orange (or Alexa488) donor and another labeled with an Alexa594 acceptor. These probes were targeted to regions flanking the splice sites of a pre-mRNA molecule such that before splicing the PNAs were separated by >300 nucleotides (nts) and after splicing the separation decreased to ≤12 nts. The decrease in separation led to an increase in fluorescence resonance energy transfer (FRET). Using these probes, we were able to show that fluorescent PNA probes can bind to a large RNA target and produce changes in fluorescence that correspond to hybridization events. In addition, the two probes can be used to demonstrate a difference in FRET efficiency between a spliced and unspliced mRNA.; In another effort to provide new probes for imaging and detection, fluorescent nucleic acid aptamers were developed. These aptamers utilize a fluorescent labeled PNA probe and appear to bind their target (thrombin) with high affinity. Although these aptamers showed small changes in fluorescence, with further modifications, this technique could prove to be a promising way to incorporate fluorophores into the selection process.; In a similar direction, an in vitro selection for aptamers that bind a fluorogenic dye (dimethyl indole red) in the presence of a small molecule (GTP) was performed. This type of selection would be a direct method for selecting allosteric aptamerc biosensors. The selections were designed such that the binding of the GTP would cause a conformational change in the aptamer and allow the fluorogenic dye to bind thereby producing an increase in the fluorescence. Aptamers were isolated that appear to only bind to the fluorogenic dye, however, the aptamers found here could prove useful in many biosensor and imaging techniques.; In an attempt to assemble a catalogue of dye-RNA aptamer pairs, aptamers were selected for the environmentally sensitive dapoxyl dye. It was found that the aptamers isolated were the same or similar to the DIR-binding aptamers described above. In addition, the dapoxyl-binding aptamers have a low affinity for dapoxyl and a high affinity for DIR. Although high affinity dapoxyl-binding aptamers were not isolated, this selection provided insight into the future design of selection libraries and protocols.