One post-transcriptional mechanism that regulates the progression of cancer and other diseases involves small 22-23 nucleotide sequences called microRNA (miR). Early detection of small changes in concentration of these biomarkers holds potential to diagnose diseases at their earliest stages. Use of current nucleic-acid based biosensors, like molecular beacons, for in situ cellular and tissue analysis is hindered by false signals from nuclease degradation and off-analyte binding. For this reason, this work describes a new miR biosensor that was created to improve upon existing miR biosensors and overcome some of their weaknesses.
The reporter+probe biosensor that was developed consists of two partially complementary strands of DNA that form a double-stranded complex. One strand, called the reporter, contains two dyes capable of energy transfer located on opposite ends of the strand. The other strand, called the probe, is partially complementary to the reporter and is fully complementary to the miR of interest. When the miR of interest is presented to the reporter-probe complex, the reporter is displaced and a probe-target complex is formed. The displaced reporter forms a hairpin conformation to bring the dyes together. This causes a quantifiable change in analytical signal dependent on miR concentration. In the first portion of this research, a reporter+probe biosensor was developed for miR let-7a. This biosensor showed improved defense against false positive signal generation from nuclease degradation when compared to a molecular beacon. The biosensor was shown to have low nM LODs for analyte let-7a.
In the second part of this research, reporter+probe biosensors were developed for two miR analytes, miR-26a and miR-27a. It was discovered that there are numerous design parameters that need to be considered when making a reporter+probe biosensor for a given analyte. Among the discovered parameters, limiting the number of ‘non-ideal hairpin’ conformations that the reporter can sample maximizes the signal change upon analyte binding. Low nM LODs were found for miR-26a and miR-27a with their respective reporter+probe biosensors.
In the third portion of this research, a miR-26a reporter from the previous study was used to test whether hexaethylene glycol spacer molecules could be used between the reporter nucleic acid sequence and the fluorescent dyes to allow FRET (Förster Resonance Energy Transfer) enhancement to occur. It was found that the hexaethylene glycol spacers did increase FRET enhancement, and that the 6-FAM|ATTO 633 dye pair was superior to Cy3|Cy5 for FRET enhancement.
In the final stage of this research, directionality of the reporter+probe biosensor was studied to determine if it would increase specificity for the analyte by limiting off-analyte binding. It was found that taking into consideration where the off-analytes will bind to the probe allow for a more selective biosensor to be developed. In this study, the two reporter+probe biosensors were developed for miR-146a. Low pM LODs for miR-146a were obtained.
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