Design and optimization of nucleic acid probes for intracellular and biosensor development

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The detection of DNA and mRNA plays an important role in disease diagnostics, bioanalysis study and gene expression studies. Currently, there are many techniques that can help decipher some of the mechanisms occurring inside the cell including, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and Northern or Southern blotting. These techniques are reliable and sensitive but are time consuming and often fail to give real time data. The key for rapid, selective and sensitive analysis is the use of molecular probes based on nucleic acids. However, currently there are limitations that hinder the use of molecular probes for bioanalysis, including low sensitivity, reduced selectivity, and poor stability. The focus of this work has been the development and optimization of molecular probes for intracellular imaging and biosensor applications.

Recently, we introduced the hybrid molecular probe (HMP) as a novel probe for mRNA and DNA monitoring in solution and in living cells. A series of spectroscopic experiments was conducted to fully characterize and optimize HMP for both in vitro and in vivo analysis and as a tool for hybridization studies. The results proved that HMP enables very sensitive analysis at low concentrations and sample volumes. In addition, HMP was able to overcome some of the problems associated with traditional methods of gene expression analysis. Specifically HMP achieved a more than 20 fold signal enhancement compared to ∼6 of linear probes, was very stable in a cell-like environment, and was less prone to generate false positive signal when compared with molecular beacons (MBs).

MBs are well-known molecular probes that are currently used in bioanalysis. Although, MBs have previously been used for surface hybridization studies, their potential has not fully exploited, primarily because of the poor stability of the beacon after immobilization onto a surface, resulting in a low signal enhancement. By incorporating locked nucleic acid (LNA) bases into the MB sequence, a considerable improvement in the stability and therefore in the overall efficiency in the signal can be achieved for surface hybridization. As part of this research, locked molecular beacons (LMB) have been evaluated and compared to regular molecular beacons (RMB) in terms of selectivity, sensitivity, thermal stability, hybridization kinetics and robustness for the detection of target sequences. The experiments were performed using biotinylated beacons immobilized onto avidin-coated microscope slide surface. After incubation with the target DNA sequence, a 25-fold enhancement has been achieved for LMB, with detection limits extending down to the low nanomolar range. In addition, the LMB-based biosensor possesses better stability, reproducibility, selectivity and robustness when compared with the RMB.

These results demonstrate the potential of the newly designed HMP and LMB probes as prospective tools in the development of DNA microarrays and for bioanalysis study , disease diagnosis and biotechnology applications.