1 research outputs found
Hairpin–Hairpin Molecular Beacon Interactions for Detection of Survivin mRNA in Malignant SW480 Cells
Cancer
biomarkers offer unique prospects for the development of
cancer diagnostics and therapy. One of such biomarkers, protein survivin
(Sur), exhibits strong antiapoptotic and proliferation-enhancing properties
and is heavily expressed in multiple cancers. Thus, it can be utilized
to provide new modalities for modulating the cell-growth rate, essential
for effective cancer treatment. Herein, we have focused on the development
of a new survivin-based cancer detection platform for colorectal cancer
cells SW480 using a turn-on fluorescence oligonucleotide molecular
beacon (MB) probe, encoded to recognize Sur messenger RNA (mRNA).
Contrary to the expectations, we have found that both the complementary
target oligonucleotide strands as well as the single- and double-mismatch
targets, instead of exhibiting the anticipated simple random conformations,
preferentially formed secondary structure motifs by folding into small-loop
hairpin structures. Such a conformation may interfere with, or even
undermine, the biorecognition process. To gain better understanding
of the interactions involved, we have replaced the classical Tyagi–Kramer
model of interactions between a straight target oligonucleotide strand
and a hairpin MB with a new model to account for the hairpin–hairpin
interactions as the biorecognition principle. A detailed mechanism
of these interactions has been proposed. Furthermore, in experimental
work, we have demonstrated an efficient transfection of malignant
SW480 cells with SurMB probes containing a fluorophore Joe (SurMB-Joe)
using liposomal nanocarriers. The green emission from SurMB-Joe in
transfected cancer cells, due to the hybridization of the SurMB-Joe
loop with Sur mRNA hairpin target, corroborates Sur overexpression.
On the other hand, healthy human-colon epithelial cells CCD 841 CoN
show only negligible expression of survivin mRNA. These experiments
provide the proof-of-concept for distinguishing between the cancer
and normal cells by the proposed hairpin–hairpin interaction
method. The single nucleotide polymorphism sensitivity and a low detection
limit of 26 nM (S/N = 3σ) for complementary targets have been
achieved