Abstract
The characteristic features of stem-loop structured probes make them robust tools to detect targets with high sensitivity and selectivity. The basis of the hairpin based sensors operation is a conformational change that occurs upon hybridization of target with stem-loop probe. The design of the stem-loop probe has an important role in target recognition. Therefore, we designed a label-free stem loop probe for targeting miR-21 as a cancer biomarker investigated by web-based tools; its thermodynamic parameters obtained by thermal UV spectroscopy. The efficiency of stem-loop structure opening in the presence of target and non-target sequences was evaluated by fluorescence spectroscopy and circular dichroism spectro-polarimetry. The results showed that the target sequence opens the structure of hairpin efficiently in comparison to non-target sequences. To optimize the stem-loop hybridization to its target, the buffer ionic strength was changed by adding different concentrations of NaCl, KCl and MgCl2. It was shown that buffering conditions have a significant role in loop structure opening and its optimization, led to an increase in sensitivity detection and have improved LOD from 60 pM to 45 pM.
Highlights
The use of stem-loop structure that can be opened in the presence of specific nucleic acids, were first reported by Tyagi and Kramer as “molecular beacon” (MB) in 199611
To increase the efficiency of the biosensors based on the hairpin structures in detecting the target oligonucleotides, a stem-loop DNA structure was designed for targeting miR-21, which is a valuable marker for a number of diseases such as several cancers
In this study we have considered all the points mentioned by other researchers[22,29] who had worked on designing stem-loop and MB structures
Summary
The use of stem-loop structure that can be opened in the presence of specific nucleic acids, were first reported by Tyagi and Kramer as “molecular beacon” (MB) in 199611. If we have just a label-free stem-loop DNA sequence, how we can find its thermodynamic properties, investigate the efficiency of its hybridization to target, assess its operation when exposed to the target and non-target agents and evaluate optimum conditions for its performance, with the application of the minimum technical cost and time and before applying it in the final structure of the sensor?. In response to these questions, we designed a stem-loop structured DNA (capture probe) for targeting miR-21 as a cancer biomarker and used fluorescence spectroscopy and circular dichroism spectropolarimetry techniques as simple and fast techniques for investigation of the stem-loop structure opening in the presence of target and non-target sequences. Since the capture probe was label free, PicoGreen was used as an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded oligonucleotide. The fluorescence intensity will reduce as a result of less amount of double-stranded DNA
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