Design & Modelling
In order to achieve our goal of rapidly detecting highly specific miRNA, we had to create a novel probe.
DNA probes are introduced into a solution containing target miRNA, and undergo a conformational change. Then, an enzyme catalyzes amplification of the DNA probes. Finally, a nucleic acid stain is added to the system, which produces a measurable fluorescence. This allows us to quantify the original concentration of miRNA.
The detection of miRNA will be facilitated by specific binding with DNA structures shaped like dog-bones. Once the miRNA-DNA complexes are created, the DNA portion undergoes a conformational change in which the pinch point between the two outer loops widens, and the entire structure becomes circular. Next, a polymerase is added which amplifies the structure through the rolling circle mechanism (RCA). If none of the target miRNA is present, the dogbone structure does not open, and rolling circle amplification is unable to continue. The amplified DNA is clipped with restriction enzymes so that the fragments disperse throughout the solution. Finally, SYBR Gold, a nucleic acid stain is added to the solution, which will fluoresce with intensity depending on the concentration of DNA fragments. The concentration of those fragments depend on the concentration of amplified, which itself depends on the initial concentration of miRNA.
This DNA probe is designed to be exclusively sensitive to specific miRNAs that are up-regulated in lung cancer. The DNA probe opens up to form a circular template when the miRNA strand hybridizes to it.
The dog-bone probe is a 100 nt strand of circular DNA that has a secondary structure containing two 36 single-stranded nucleotide loops separated by a 14 nucleotide double-stranded stem. It is very stable in its “locked” form (-G<10 kcal/mol) (NUPACK).
When the miRNA is in proximity of the probe it will bind to a 9 nucleotide complementary “toehold” section of the dog bone.. The stem and the unbound miRNA part are complementary. Once the miRNA binds to the toehold section of the dog bone, the rest of the miRNA will break open the stem through a strand displacement mechanism , turning the dog bone into an open single-stranded loop with one section of double stranded DNA-RNA hybrid.
One benefit of a dog-bone probe is that it does not need a ligation step during the assay compared to other RCA methods for detecting oligonucleotides. Thus, our detection system does not have to deal with ligation inefficiencies which will affect the sensitivity of our system. In addition, the toe-hold initiated attachment of miRNA to DNA and subsequent strand displacement are highly specific to the miRNA, making this probe robust and resistant to false positives .
1. Toehold-initiated rolling circle amplification for visualizing individual microRNAs in situ in single cells.
2. A new class of homogeneous nucleic acid probes based on specific displacement hybridization.
Typically, miRNA concentration in blood is quite low, on the order of nano-grams per microlitre, even when overexpressed due to cancer . To achieve detection, signal amplification in needed. In our design, Rolling Circle Amplification is used to increase the amount of DNA in solution, which leads to increased fluorescence in the presence of SYBR gold.
Rolling Circle Amplification (RCA) is an enzymatic process that has the ability to rapidly and effectively amplify circular DNA template. The processes is isothermal, which is an advantage over other conventional amplification processes as it reduces the reliance of heating and cooling cycles . The polymerase complex (phi29) moves along the template strand adding nucleotides to the daughter strand. Once initiated, the reaction continues until about 10,000 copies are made. The amplified DNA consists of a long DNA chain containing repeated sequences. The secondary structure of the concatemer is a repeated stem loop shape where the stem is identical to the stem of the dog-bone structure.
Phi-29 protein model (source)
After rolling circle amplification with phi29 polymerase, a large number of DNA concatemers are produced. A selected restriction enzyme cuts the concatemer and disperses the smaller DNA fragments. This results in a uniform solution of DNA fragments that will be stained for fluorometry.
The role of the restriction enzyme in our system is to cut the DNA concatemers into amplicons for fluorometry. Fluorometers emit a monochromatic wavelength of light that is absorbed by our DNA stain and subsequently reemitted. The restriction enzyme cuts and disperses DNA fragments to form a homogeneous solution. Without the restriction enzyme, the analyte will contain localized, clumped DNA concatemers that are unevenly distributed throughout the well. The fluorometer provides more consistent and accurate results for the homogeneous solution versus the solution that contains DNA clumps. The restriction enzyme chosen for this miRNA is HAEIII.
HaeIII is a type II restriction enzyme with the recognition site of 5’-GG/CC-3’ . HaeIII is chosen as the restriction enzyme for miRNA-193b because the miRNA-193b sequence contains the recognition site for HaeIII. Thus by design, the stem section of our dog bone structure contains the recognition site for HaeIII. This provides a consistent location in the amplified DNA concatemer for the enzyme to cleave.
5. Sigma HAEIII Product Information
Once the concatemer has been fragmented, a nucleic acid stain, SYBR Gold, is added to the system. SYBR Gold fluoresces, and the magnitude of the fluorescence scales with the amount of DNA in the system. The fluorescence is measured with a spectrophotometer, and the results can be used to determine the amount of target miRNA in the sample.
SYBR Gold is a nucleic acid stain that changes fluorescence magnitude in the presence of nucleic acids. Fluorescence is the property of a substance to absorb and re-emit the light energy at different wavelengths. SYBR Gold absorbs light at 485 nm, and emits light at 537nm . Using a spectrophotometer, it is possible to detect the intensity of the re-emitted light. A larger concentration of DNA will yield a larger intensity, and therefore fluorescence analysis can be used to quantify the amount of DNA in the system. To determine the miRNA concentration, a calibration curve is needed. Known concentrations of the target miRNA are subjected to the binding, amplification and fragmentation process. Then, the stain is added and the fluorescence is recorded. Once complete, it is possible to determine target miRNA concentration from the fluorescence intensity.
6. Envitrogen SYBR® Gold Nucleic Acid Gel Stain