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RNA Aptamer Probe for N9 (545-566)

This biobrick is a miniSpinach-based RNA Aptamer Probe (RAP). It targets the influenza Neuraminidase subtype 9 (Gene accession number: CY235364.1) by nucleotide 545-566.

Illustration of the design

Usage and Biology

The full miniSpinach allows the docking of fluorogen 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and increases its quantum yields by hundreds of folds. By truncating the critical P1 arm and attaching it to a 22-bp RNA-specific probe, the RNA Aptamer Probe can bind to a RNA sequence and enhances the fluorecence level of the docking DFHBI subsequently. In our work, this probe is demonstrated to be functional in vitro, targeting influenza RNA fragments. It also has the potential for live-cell reporting of mRNA level (Ong. et. al. 2017.).

The probe can be used for on-site, rapid and sequence-specific influenza detection. Its target neuraminidase gene encodes for one of the influenza viral protein that determines the subtype of the virus, which is a important factor for epidemic monitoring and some treatment schemes.

Overlapping DNA oligos can be ordered as template for in vitro transcription for production of this probe.

Characterization

Sensitivity & Specificity

By hybridizing in silico designed RNA probes and its complemntary target RNA, we successfully screened a probe with high ON/OFF ratio and visible difference.

Probe screening was performed by thermal refolding of the 1uM probes and their 22-bp targets in aptamer refolding buffer. The aptamer-target pairs (A+T) were significantly brighter than its aptamer only (A) counterpart. Using Chemidoc Blue Light Excitation / SYBR Green Mode, the difference in their fluorescence level is visible. N=3. *:p<0.05, **:p<0.01, ***:p<0.005, ****:p<0.0001.

Cross-reactivity is checked by hybridizing non-complementary aptamer-target pairs with other successful probes. It is shown that the probe is specific and aptamer-target pairs are statistically orthogonal.

Heatmap and its 2-way ANOVA showing the microplate reader data from hybridizing different targets and aptamers. This shows that our aptamer-target interaction pairs are orthogonal. N=3.

Time & Temperature

Our probe has to be first heated up to 95 degrees Celsius before slowly cooling down for aptamer refolding and fluorescence readout. To determine the time required for signal development, we used the Bio-Rad Real-time PCR system to simulate the dropping temperature and measured the fluorescence intensity of the reaction mixtures. It was shown that our aptamer-probe pairs generally require only 10 minutes of cooling to give out a detectable signal, which is around 75 degrees Celsius.

Samples were heated at 95 degrees Celsius for 5 minutes, then cooled to 25 degrees Celsius over the time course of 35 minutes. N=1. More replicates are needed.

After refolding is completed, we performed melt curve analysis with the same real-time PCR system. Surprisingly, the melting curve shows that the fluorescence intensity drops quickly at 18-35 degrees Celsius, which does not correspond to the previous data. However, this might be caused by the assymetry of association and dissociation kinetics of DFHBI docking, which was never studied in the previous literature. Another interesting point is that both miniSpinach and aptamer-target pairs share 2 melting temperatures, suggesting a two-step mechanism of DFHBI docking. Nevertheless, the temperature where the probe achieves optimal performance is around 18 degrees Celsius.

After refolding, samples were heated from 4 degrees to 95 degrees over the course of one hour. N=1. More replicates are needed.

Limit of Detection

Performing the aptamer-target refolding assays with different concentrations of targets, we calculated the fluorescence value of aptamer only + 3 standard deviations, then plotted onto the graph. It is discovered that the limit of detection for this probe is around 50nM.

Red line represents the fluorescence level of Aptamer only plus 3 standard deviations. N=3.

Ion Concentration

Our probe is applicable in nasal fluid. To test the ion dependency of our RNA aptamer probes, we performed the aptamer refolding assay over buffers of varying ion concentrations, based on the aptamer refolding buffer (10mM Tris-HCl pH 7.5, 100mM KCl, 5mM MgCl2). Briefly, ions from the original buffer (K+, Mg2+) were changed to other concentrations, while different concentrations of absent ions (Na+, Ca2+) were added in the refolding buffer. Based on the data, it is observed that while our probe can withstand excessive of Na+/K+ ions, some level of monovalent ions is needed for its function. Our probe is inhibited by Ca2+ ions and excessive Mg2+ ions, although low level of Mg2+ ions is necessary for its function.

Thermal refolding of 1uM probes and their 22-bp targets in aptamer refolding buffer of different ion concentrations were performed. Ca2+: N=3. *:p<0.05, **:p<0.01, ***:p<0.005, ****:p<0.0001. Other ions: N=1. More replicates are needed.

Sequence and Features