RNA

Part:BBa_K2582009

Designed by: Lo Ho Sing   Group: iGEM18_Hong_Kong-CUHK   (2018-10-07)

RNA Aptamer Probe for PB2 (2247-2268)

This biobrick is a miniSpinach-based RNA Aptamer Probe (RAP). It targets the universal region of the influenza Polymerase Basic 2 (By Marsh et. al. 2008. J. of Virology) by nucleotide 2247-2268.

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 polymerase basic 2 gene encodes for one of the influenza viral protein that is ubiquitous across almost all influenza genomes.

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.

Fluorescence data obtained from plate reader. 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.

Fluorescence data obtained from plate reader. 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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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