Aptamer-based RNA Imaging

Light-up aptamers or Fluorescent light-up aptamers (FLAPs) are a genetically-encoded RNA imaging platform. They are designed to bind specific fluorogenic dyes that 'light-up' only in the bound state. This property of 'fluorogenicity' means that fluorescence can be 'switched on' upon RNA expression. Light-up aptamers can be thought of as the RNA counterpart to fluorescent proteins, such as GFP. Commonly used light-up aptamers include Spinach, Mango, Corn and Broccoli, named for their vibrant colors.

Products
Background
Literature (1)
Cat. No. Product Name / Activity
5609 DFHBI
GFP fluorophore mimic for imaging RNA in living cells; activated by binding Spinach2 and Broccoli aptamers
5610 DFHBI 1T
GFP fluorophore mimic for imaging RNA in living cells; activated by binding Spinach2 and Broccoli aptamers
6434 DFHO
RFP fluorophore mimic for imaging RNA in living cells; activated by binding Corn aptamers

RNA Imaging using Light-up Aptamers

An RNA-based fluorogenic complex or module is made up of two parts, a light-up RNA aptamer, and a fluorogenic cognate dye, the "fluorogen", which binds the light-up aptamer with high affinity. Once bound, the complex becomes highly fluorescent. The light-up aptamer and fluorogen do not fluoresce unless bound to each other, making this system highly effective for RNA imaging.

Light-up Aptamer Principles

A light-up aptamer sequence is genetically engineered into an RNA sequence of interest, e.g. by in vitro transcription. This is then transcribed into RNA by the host cell machinery. A high affinity cognate fluorogen is then added, which binds the Light-up Aptamer and fluoresces brightly upon stimulation at the appropriate wavelength.

A fluorogen is nonfluorescent in the unbound state; energy from excitation is dissipated via non-radiative pathways such as molecular vibration (e.g. heat). When the fluorogen binds an aptamer, the conformational change imposed on the fluorogen leads to suppression of the non-radiative pathways, and bright fluorescence is produced as the excitation energy is dissipated via photons. See figure below.

 Principles of Light-up Aptamer

Figure 1- Light-up Aptamer Principles: A correctly folded Light-up Aptamer conformationally constrains a cognate fluorogen (e.g. DHFO Cat. No. 6434) to become highly fluorescent.

Advantages of Light-up Aptamer Technology

Studying transcription and translation as well as cellular trafficking at an RNA level has traditionally been done using the RNA tagging system MS2. This system genetically tags RNA molecules with a MS2 stem loop (MS2-SL), which binds fluorescent protein MS2 coat protein (MCP). This system has several drawbacks, however, including a relatively large RNA tag size (with negative effects on RNA processing), and purported blocking of 5'- and 3'-exonuclease activity due to the MS2 arrays. A direct comparison of Mango II (a light-up aptamer) and MS2- tdMCP-mCherry dual-labeled mRNAs, demonstrated that fluorogenic Mango aptamers provide a much greater signal-to-noise ratio. Read more in Cawte et al (2020).

Light-up Aptamer systems offer several advantages over MS2 and GFP:

  • Fluorogenic nature produces exceptionally high signal-to-noise ratio
  • Very bright fluorescent signal
  • Light-up aptamers are small RNA tags, thus have a lower propensity to interfere with cellular functions
  • They enable direct, fast measurement of gene transcription at the RNA level, providing a more accurate real time observation of RNA localization and promoter activity; GFP can take up to 30 minutes after stimulation to be translated into protein.

Light-up Aptamer Range

Since the initial development of the green Broccoli and Spinach aptamer systems, more aptamers and fluorogen pairs have been developed, to span the color range of the visible spectrum.

Fluorogen Light-up Aptamer Kd (nM) λEx (nM) λEm (nM) Absorption coefficient (ε) (M-1/cm) Complex quantum yield (φ) Brightness
DFHBI #5609 Spinach 540 469 501 24,300 0.72 17.5
DFHBI 1T #5610 Spinach 2 560 482 505 31,000 0.94 29.1
DFHBI #5609 Broccoli 360 472 507 29,600 0.94 27.8
DFHBI 2T Spinach2 1300 500 523 29,000 0.12 3.48
TO-1 Mango 3 510 535 77,500 0.14 10.85
DFHO #6434 Corn 70 505 545 29,000 0.25 7.25
DFHO #6434 Red-Broccoli 206 518 582 35,000 0.34 11.90

Table 1: Common RNA Aptamer-dye complex spectral data; adapted from Bouhedd et al, 2018. λEx= excitation wavelength, λEm= emission wavelength, Brightness calculated as Brightness = (ε × φcomplex)/1000)

Light-up Aptamer Applications

Light-up aptamer systems are suitable for imaging and functional characterization of coding and non-coding RNA in live cells. They have been used to detect and analyze mRNA, miRNA, tRNA and snoRNA among others. Through systematic evolution of ligands by exponential enrichment (SELEX), Light-up Aptamers have been engineered to work in a wide range of applications including: monitoring real time RNA gene transcription and RNA trafficking; metabolite and protein sensing; RNA and ribonucleoprotein (RNP) purification; as well as high-throughput drug screening.

Biological Applications of Light-up Aptamer

Figure 2: Examples of Light-up Aptamer Applications: A) Monitoring gene expression: RNA with a light-up aptamer (light blue structure) coded is expressed, a fluorogen (orange pentagon) binds and becomes highly fluorescent. B) Metabolite sensor or riboswitch: a metabolite (green heptagon) binds an aptamer, stabilizing the light-up aptamer conformation, allowing the fluorogen to bind. Fluorogen becomes highly fluorescent. C) RNA sensor: RNA binds and stabilizes the light-up aptamer conformation, allowing the fluorogen to bind. Fluorogen becomes highly fluorescent. Image adapted from Neubacher and Hennig (2019)

COVID-19 (coronavirus) Testing Kits using RNA Aptamers

Researchers from Simon Fraser University (SFU) Vancouver, have developed a Mango aptamer-based platform for detecting COVID-19. They developed Mango II aptamer arrays, which worked well in live and fixed cells with single-molecule sensitivity. They found the signal-to-noise ratio was significantly improved when compared to the traditional RNA monitoring system MS2 (MS2-tdMCP-mCherry dual-labeled mRNAs). For more information on this kit, read the Covid-19 test kit article article from GEN news.

References

For further information on Light-up Aptamer systems, see references below:

Literature for Aptamer-based RNA Imaging

Tocris offers the following scientific literature for Aptamer-based RNA Imaging to showcase our products. We invite you to request* or download your copy today!

*Please note that Tocris will only send literature to established scientific business / institute addresses.


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