Aptamer-based RNA Imaging

Light-up RNA 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
7466 BI dihydrochloride
DFHBI derivative for imaging of RNA in living cells that bind Broccoli aptamers
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
7277 HBC 530
GFP fluorophore mimic for imaging RNA in live cells; activated by binding to Pepper aptamers; suitable for confocal and two-photon microscopy
7660 TBI
Fluorogenic ligand for Broccoli RNA aptamer

RNA Imaging using Light-up RNA 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 RNA aptamer with high affinity. Once bound, the complex becomes highly fluorescent. The light-up RNA aptamer and fluorogen do not fluoresce unless bound to each other, making this system highly effective for RNA imaging.

Light-up RNA Aptamer Principles

A light-up RNA aptamer sequence is genetically engineered into an RNA sequence of interest, by commonly used techniques for recombinant DNA. This is then transcribed into RNA by the host cell machinery. A high affinity cognate fluorogen is then added, which binds the Light-up RNA 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.

Spinach aptamer and the fluorogen DFHBI are examples, discovered in the laboratory of S. R Jaffrey (see Paige et al, 2011). 4-hydroxybenzylidene imidazolinone (HBI) only produces fluorescence when bound to a scaffold to form a specific tertiary interaction that stabilizes the molecule. The difluoro derivative of HBI (DFHBI) shows a remarkably enhanced green fluorescence when in presence of a 98 nucleotide-long RNA named as Spinach aptamer. DFHBI is cell permeable and does not induce cytotoxicity or phototoxicity. When incubated in living mammalian cells, the trafficking of 5S rRNA with a Spinach aptamer tag may be observed by fluorescence microscopy. Spinach 2 aptamer and Broccoli aptamer have subsequently been developed by systematic mutagenesis of the Spinach aptamer to improve intracellular folding of the RNA aptamers.

 Principles of Light-up Aptamer

Figure 1- Light-up RNA 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 RNA Aptamer Technology

Studying transcription and translation at an RNA level has traditionally been done using fluorescent in situ hybridization (FISH), which requires chemically fixing the sample. The RNA tagging system MS2 was developed for RNA imaging in live cells. 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 RNA 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, so 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 RNA Aptamer Range

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

Fluorogen Light-up RNA Kd (nM) λEx (nM) λEm (nM) Absorption coefficient (ε) (M-1/cm) Complex quantum yield (φ) Brightness
DFHBI #5609 Spinach Aptamer 540 469 501 24,300 0.72 17.5
DFHBI #5609 Squash Aptamer 45 452 503 24,200 0.71 17.18
DFHBI #5609 Broccoli Aptamer 360 472 507 29,600 0.94 27.8
DFHBI 1T #5610 Spinach 2 Aptamer 560 482 505 31,000 0.94 29.1
DFHBI 2T Spinach2 Aptamer 1300 500 523 29,000 0.12 3.48
TO-1 Mango Aptamer 3 510 535 77,500 0.14 10.85
BI #7466 Broccoli Aptamer 51 470 505 33,600 0.67 22.5
OBI Red Broccolli Aptamer 23 541 590 47,300 0.67 31.7
DFHO #6434 Corn Aptamer 70 505 545 29,000 0.25 7.25
DFHO #6434 Squash Aptamer 54 495 562 24,600 0.60 14.76
DFHO #6434 Orange Broccoli Aptamer 230 513 562 34,000 0.28 9.5
DFHO #6434 Red-Broccoli Aptamer 206 518 582 35,000 0.34 11.90
SiRA 2 SiRA 430 649 662 86,000 0.98 84.3
TMR-DN RhoBAST 15 564 590 96,000 0.57 54.7
HBC 530 #7277 Pepper Aptamer 3.5 485 530 65,300 0.66 43.1

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 RNA Aptamer Applications

Light-up RNA technologies are suitable for RNA imaging and functional RNA analysis of mRNA, miRNA, tRNA and snoRNA in live cells. Light-up RNA 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 RNA Aptamer Applications: A) Monitoring gene expression and RNA imaging: the RNA of interest is tagged with a light-up RNA aptamer (light blue structure) and it is expressed in cells; a fluorogen (orange pentagon) binds to the RNA aptamer tag and becomes highly fluorescent for RNA imaging and RNA analysis. 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: the RNA of interest binds and stabilizes the light-up RNA aptamer conformation, allowing the fluorogen to bind; the 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* your copy today!

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


Fluorescent Probes and Dyes Research Product Guide

Fluorescent Probes and Dyes Research Product Guide

This product guide provides a background to the use of Fluorescent Probes and Dyes, as well as a comprehensive list of our

  • Fluorescent Dyes
  • Dyes for Flow Cytometry
  • Fluorescent Probes
  • Anti-fade Reagents
  • Bioluminescent Substrates
  • Fluorogenic Dyes for Light-Up Aptamers
  • Fluorescent Probes for Imaging Bacteria
  • TSA Reagents for Enhancing IHC, ICC & FISH Signals