A New Paradigm for the Development of Fluorescent RNA Imaging Technology Using Synthetic Molecules

In vivo protein imaging technology has been greatly advanced over the last decade by the development of green fluorescent protein (GFP) and its derivatives as investigational tools. Typical applications of GFP involve tagging a protein of interest so that the protein expression and localization can be imaged as the fusion protein is being expressed in live cells.

In contrast to protein imaging, RNA imaging in live cells lags far behind. The currently available in vivo RNA imaging methods are 1) fluorescence in situ hybridization,1,2 involving fluorescently labeled oligonucleotides as probes that bind to complementary RNA sequences in fixed cells; 2) a molecular beacon, an alternative oligonucleotide-based probe labeled with a fluorophore and a quencher at each end;3,4 3) a protein-based RNA biosensor system that requires that GFP be fused to the RNA-binding protein MS2 and that the RNA of interest be linked to many copies of MS2-binding RNA sequence;5 and 4) the quenched autoligating fluorescence resonance energy transfer (FRET) probes.6 All of these methods involve the use of biopolymers as fluorescent probes, which may be a major concern since biopolymers are prone to rapid degradation.

New RNA imaging technology would significantly enhance our understanding of rarely investigated transcription kinetics and RNA trafficking in live cells. The focus of this article is the development of a new paradigm for RNA imaging technology. The long-term goal of the research program described is to tag an RNA of interest with a fluorescence-inducing RNA aptamer (reporter RNA) at the DNA level and investigate its transcription and RNA trafficking using RNA chemosensors (small molecules that report specific RNA) in live cells. Analogous to GFP, this fluorescence-inducing aptamer will be used universally as a tagging (investigational) tool to study various genes.

RNA imaging with small molecules

A desirable RNA sensor should be cell permeable, stable, and fluorescent only when bound to specific RNA. Some of the benefits of such a chemosensor are as follows:

  • Organic synthesis allows for tuning chemical and photophysical properties of chemosensors
  • Chemosensors enable better quantification of RNA than biosensors due to the linear correlation between the emission signal and quantity of RNA–sensor complex
  • Cell-permeable RNA chemosensors allow for both noninvasive introduction of the molecule to live cells and animals and microinjections to a specific area inside cells
  • Chemosensors are less likely to be degraded in live cells than biosensors, permitting longer time-scales for live cell imaging; of particular interest is the potential use of an RNA chemosensor to monitor the dynamics of RNA concentrations during apoptosis, which presumably cannot be studied using currently available technology
  • The stability and cell permeability of chemosensors provide a more steady concentration in biological samples
  • Chemosensor-bound RNA is expected to behave more similarly to natural RNA than biomacro-molecule-bound RNA.

Figure 1 - Overall strategy for developing reporter RNA systems.

Figure 1 illustrates a new paradigm for small-molecule-based RNA sensors developed in the University of Pittsburgh Department of Chemistry laboratory (Pittsburgh, PA).7 In step 1, using synthetic organic chemistry, a quencher (Q) is covalently linked to a fluorophore (F) to provide a fluorogenic compound (F-Q). By virtue of the photoinduced electron transfer (PET; for a description of PET, please see below) process from Q to F, the fluorescence of F-Q is quenched. In step 2, RNA aptamers for Q (but not for F) are raised and isolated by means of in vitro RNA selection. In step 3, fluorescence spectroscopic analysis of the aptamer–F-Q mixture is performed; these aptamers suppress the quenching function of Q, thereby restoring the fluorescence signal from F.

The physical chemistry underlying the paradigm: PET

Figure 2 - Ability of PET to quench fluorescence.

A desirable probe fluoresces when bound to a specific biomolecule, while generating no signal when unbound. This fluorescence on-off switch can be implemented by the PET mechanism (Figure 2).8 When a quencher is proximate to a fluorophore and its highest occupied molecular orbital (HOMO) energy level is sufficiently high (Figure 2, left), PET occurs from the quencher to the excited fluorophore without fluorescence emission. According to the Rehm-Weller equation (Eq. [1]),9 lowering Eox increases the fluorophore’s fluorescence signal (Figure 2, right). This theory is well-established10 and has been used to rationally design chemosensors for biologically important ions.11–16

ΔGPET = EoxEred – ΔE00wp       (1)

where Eox = oxidation potential of a quencher, Ered = reduction potential of a fluorophore, ΔE00 = the singlet excited energy, and wp = the work term for the charge separation state.

Fluorescence quenching is controlled not only by potential energy but also by the concentration of quencher as indicated by the Stern-Volmer equation (Eq. [2]). This equation shows that as [Q] increases, fluorescence quantum yield Φ drops dramatically. This was taken into account and it was decided to append two quenchers to a fluorophore.

Φ0/Φ = (1 + KD[Q]) (1 + Ks[Q])        (2)

where Φ0 and Φ are the quantum yields in the absence and presence of quencher, respectively; KD and KS are the Stern-Volmer constants of dynamic and static quenching, respectively; and [Q] is the concentration of quencher.

Results and discussion

Synthesis of 2′,7′-dichlorofluorescein derivatives

Figure 3 - Synthesis of compound 1 from DCF.

As part of step 1 (Figure 1), commercially available 2′,7′-dichlorofluorescein (DCF) was converted to compound 1 and others (not shown) in one step (Figure 3), and their quantum yields were determined.17 From these studies, compound 1 emerged as a potential sensor due to its lowest background emission (Φ1 = 0.025) presumably by the PET process from aniline nitrogen atoms.

In vitro RNA selection and fluorescence analysis

Figure 4 - a) Structure of affinity column. b) Concentration-dependent fluorescence induction of compound 1 (1 μM) with its aptamer (graphs taken from the literature). (Reproduced with permission from Ref. 7.)

The approach depicted in Figure 1 is unique because it requires RNA that binds to a quencher and not to a fluorophore. To identify such aptamers, the quencher of 1, N-(p-methoxy-phenyl)piperazine was linked to agarose resin (Figure 4a).7 The resulting matrix was used as bait in a series of in vitro RNA selections using an N70 RNA library (70 nucleotides were randomized), and three RNA aptamers were characterized.7 It was gratifying to see that one of the three RNA aptamers enhanced the fluorescence of compound 1 in a concentration-dependent manner (Figure 4b). The addition of N-(p-methoxyphenyl) piperazine was found to antagonize this fluorescence induction effect, suggesting that the aptamer induces the fluorescence of compound 1 by the mechanism envisioned (Figure 1, step 3). The mixture of the N70 RNA library did not enhance fluorescence, excluding nonspecific fluorescence enhancement by RNA. Despite the high concentrations of RNA used in the titration experiments (20 μM to induce threefold fluorescence enhancement), this result provides the proof of concept for the approach illustrated in Figure 1.7