A Lucifer‐Based Environment‐Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs

Abstract Fluorescence‐based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment‐sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore (1,8‐naphthalimide) at the 5‐position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells—poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.


Photophysical characterization of PNA base analog 5 UV-Vis absorption:
Samples of analog 5 (25.0 µM) were prepared in water, dioxane and their mixtures (25% dioxane, 50% dioxane, 75% dioxane in water). All solutions contained 2.5% DMSO. Steady-state fluorescence: Emission spectra of analog 5 (5.0 µM) in water, dioxane and their mixtures (25% dioxane, 50% dioxane, 75% dioxane in water) were recorded by exciting the samples at respective longest absorption maximum (Table 1). Excitation and emission slit widths were kept at 1 and 3 nm, respectively. All solutions contained 0.5% DMSO. Time-resolved fluorescence: Excited-state lifetime of Lucifer PNA analog 5 (5.0 μM) in various solvents was determined using TCSPC instrument (Horiba Jobin Yvon, Fluorolog-3). Samples were excited using a 371 nm LED source (IBH, UK, NanoLED-371L) and fluorescence signal at respective emission maximum was collected. Lifetime measurements were performed in duplicate and decay profiles were analyzed using IBH DAS6 analysis software. Fluorescence intensity decay profiles were found to be biexponential with  2 (goodness of fit) values very close to unity.

Quantum yield determination of emissive PNA base analog 5
The quantum yield of emissive PNA analog 5 in different solvents relative to cumarin 153 standard was determined using the following equation. [S4] Φ F(x) = (A s /A x ) (F x /F s ) (n x /n s ) 2 Φ F(s) Where s is the standard, x is PNA analog (5), A is the absorbance at excitation wavelength, F is the area under the emission curve, n is the refractive index of the solvent, and Φ F is the quantum yield. Quantum yield of cumarin 153 in acetonitrile is 0.56. [S5] Quantum yield of  Table 1 for details.

Solid-phase synthesis of control unmodified and naphthalimide-modified PNA oligomers
Model 15mer control unmodified (69) and fluorescently-modified (6X9X) PNA oligomers were synthesized on MBHA resin using Boc-protected PNA monomers and Boc-protected fluorescent naphthalimide PNA acid 4 (Scheme S1) according to our reported protocol. [S6] Fluorescent poly(T) PNA probe 10 was synthesized by SPPS protocol on Rink amide resin using Fmoc-protected aeg-thymine and fluorescent naphthalimide-modified uracil PNA acid f (Scheme S2). The detailed protocol for the synthesis of poly(T) PNA probe 10 and unmodified poly(T) PNA 13 using Fmoc-chemistry is given below. To enhance aqueous solubility of the PNA oligomers, two lysine residues were attached at the C-terminus of all the PNA oligomers. In a glass sintered flask, Rink-amide resin (250 mg, 0.65 mmol/g) was swelled in CH 2 Cl 2 (8 mL) for 12 h. The solvent was then removed, and the resin was treated with 20% piperidine in DMF (6 mL) for 10 min to remove Fmoc-group from the resin. This step was repeated two more times. The resin was then washed sequentially with DMF (3 x 3 mL), CH 2 Cl 2 (3 x 3 mL), and DMF (3 x 3 mL). The resin was dried under nitrogen flow for few minutes. The coupling reaction was performed in dry DMF (1.8 mL) with Fmoc-Lys(Boc)-OH (36 mg, 1.0 equiv. to obtain a loading of 0.35 mmol/g) in the presence of HOBt (1.0 equiv.), HBTU (1.0 equiv.) and DIPEA (1.0 equiv.) for 7-9 h at RT. The resin was further washed with DMF (3 x 3 mL), CH 2 Cl 2 (3 x 3 mL), and DMF (3 x 3 mL). Next, remaining amino groups on the resin was capped with acetic anhydride (1.0 mL) in pyridine (1.0 mL) for 1 h at RT. This step was repeated two more times, and the resin was then washed with DMF (3 x 3 mL), CH 2 Cl 2 (3 x 3 mL), and DMF (3 x 3 mL) and dried under nitrogen flow for few minutes. The resin was treated with 20% piperidine in DMF (5 mL) for 10 min to remove Fmoc-group. This step was repeated two more times, and the resin was washed with DMF (3 x 3 mL), CH 2 Cl 2 (3 x 3 mL), and DMF (3 x 3 mL). Then coupling reaction was performed in dry DMF (1.8 mL) with Fmoc-Lys(Boc)-OH (3.0 equiv.) in the presence of HOBt (3.0 equiv.), HBTU (3.0 equiv.) and DIPEA (3.0 equiv.) for 7-9 h at RT. The resin was then washed with DMF (3 x 3 mL), CH 2 Cl 2 (3 x 3 mL), and DMF (3 x 3 mL). The resin was dried under nitrogen flow for few minutes.
Above lysine loaded Rink-amide resin (25 mg, 0.35 mmol/g) was swelled in CH 2 Cl 2 (2 mL) for 2 h in glass sintered flask. The solvent was removed and the resin was treated with 20% piperidine in DMF (1.0 mL) for 10 min to remove Fmoc-group as mentioned above. The resin was then washed with DMF (3 x 3 mL), CH 2 Cl 2 (3 x 3 mL), and DMF (3 x 3 mL). The coupling reaction was performed in dry DMF (0.6 mL) with Fmoc-thymine PNA monomer (3.0 equiv.) in the presence of HOBt (3.0 equiv.), HBTU (3.0 equiv.) and DIPEA (3.0 equiv.) for 6 min at 65 °C using microwave peptide synthesizer (Note: coupling reaction for fluorescent PNA monomer f was carried out at RT for 7-9 h). The resin was washed again, and the coupling, Fmoc deprotection, and washing steps were repeated in the cycle as mentioned above to synthesize the desired PNA sequence. Also, a double-coupling reaction was performed for only fluorescently-modified PNA monomer f in the presence of HOBt (3.0 equiv.), HBTU (3.0 equiv.) and DIPEA (3.0 equiv.) for 7-9 h at RT.
Cleavage procedure: The dried resin (20 mg) was transferred to a glass vial and treated with H 2 O (10 L) and anisole (10 L) in an ice bath for 10 min. TFA (380 L) was then added to the above mixture and was stirred for 1.5 h at room temperature. The resin was filtered, and the filtrate was concentrated and precipitated as a white solid by adding cold diethyl ether (1 mL). The solvent was decanted, and the crude product was dissolved in autoclaved water and purified by RP-HPLC.

S18
Gel shift assay: A series of solution of Cy5-(dT) 30 (1.0 µM) containing increasing concentrations of poly(A) RNA ON 12 (100 nM to 5 µM) was prepared as mentioned above. The volume of samples was 10 μL. To the samples was added 10 μL of loading buffer (10 mM Tris-HCl, pH 7.2 and 10% glycerol). The samples were loaded onto a 15% nondenaturing polyacrylamide gel containing 100 mM NaCl. A peristaltic pump was used to maintain the ionic conditions in both the chambers. The gel electrophoresis was performed at ~4 C with a constant power supply of 14 W for ~10 h. The bands were visualized by using Typhoon-TRIO+ imager by illuminating the gel using fluorescence of Cy5. The bands were quantified using GeneTools image analysis software (Syngene) and K d was determined as explained below. K d determination: Fraction bound (F N ) versus log of poly(A) RNA 12 concentration plots were fitted using Hill equation (OriginPro 8.5.1) to determine the apparent binding constant K d for the binding of 12 to Cy5-(dT) 30 . [S8] F N = F 0 F i F 0 and F i are band intensity in the absence of RNA ON 12 and at each titration point, respectively. Normalized F N was used in the plot. F s is band intensity at saturated amount of RNA ON 12. L is concentration of ON 12. n is the Hill coefficient or degree of cooperativity associated with the binding.