Impacts of sequence and structure on pyrrolocytosine fluorescence in RNA

Abstract

Fluorescence spectroscopy encompasses many useful methods for studying the structures and dynamics of biopolymers. Applications to nucleic acids require the use of extrinsic fluorophores such as fluorescent base analogs (FBAs), which mimic the native bases but have enhanced fluorescence quantum yields. In this work, we use multiple complementary methods to systematically investigate the sequence- and structure-dependence of the fluorescence of the FBA pyrrolocytosine (pC) within RNA. We demonstrate that pC is typically brightest in conformations in which it is base-stacked but not base-paired, properties that distinguish it from more widely used FBAs. This effect is strongly sequence-dependent, with adjacent adenosine and cytidine residues conferring the greatest contrast between stacked and unstacked structures. Structural heterogeneity was resolved in single-stranded RNA and fully complementary and mismatched double-stranded RNA using time-resolved fluorescence measurements and fluorescence-detected circular dichroism spectroscopy. Double-stranded contexts are distinguished from single-stranded contexts by the presence of inter-strand energy transfer from opposing bases, while base-paired pC is distinguished by its short excited state lifetime. This work will enhance the value of pC as a structural probe for biologically and medicinally significant RNAs by guiding the selection of labeling sites and interpretation of the resulting data.

Graphical Abstract

Figure 1.

(A) Structures of cytosine (top) and pC (bottom) forming Watson–Crick base pairs with guanine. (B) Normalized fluorescence emission spectra of pC riboside (pCr), pCC, CpCC, and Strand 1. (C) FQY of all oligonucleotides, normalized to value of 1 for pCr in buffer. Error bars show the standard deviation across three separately prepared samples. “Di” indicates a dinucleotide with the base indicated by “X” to the 3′ side of pC. “Tri” indicates a trinucleotide with the base indicated by “X” to the 5′ and 3′ sides of pC. Samples prepared in 30% ethanol are indicated by blue. (D) Normalized fluorescence excitation spectra of pCr, ApCA, CpCC, GpCG, and UpCU. Approximate peak absorbance wavelengths of each native base (260, 271, 254, and 262 nm for A, C, G, and U, respectively) and pC itself (265 nm) are indicated by the tips of the arrows.

Figure 2.

TCSPC measurements of pCr and di- and trinucleotides. (A) pCA and ApCA, (B) pCC and CpCC, (C) pCG and GpCG, (D) pCU and UpCU. Bi-exponential (A–C) or tri-exponential (D) fits are overlaid in light gray. A typical IRF is plotted in gray.

Figure 3.

(A) Fluorescence per unit absorbance of pCr and CpCC in buffer (lighter shades) or buffer + 30% EtOH (darker shades). (B) TCSPC of pCr in buffer or buffer + 30% EtOH. (C) TCSPC of CpCC in buffer or buffer + 30% EtOH. Multi-exponential fits are overlaid in light gray (mono-exponential for pCr in 30% EtOH and bi-exponential for all others).

Figure 4.

CD and processed FDCD spectra of di- and trinucleotides. (A) Spectra of pCA. (B) ApCA. (C) pCC. (D) CpCC. (E) pCG. (F) GpCG (inset: close-up of long-wavelength region). (G) pCU. (H) UpCU (inset: close-up of long-wavelength region).

Figure 5.

Steady-state fluorescence spectra and TCSPC of single-stranded (“Strand 1″), double-stranded (“1:2 GGG”) and mismatched (“1:2 GUG”) 21-mers. (A) Normalized fluorescence excitation spectra of ss, ds, and mm RNA at 20°C. Emission was detected at 460 nm. The approximate peak absorption wavelengths of guanosine (solid arrow), cytidine (dotted arrow), and a secondary absorption peak of pC (dashed arrow) are indicated. (B) Normalized fluorescence emission spectra of ss, ds, and mm RNA at 20°C under 340 nm excitation. (C) IRF and fluorescence decays of ss, ds, and mm RNA at 20°C. Fits (bi-exponential for Strand 1 and 1:2 GGG; 1 gamma for 1:2 GUG) are overlaid in lighter color. (D) IRF and fluorescence decays of ss, ds, and mm RNA at 70°C. 1 exponential + 1 gamma fits are overlaid in lighter color.

Figure 6.

(A) CD spectra of ss, ds, and mm 21-mers. The inset shows a close-up of the 300–410 nm range. (B) Processed FDCD spectra of ss, ds, and mm (purple) 21-mers.