Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design

Abstract

Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (εΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor

“…2CNqA has a high fluorescence quantum yield as a monomer in aqueous solution (42%) as well as a high brightness value ( ϵ Φ F = 4500 M −1 cm −1 ) ( 32 ). Here, we investigate its fluorophore properties inside DNA (Table 1 ) and will return in the RNA FRET study below to a smaller photophysical characterisation of 2CNqA in RNA employing the RNA FRET sequences (Table 3 ).…”

“…The 2CNqA-DNA monomer has its lowest-energy absorption ( λ Abs,Max ) and emission maxima ( λ Em,Max ) at 356 and 480 nm, respectively ( 32 ). The corresponding absorption maxima of 2CNqA in ssDNA and dsDNA are found at virtually the same positions as for the monomer and are centered at approximately 358 and 356 nm, respectively, across the different combinations of neighbouring bases (Figure 4 , left, Table 5 and Supplementary Figures S4 and S5 ).…”

“…The molar absorptivities of DNA oligonucleotides were approximated by the linear combination of molar absorptivities of individual nucleotides and multiplied by 0.9 to account for the base-stacking interactions. The following molar absorptivities at 260 nm were used: ϵ A = 15300 M −1 cm −1 , ϵ C = 7400 M −1 cm −1 , ϵ G = 11800 M −1 cm −1 , ϵ T = 9300 M −1 cm −1 ( 39 ), ϵ 2CNqA = 14600 M −1 cm −1 ( 32 ) and ϵ qAnitro = 12900 M −1 cm −1 ( 30 ). For RNA oligonucleotides, molar absorptivities at 260 nm were calculated using the Integrated DNA Technologies (IDT) OligoAnalyzer ( http://eu.idtdna.com ), with the modified base replaced by its unmodified counterpart, and correcting for the difference in molar absorptivity at 260 nm between unmodified base and 2CNqA ( ϵ 2CNqA = 14600 M −1 cm −1 ) or tC nitro ( ϵ tCnitro = 9700 M −1 cm −1 ) ( 33 ).…”

“…Time-dependent density functional theory (TDDFT) methodology was used as described previously ( 32 ) to computationally predict the orientation of the lowest energy transition dipole moment of 2CNqA and qA nitro . These values and the experimentally determined orientation for the transition dipole moment of the tC nitro acceptor were used together with nucleic acid B- and A-form, respectively, to calculate the phase angle.…”

“…The brightness of pA inside DNA is still respectable (on average 1400 M −1 cm −1 ) and close to the highest average brightness inside DNA (2000 M −1 cm −1 , as reported for tC O ) ( 31 ), but compared with commonly used external fluorophores, which in general have brightness values well above 10 4 M −1 cm −1 , there is still room for improvement. In our recent study on outer ring substituents of qA, the 2CNqA nucleobase (Figure 1 ), bearing a cyano (CN) group in the 2-position of the outer ring, was found to possess a high and stable quantum yield in a variety of solvents ( 32 ). Therefore, in our search for an adenine analogue which, like tC O ( 31 ), would exhibit a high and stable quantum yield regardless of sequence context, rendering a high brightness and therefore applicability as a nucleic acid label and as an interbase FRET donor ( 30 , 33 , 34 ), we decided to investigate 2CNqA in nucleic acids.…”