Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation

Abstract

The ability to routinely detect fluorescent analogs of nucleobases at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylaminesubstituted extended-6-aza-uridine (DMA th aU). We show that DMA th aU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMA th aU undergoes two-photon (2P) absorption at low excitation powers, switching to threephoton (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analog, which facilitated the first singlemolecule detection of an emissive nucleoside with multiphoton excitation.

Mechanical characterization of base analogue modified nucleic acids by force spectroscopy

“…Moreover, by combining base analogues and force microscopy we have pointed out the direction towards interbase singlemolecule FRET for nucleic acids in optical tweezers experiments with fluorescence readout. We have previously shown this to be a promising technique to study structure and dynamics of nucleic acids in bulk [41][42][43] and with the improved photophysics of fluorescent base analogues recently reported, for example pA and 2CNqA, 32,44,45 we suggest that interbase-FRET in optical tweezers will soon become an important tool in single-molecule studies. FR, FW and MW designed the project.…”

“…23,24 Moreover, base modifications and mismatches were recently studied using force spectroscopy. [25][26][27][28][29] The literature on using synthetic base analogues in single-molecule experiments is very limited, [30][31][32] but we envision that it will be a powerful tool for studying structure and dynamics of nucleic acids. Here, we combine optical tweezers and thermal melting to study how structural stability, (un)folding thermodynamics and intermolecular forces are affected by substitution of C with tC.…”

Single-molecule fluorescence detection of a tricyclic nucleoside analogue

“…Recent studies have demonstrated multiphoton excitation as a promising approach to the sensitive detection of fluorescent base analogues. 19,20 Two-photon excitation of pA in oligonucleotides allowed detection close to the single-molecule level, whereas one-photon (1P) excitation resulted in rapid photobleaching. 19 In a later study, the base analogue DMA th aU was detected as a free nucleoside at the single-molecule level for the first time via multiphoton excitation with a brightness of 7 kHz/molecule following threephoton excitation.…”

“…19 In a later study, the base analogue DMA th aU was detected as a free nucleoside at the single-molecule level for the first time via multiphoton excitation with a brightness of 7 kHz/molecule following threephoton excitation. 20 Using an experimental setup consisting of a broadband ultrafast laser with dispersion compensation, 19,20 we found that ABN could be optimally excited via a 2P process ( Figure 5A). The 2P brightness was measured using fluorescence correlation spectroscopy (FCS) and found to match that of DMA th aU at 7 kHz/molecule ( Figure 5B).…”

“…8,9,[15][16][17] This lack of brightness has rendered them largely unsuitable for singlemolecule fluorescence studies. [18][19][20] Furthermore, with the current rapid development of spatially-resolved transcriptomics and genomics, there is clearly a future need for fluorescent analogues that can act as effective single-molecule probes. 21,22 Conventional fluorophores typically exhibit superior optical properties to fluorescent nucleobase analogues, primarily because of their larger extinction coefficients-sometimes in excess of 10 5 -but they need not be larger molecules.…”