Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle

Abstract

Fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and pyrrolo-C (PyC), have been extensively used to study nucleic acid local conformational dynamics in bulk experiments. Here we present a proof-of-principle approach using 2AP and PyC fluorescence at the single-molecule level. Our data show that ssDNA, dsDNA, or RNA containing both 2AP and PyC can be monitored using single-molecule fluorescence and a click chemistry immobilization method. We demonstrate that this approach can be used to monitor DNA and RNA in real time. This is the first reported assay using fluorescent nucleotide analogs at the single-molecule level. We anticipate that single 2AP or PyC fluorescence will have numerous applications in studies of DNA and RNA, including protein-induced base-flipping dynamics in protein-nucleic acid complexes.

Scheme 1

Figure 1

Single-molecule 2AP fluorescence. (a) Prism-based, total internal reflection microscope with 325 nm excitation for 2AP fluorescence. 2AP is linked through a long, flexible linker to surface-immobilized DNA. (b) Fluorescence image of individual FREE-2AP molecules (bright spots). (c) Fluorescence trajectory of FREE-2AP reveals the absence of blinking and single-step photobleaching. (d) Histogram of photobleaching times (t_Phb).

Figure 2

Fluorescence of single 2AP molecules incorporated into DNA. (a) Three fluorescence trajectories of 2AP in ssDNA. Fluorescence intensity jumps between a bright and a dark state. (b) Dwell-time histograms of 2AP-ssDNA ON (τ_ON) and OFF (τ_OFF) states (112 molecules). (c) Experimental setup combining 532 and 325 nm excitation for Cy3 and 2AP, respectively. (d) First, Cy3 is excited at 532 nm and fluorescence is monitored to localize dsDNA molecules. Next, 2AP is excited at 325 nm. (e) SM trajectories of 2AP in dsDNA reveal a small subpopulation of molecules with ON–OFF transitions. (f) Dwell time histograms of 2AP-dsDNA ON (τ_ON) and OFF (τ_OFF) states (182 molecules).

Figure 3

Single-molecule fluorescence of 2AP across an abasic site in dsDNA. Two populations are observed: (a) a major population exhibits ON–OFF (blinking) fluorescence transitions and (b) a minor population exhibits static fluorescence with single-step photobleaching. (c) Static population fraction increases with Mg²⁺ ion concentration.

Figure 4

Single-molecule fluorescence of PyC in tRNA^Pro. (a) PyC-labeled tRNA^Pro is composed of a synthetic 3′-16-mer (red) annealed to a transcribed 5′-57-mer (black). The 16-mer strand includes PyC (C74) and 5′-N₃ for surface immobilization. (b) Fluorescence image of the surface-immobilized 16-mer strands. Each bright spot corresponds to an individual PyC-labeled 16-mer. (c) SM trajectory for a single PyC-16-mer molecule and (d) for an annealed/folded PyC-tRNA^Pro.