Fluorescent nucleobases as tools for studying DNA and RNA

Abstract

Understanding the diversity of dynamic structures and functions of DNA and RNA in biology requires tools that can selectively and intimately probe these biomolecules. Synthetic fluorescent nucleobases that can be incorporated into nucleic acids alongside their natural counterparts have emerged as a powerful class of molecular reporters of location and environment. They are enabling new basic insights into DNA and RNA, and are facilitating a broad range of new technologies with chemical, biological and biomedical applications. In this Review, we will present a brief history of the development of fluorescent nucleobases and explore their utility as tools for addressing questions in biophysics, biochemistry and biology of nucleic acids. We provide chemical insights into the two main classes of these compounds: canonical and non-canonical nucleobases. A point-by-point discussion of the advantages and disadvantages of both types of fluorescent nucleobases is made, along with a perspective into the future challenges and outlook for this burgeoning field.

Figure 1. Structures of natural nucleobases and fluorescent analogues

a, Nucleobases carry hereditary information through specific hydrogen bonding and steric interactions; that is, adenine with thymine/uracil and guanine with cytosine. Adjacent nucleobases interact with each other via π-stacking, thus forming the double-helix conformation. b, Potential chemical modification sites of canonical pyrimidines. Examples cover ring substitution, conjugated linker extension and ring fusion. Yellow shading highlights modified structures and the purple bonds and atoms are base-pairing moieties. c, Potential chemical modification sites of canonical purines. Examples include ring structure modification, substitution and ring fusion. d, Examples of non-canonical fluorescent nucleobases, showing larger sizes that allow coverage of the redder end of the spectrum. Colours indicate approximate emission hue: ~370–390 nm, violet; 410–430 nm, blue; 440–470 nm, cyan; 480–510 nm, green; 520–540 nm, yellow; 550–600 nm, orange; >600 nm, red.

Figure 2. A chronicle of fluorescent nucleobase development

In 1969, Stryer reported the first canonical fluorescent nucleobases, featuring 2AP and formycin; in 1972, Leonard synthesized the first non-canonical fluorescent nucleobase, etheno-dA. The fluorescent nucleobase family gradually expanded during the following two decades, including m⁵K reported in 1990 by McLaughlin and pteridines by Hawkins in 1995. C-glycosidic nucleobases with hydrocarbon fluorophores directly attached to the sugar were first reported by Kool in 1996. Since 2000, the number of fluorescent nucleobases has increased markedly, featuring notable examples such as BPP by Saito and Okamoto, tC by and Wilhelmsson, dU^Phen (Hocek) and thieno-appended analogues of Tor. Dozens of non-canonical nucleobases have been introduced since 2010 (examples by Kool shown here). Hundreds of fluorescent nucleobases have been reported thus far and their numbers and applications are still rapidly expanding.

Figure 3. Molecular strategies for design of fluorescent purine analogues

a, Numbering of the purine skeleton. b, Potential modification sites of adenine and guanine. Expansion and modification mostly occur on positions 2, 5 and 8 of adenine and positions 5, 6, 7 and 8 of guanine, as indicated by the arrows. c, Examples of purine ring fusion modifications. d, Examples of extending the purine scaffold through conjugated linkers. e, Examples of purine substituent modifications. f, Examples of purine ring fusions. Native base-pairing groups are rendered in pink, whereas fluorescence modifications are shown in blue.

Figure 4. Molecular strategies for design of fluorescent pyrimidine analogues

a, Numbering of the pyrimidine scaffold. b, Potential modification sites of thymine and cytosine. Expansion and modification mostly occur on positions 5 and 6 of thymine and positions 4, 5 and 6 of cytosine, as indicated by the arrows. c, Modification of the pyrimidine substituents at position 5. This category includes changes at only one especially versatile chemical position. d, Modification of the pyrimidine substituents at positions 5 and 6. The extensions form a new six-membered ring containing heteroatoms, which can be further expanded for fluorescent modification. e, Extension of pyrimidines via conjugated linkers. f, Pyrimidine ring fusion.

Figure 5. Molecular features and examples of non-canonical fluorescent nucleobases

a, Nucleobases composed of aromatic hydrocarbons. b, Nucleobases composed of planar heterocyclic fluorophores. The lack of hydrogen bonding and weaker π-stacking are compensated by versatile energy states brought from the heteroatoms. These fluorophores contribute to a broader spectrum of emission wavelengths. Functional groups can be added to expand functionality, such as metal binding. c, Examples of nucleobases based on hydrocarbons. d, Photoreaction of adjacent phenethynylpyrene nucleobases yields a colour change in emission. The left image shows phenylalkynylpyrene excimer emission whereas the right image shows pyrene monomer emission, both excited at 360 nm. e, C-glycosidic nucleobases based on known fluorophores. f, Simple heterocyclic nucleobases used in the detection of DNA repair activity. g, Nucleobase pairs based on shape complementarity. Although they lack hydrogen bonding, the conformation of these bases counterpart each other, thus forming unnatural base pairing.

Figure 6. Methods for incorporating fluorescent nucleobases into DNA or RNA

a, Direct oligonucleotide synthesis via synthesizer and phosphoramidite chemistry. The main steps of DNA synthesis are: (1) removal of 4,4′-dimethoxytrityl (DMT) under acidic conditions; (2) coupling of the nucleoside phosphoramidite with the growing chain; and (3) oxidation of the phosphorus linkage. b, Post-synthesis modification using mild coupling methods or gene-editing methods. Fluorescent nucleobases equipped with organoboron or organostannane groups are coupled to halogen-labelled nucleobases in DNA strands. Gene-editing and ligation methods enzymatically join smaller labelled strands to make longer ones. c, Direct enzymatic incorporation using fluorescent nucleoside triphosphate derivatives. When the fluorescent nucleobases are labelled in the primers or supplied as free nucleobases in the pool, polymerases that recognize them can incorporate the fluorescent nucleobases into DNA sequences. TP, triphosphate; dNTP, deoxynucleoside triphosphate.

Figure 7. Examples of applications of fluorescent nucleobases

a, Fluorogenic sensing of a demethylation enzyme, ALKBH3. The emission of pyrene is initially quenched by the positive charge of 1-methylated adenine (m1A). When ALKBH3 demethylates m1A, the quenching effect is removed and a signal is generated. b, Fluorogenic analysis of adenine-to-inosine RNA editing enzyme. The emission maxima of the thiolated adenine (^thA) and inosine (^thI) are different. Hence by measuring the intensity of ^thA and ^thI at their respective maximal wavelengths, the activity of the A-to-I enzyme can also be measured. dsRNA, double-stranded RNA; ssDNA, single-stranded DNA. c, Kinetic and thermodynamic investigation of the effects of mercury on DNA metabolism. The fluorescent thymine can chelate mercury with another thymine ring and link DNA strands. This can be used to probe mercury metabolism in vivo and to study the effects of mercury on DNA status. k₁, strand displacement rate constant; k₋₁, reverse reaction rate constant. d, Visualization and analysis of human concentrative nucleoside transporters (hCNTs). The fluorescent nucleoside can enter the plasma membrane through the transporters, thus allowing the measurement of the transport activity. TGF-β1, transforming growth factor β1.