Enzymatic Incorporation of a Coumarin–Guanine Base Pair

Abstract

Previous expansions beyond nature's preferred base‐pairing interactions have utilized either nonpolar shape‐fitting interactions or classical hydrogen bonding. Reported here is a hybrid of these systems. By replacing a single N−H with C−H at a Watson–Crick interface, the design space for new drug candidates and fluorescent nucleobase analogues is dramatically expanded, as demonstrated here by the new, highly fluorescent deoxycytidine mimic 3‐glycosyl‐5‐fluoro‐7‐methoxy‐coumarin‐2′‐deoxyribose (dCC). dGTP is selectively incorporated across from a template dCC during enzymatic DNA synthesis. Likewise, dCC is selectively incorporated across from a template guanine when dCC is provided as the triphosphate dCCTP. DNA polymerase I (Klenow fragment) exhibited about a 10‐fold higher affinity for dCCTP than dCTP, allowing selective incorporation of dCC in direct competition experiments. These results demonstrate that a single C−H can replace N−H at a Watson–Crick‐type interface with preservation of functional selectivity and enhanced activity.

A Highly Fluorescent Nucleobase Molecular Rotor

“…Fluorescent nucleobase analogs (FBAs) provide powerful tools for probing nucleic acids’ structures, dynamics, and binding interactions. , Compared to conjugated fluorophores and intercalating dyes, FBAs have the advantage of highly precise positioning within RNA and DNA structures. However, fluorescence quenching of FBAs by neighboring residues via photoinduced electron transfer (PET) can dramatically limit their brightness. − For example, small electron-poor systems such as 2-aminopurine (2AP) are reductively quenched by purines, and electron-rich systems such as MD A are oxidatively quenched by pyrimidines . The brightest, previously reported fluorescent nucleobase analogs, such as tC o (ε × Φ ≈ 2,000 cm –1 M –1 ), , avoid PET quenching by having HOMO–LUMO energy levels that are intermediate between the HOMO of guanine and the LUMO of thymidine (Figure and Figure S1).…”

Enzymatic synthesis of biphenyl-DNA oligonucleotides

“…[75] Enzymatic synthesis of modified oligonucleotides is an alluring strategy since a single modified nucleotide could be employed for the construction of numerous oligonucleotides potentially containing multiple modified C-nucleotides irrespective of the length of the sequence. [76,77] Our study shows that the C-nucleotide analog dBphTP can be used as a building block for the enzymatic construction of modified oligonucleotides under certain experimental conditions. We have shown that the presence of consecutive modified residues on template oligonucleotides can partially improve the incorporation efficiency of dBphMP into DNA even though the yields remain modest (~50% conversion was obtained with the Dpo4 polymerase).…”

Single-molecule fluorescence detection of a tricyclic nucleoside analogue

“…Next to its closest competitors on brightness, pentacyclic adenine pA (ε 387 = 15,300 M −1 cm −1 and Φ em,420 = 0.66 in water) and a coumarin nucleoside (ε 315 = 38,000 M −1 cm −1 and Φ em,455 = 0.11 in water), the absorption and emission of ABN in aqueous solution are red-shifted by more than 50 and 80 nm, respectively. 16,17 Owing to the conjugated push-pull system in the design of ABN, the absorption and emission wavelengths are the longest known for a FBA designed to be capable of Watson-Crick hydrogen bonding. Recorded emission spectra in water using excitation wavelengths ranging from 310-500 nm are nearly superposable, as are excitation spectra recorded for emission wavelengths spanning 500-650 nm ( Figures S20 and S21).…”

“…[11][12][13][14] However, few nucleobase analogues have extinction coefficients > 10 4 with Φ em > 0.3; most are approximately an order of magnitude dimmer than conventional fluorophores such as Alexa Fluor 488 and rhodamine B. 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.…”