An Isosteric and Fluorescent DNA Base Pair Consisting of 4‐aminophthalimide and 2,4‐diaminopyrimidine as C‐Nucleosides

Abstract

A 13mer DNA duplex containing the artificial 4-aminophthalimide:2,4-diaminopyrimidine (4AP:DAP) base pair in the central position was characterized by optical and NMR spectroscopy. The fluorescence of 4AP in the duplex has a large Stokes shift of Δλ=124 nm and a quantum yield of Φ =24 %. The NMR structure shows that two interstrand hydrogen bonds are formed and confirms the artificial base pairing. In contrast, the 4-N,N-dimethylaminophthalimide moiety prefers the syn conformation in DNA. The fluorescence intensity of this chromophore in DNA is very low and the NMR structure shows no significant interaction with DAP. Primer-extension experiments with DNA polymerases showed that not only is the 4AP C nucleotide incorporated at the desired position opposite DAP in the template, but also that the polymerase is able to progress past this position to give the full-length product. The observed selectivity supports the NMR results.

New Size‐Expanded Fluorescent Thymine Analogue: Synthesis, Characterization, and Application

“…[40][41][42] Therefore, the intramolecular charget ransfer of diox T might contribute to the enhancemento fq uantum yield upon the formation of double-stranded DNA. [40][41][42] Therefore, the intramolecular charget ransfer of diox T might contribute to the enhancemento fq uantum yield upon the formation of double-stranded DNA.…”

“…In previousr eports, some fluorescentn ucleobases sucha s DMA T, 2PyG,4 AP,a nd 8-DEA-tC showede nhanced fluorescence depending on their location in double-stranded DNA, au nique phenomenon that could be explained by ac harge-separated excited state. [40][41][42] Therefore, the intramolecular charget ransfer of diox T might contribute to the enhancemento fq uantum yield upon the formation of double-stranded DNA. Both this enhancement and the high quantum yield of diox T-containing DNA duplexes will allow the development of many useful tools.…”

Energetics and dynamics of the non-natural fluorescent 4AP:DAP base pair

“…12,13,[25][26][27] In this context, 4-aminophthalimide (4AP), a well-known organic dye, 28 has also been introduced as a covalent base modification in DNA since 2013, 29 receiving however increased attention only recently. 30,31 4AP is highly fluorescent and exhibits strong solvatofluorochromism. 32 It has indeed been used as a solvatofluorochromic probe in polymers, enzyme complexes, and cyclodextrins.…”

“…30 To circumvent this problem, in a recent study, 4AP was incorporated into the same 13-mer DNA duplex by shifting the attachment of the sugar moiety from the C6 to the C5 position. 31 4AP attached through C5 was annealed both with DAP and with the four natural DNA bases (A/T/G/C). 31 Notably, changing the glycosidic connection of 4AP from C6 to C5 significantly improves its structural and functional compatibility with DNA by (i) preventing the formation of different H-bonding conformations for the 4AP:DAP base pair; 31 (ii) increasing the melting temperature of the DNA duplex by roughly 1 degree as compared to the C6-attached 4AP (from 53.6 to 54.4 1C); (iii) making the DNA duplex with a central 4AP-DAP pair more stable (in terms of melting temperature) than analogous duplexes with a central 4AP paired to natural bases; (iv) making the 4AP nucleotide efficiently recognized and incorporated by DNA polymerases, which can also progress past 4AP to give full-length products.…”

“…31 Notably, changing the glycosidic connection of 4AP from C6 to C5 significantly improves its structural and functional compatibility with DNA by (i) preventing the formation of different H-bonding conformations for the 4AP:DAP base pair; 31 (ii) increasing the melting temperature of the DNA duplex by roughly 1 degree as compared to the C6-attached 4AP (from 53.6 to 54.4 1C); (iii) making the DNA duplex with a central 4AP-DAP pair more stable (in terms of melting temperature) than analogous duplexes with a central 4AP paired to natural bases; (iv) making the 4AP nucleotide efficiently recognized and incorporated by DNA polymerases, which can also progress past 4AP to give full-length products. 31 To complement the reported experimental studies and rationalize the impact of the above non-natural bases on the structure, stability and dynamics of nucleic acid structures, we decided to perform quantum mechanics (QM) calculations along with classical molecular dynamics (MD) simulations, which both proved extremely useful to characterize base pairing and stacking in natural nucleic acids. [39][40][41][42][43][44][45][46][47][48][49][50][51] QM calculations were initially focused on the geometry and energetics of the 4AP:DAP non-natural pair, which were compared to those of standard A:T and G:C Watson-Crick pairs.…”