Synthesis, oligonucleotide incorporation and fluorescence properties in DNA of a bicyclic thymine analogue

Abstract

Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids. In our overall mission to develop bright and useful FBAs for all natural nucleobases, herein we describe the synthesis and thorough characterization of bicyclic thymidine (bT), both as a monomer and when incorporated into DNA. We have developed a robust synthetic route for the preparation of the bT DNA monomer and the corresponding protected phosphoramidite for solid-phase DNA synthesis. The bT deoxyribonucleoside has a brightness value of 790 M−1cm−1 in water, which is comparable or higher than most fluorescent thymine analogues reported. When incorporated into DNA, bT pairs selectively with adenine without perturbing the B-form structure, keeping the melting thermodynamics of the B-form duplex DNA virtually unchanged. As for most fluorescent base analogues, the emission of bT is reduced inside DNA (4.5- and 13-fold in single- and double-stranded DNA, respectively). Overall, these properties make bT an interesting thymine analogue for studying DNA and an excellent starting point for the development of brighter bT derivatives.

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

“…In water, diox T showed absorption at 325 nm and fluorescence at 388 nm with high quantum yield (0.36), resulting in very bright fluorescence ( Φϵ =3602 m −1 cm −1 ). This brightness value is higher than previous reported fluorescent thymine/uridine analogues such as DMA T ( Φϵ =87 m −1 cm −1 ), 1PydU and 2PydU ( Φϵ ≈500 m −1 cm −1 in MeOH), bT ( Φϵ =790 m −1 cm −1 ), xT ( Φϵ =1020 m −1 cm −1 in MeOH), th dT ( Φϵ =1984 m −1 cm −1 ), and TPAU ( Φϵ =2200 m −1 cm −1 ) . The absorption and emission spectra of diox T change depending on the solvent.…”

“…It is generally known that fluorescent nucleobases are strongly influenced by their nearest‐neighbor bases . To select suitable sequences for the application, the characteristics of diox T ‐containing single‐ and double‐stranded DNA (5′‐CGTCCGTNXNACGCACGC‐3′, where X= diox T , Table and Table S1 in the Supporting Information) were investigated.…”

“…The comparable thermal stability, hypochromic effect, and redshift of diox T in duplex DNA indicate that diox T is well stacked between base pairs and does not flip to the outside of the duplex. Furthermore, the overall brightness of diox T in ds DNA shows an average of 622 m −1 cm −1 (Table S2 in the Supporting Information), and this value is significantly higher than that of bT (average Φϵ =28 m −1 cm −1 in duplex) and th dT ( Φϵ =90 m −1 cm −1 in ds DNA, 5′‐GCGCGA th dT A th dT A th dT AGGAGC‐3′), but less than fluorescent adenine and guanine analogues such as tC° ( Φϵ =2000 m −1 cm −1 ) and 6MI ( Φϵ =1700 m −1 cm −1 ) …”

“…This may be attributed to the good integration of diox T because of strong stacking interactions between neighboring bases,e ven thought he T m is al ittle lower than that of the native duplex because of electronic repulsion between the phosphate group and dioxole ring of diox T ( Figure S10 in the Supporting Information). The comparable thermal stability, hypochromic effect, and redshift of diox T in duplex DNA indicatet hat diox T is well stacked between base pairs and does not flip to the outside of the duplex.F urthermore, the overall brightnesso f diox T in ds DNA shows an average of 622 m À1 cm À1 (Table S2 in the Supporting Information), and this value is significantly higher than that of bT (average Fe = 28 m À1 cm À1 in duplex) and th dT (Fe = 90 m À1 cm À1 in ds DNA, 5'-GCGCGA th dTA th dTA th dTAGGAGC-3'), [20,52] but less than fluorescent adenine and guaninea naloguess uch as tC8 (Fe = 2000 m À1 cm À1 )a nd 6MI (Fe = 1700 m À1 cm À1 ). [35] It is generally known that fluorescent nucleobases are strongly influenced by their nearest-neighbor bases.…”

“…The modified duplex containing diox T had as lightly lower T m (average 1.9 8C) than the unmodified duplex. [36][37][38][39] To select suitables equences for the application,t he characteristics of diox T-containing single-and double-stranded DNA (5'-CGTCCGTNXNACGCACGC-3',w here X = diox T,T able 3 and Ta ble S1 in the Supporting Information) were investigated. This may be attributed to the good integration of diox T because of strong stacking interactions between neighboring bases,e ven thought he T m is al ittle lower than that of the native duplex because of electronic repulsion between the phosphate group and dioxole ring of diox T ( Figure S10 in the Supporting Information).…”