Benzoquinazoline Derivatives as Substitutes for Thymine in Nucleic Acid Complexes. Use of Fluorescence Emission of Benzo[g]quinazoline-2,4-(1H,3H)-dione in Probing Duplex and Triplex Formation

Abstract

Triple helix formation obeys structural features that do not allow accommodation of every double-stranded sequence; it requires the occurrence of homopurine stretches. A further constraint comes from the weak energy of interaction between the third strand and the double-stranded target. In an attempt to design bases leading to increased stability of triplexes, we explored the ability of modified bases with an extended aromatic domain to increase third strand binding through stacking interactions. We report here the use of benzo[g]- and benzo[f]quinazoline-2,4-dione-(1H,3H)-dione as substitutes for thymine in the canonical TAT triplet. The synthesis and characterization of the beta nucleoside derivatives of benzoquinazolines are described. Triplex-forming oligonucleotides containing these modified bases have been prepared, and their ability to form triplexes has been evaluated by UV absorption-monitored thermal denaturation measurements. Benzo[g]quinazoline and benzo[f]quinazoline formed triple-stranded structures with slightly decreased stabilities. In addition, benzo[g]quinazoline revealed strong fluorescence emission properties which can be used to monitor selectively the formation of triple-helical structures. Annealing of benzo[g]quinazoline to complementary strands did not produce any fluorescence modification. But when it was introduced into the Hoogsteen strand of PyPuPy complexes, the fluorescence intensity was reduced and the emission maximum was shifted to short wavelengths.

Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation

“…It specifically Watson-Crick base-pairs with thymine or uracil and has proven its utility as a probe to monitor local conformational changes in nucleic acid structure, for example, in catalytic RNA and upon formation of nucleic acid-protein complexes (Millar 1996;O'Neill and Barton 2002;Walter et al 2002;Patel and Bandwar 2003;Roy 2003;Bradrick and Marino 2004;Clerte and Hall 2004). Several fluorescent pyrimidine analogs also have been developed, although they have not yet reached a similar level of popularity (Inoue et al 1985;Wu et al 1990;Godde et al 1998;Wilhelmsson et al 2001).…”

Photophysical Characterization of Fluorescent DNA Base Analogue, tC

“…[21][22][23][24] Like 2-AP, these probes have a high fluorescent quantum yields in their free form, 88% and 70% respectively, 22 but exhibit a substantial reduction in quantum yields, up to 25-fold, upon incorporation into oligonucleotides. Another new group of fluorescent base analogues with promising properties are the benzoquinazolines, [25][26][27] which were originally designed to increase the stability of triple helices. Interestingly, they display high fluorescence quantum yield both free and when included in oligonucleotide duplexes.…”

Use of the Fluorescent Nucleoside Analogue Benzo[g]quinazoline 2′-O-Methyl--D-ribofuranoside to Monitor the Binding of the HIV-1 Tat Protein or of Antisense Oligonucleotides to the TAR RNA Stem-Loop

“…Unfortunately, neither of these techniques is a measure of the true binding constant in solution under equilibrium conditions. We have now developed a new assay for the measurement of Tat binding to TAR in solution based on the change of fluorescence of the base analogue benzo[g]quinazoline-2,4(1H,3H)-dione (BgQ; see 1) [30] [31] incorporated into the chemically synthesized model TAR stem-loop 2.…”

“…Thus U 24 is an obvious site for incorporation of a fluorescent base. BgQ is a thymine analogue that has a strong fluorescence-emission intensity which becomes quenched upon stacking within DNA duplexes and triplexes [30a] [31]. The excitation spectrum of BgQ as a 2'-deoxynucleoside shows three maxima at 253 (major), 294, and 360 nm and a broad emission spectrum centered between 420 and 440 nm [30a].…”