Synthesis, oligonucleotide incorporation and base pair stability of 7-methyl-8-oxo-2′-deoxyguanosine (original) (raw)
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Nucleic Acids Research, 2007
This article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tC O , is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tC O is thoroughly characterized with respect to its photophysical properties and structural performance in single-and double-stranded oligonucleotides. The lowest energy absorption band at 360 nm (e ¼ 9000 M 21 cm 21 ) is dominated by a single in-plane polarized electronic transition and the fluorescence, centred at 465 nm, has a quantum yield of 0.3. When incorporated into doublestranded DNA, tC O shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tC O , on average, the brightest DNA-incorporated base analogue so far reported. Furthermore, it base pairs exclusively with guanine and causes minimal perturbations to the native structure of DNA. These properties make tC O a promising base analogue that is perfectly suited for e.g. photophysical studies of DNA interacting with macromolecules (proteins) or for determining size and shape of DNA tertiary structures using techniques such as fluorescence anisotropy and fluorescence resonance energy transfer (FRET).
Nucleic Acids Research, 1999
We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (U P). The intramolecular triplex A 6-L-T 6-L-(U P) 5 T (L = two octanediol residues) shows a single UV-melting transition which is >20_ higher than that of the parent triplex A 6-L-T 6-L-T 6 at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by U P , in a pH dependent fashion. Triplex stability increases by ∼12 K for each T to U P substitution. Quantitative footprinting studies have examined the interaction of three U P-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5′-AGGAAG-AGAAAAAAGAA. At pH 5.0, the U P-containing oligonucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (U P) 6 CU P T binds ∼1000-fold more tightly than the unmodified oligonucleotide T 6 CTT. Oligonucleotides containing fewer U P residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.
Synthesis and site-specific incorporation of a simple fluorescent pyrimidine
Nature protocols, 2007
We describe procedures for the synthesis of a fluorescent pyrimidine analog and its site-specific incorporation into a DNA oligomer. The 5'-protected and 3'-activated nucleoside 4 is synthesized in three steps with an overall yield of 40%. Site-specific incorporation into a DNA oligomer occurs with greater than 88% coupling efficiency. This isosteric fluorescent DNA analog can be used to monitor denaturation of DNA duplexes via fluorescence and can positively detect the presence of abasic sites in DNA duplexes. The total time for synthesis of the phosphoramidite 4 is about 75 h, whereas the total time for site-specific incorporation of nucleoside 2 into an oligonucleotide and purification of the corresponding oligonucleotide is about 114 hours.
2011
To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (A T) and its photophysical characterization inside DNA. A T shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single-and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, A T shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that A T only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that A T shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, A T shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.
Novel Nucleoside Analogues with Fluorophores Replacing the DNA Base
Helvetica Chimica Acta, 1999
We describe the preparation and fluorescence properties of a set of new nucleosides in which a known hydrocarbon or oligothiophene fluorophore replaces the DNA base at C(1) of the deoxyribose moiety (see 3a-f). These compounds are potentially useful as probes in the study of the structure and dynamics of nucleic acids and their complexes with proteins. In addition, they may find use as fluorescent labels for nucleic-acid-based biomedical diagnostics methods. The fluorophores conjugated to deoxyribose at C(1) in the α-D-form include terphenyl, stilbene, terthiophene, benzoterthiophene, and pyrene. Also included is a non-fluorescent spacer in which cyclohexene replaces the DNA base. The nucleosides are derived from brominated fluorophore precursors and Hoffer's 2-deoxy-3,5-di-O-(p-toluoyl)-D-ribofuranosyl chloride. The emission maxima of the free nucleosides range from 345 to 536 nm. Also described are the 5′-(dimethoxytrityl) 3′-O-phosphoramidite derivatives 5a-f, suitable for incorporation into oligonucleotides by automated synthesizers. We have taken a related approach to developing new fluorescent labels for DNA [10]. Rather than modifying an existing DNA base, however, we have simply replaced it by another flat aromatic structure, i.e., by a hydrocarbon rather than by a heterocyclic N