Base-specific photocleavage of DNA induced by nanosecond U.V. pulsed laser radiation or methylene blue sensitisation

Abstract

Excitation of DNA in neutral solution either with short-duration, intense pulses of excimer laser (248 nm) radiation or with l o w intensity red-light (633 nm) sensitisation by methylene blue causes cleavage of the DNA backbone selectively at guanine.The development of photochemical procedures which could complement (or even replace) current DNA sequencing methods is of considerable interest. One appoach could be to use base-specific photocleavage of DNA to replace the chemical methods of Maxam and Gilbert.1 However as it is known that the photochemistry of DNA is dominated by photoreactions of the bases (e.g. thymine dimerisation) and that breaking of the sugar-phosphate backbone is a very inefficient process,2 such a strategy might seem unlikely. We report here that excitation of DNA with short-duration pulses of U.V. radiation causes such cleavage, base-selectively at guanine.Irradiation of DNA with 'picosecond' 314 or 'nanosecond' 5-7 pulses of U.V. radiation has recently been shown to cause rupture of the DNA backbone. Our present experiments have been carried out with a standard (Lambda Physik EMG 102) KrF excimer laser used to provide U.V. pulses (248 mm; pulse-width 22 ns; energy 70 mJ). The laser beam is focussed down so as to excite uniformly a small (4-50 pl) sample of DNA solution contained in a tube of diameter 1.5 or 2.3 nm. By suitable focussing and filtering of the beam it is possible to control the intensity of the pulse within the range 1 x 109-5 x 1012 W m-2. The cleavage of DNA by this radiation has been studied using covalently closed circular (plasmid pBR322) DNA. As expected from the earlier reports,sJj it was found that low intensity light caused little change in the electrophoretic behaviour of this DNA, whereas at the same fluence but at intensities greater than lo9 W m-2 single strand nicking occurred and the open circular and linear forms were produced. This intensity dependence indicates that the bond-rupture reaction is initiated by multi-photon (probably consecutive two photon) excitation, as previously observed with picosecond e~citation.3.~ At an intensity of 5 x 1010 W m-2, the quantum yield for single strand cleavage was found to be ca. 1-2 x 10-5. The base specificity of the cleavage was determined using a 5'-terminus 32P-labelled DNA sample (4.3 kbase EcoRI-Hind I11 restriction fragment of pBR322) and subsequent electrophoresis on sequencing gels in parallel with the products formed by conventional were irradiated for 30 min. Samples were then dried, resuspended in 80% formamide, denatured for 5 min at 95 "C, and electrophoresed as above. It may be noted that the denaturing conditions used (2-5 min at 90°C) might possibly be sufficient to induce some additional cleavage at sites where the bases had been modified during irradiation. before electrophoresis. Sections of the autoradiograph obtained in these experiments are shown in Figure 1 (Lanes 3-5). It may be noted from the pattern of the bands that cleavage is observed only at guanine and that a band is present for each guan...

The Reaction of Triplet 2‐Methyl‐1,4‐Naphthoquinone (Menadione) with DNA and Polynucleotides

“…Photosensitized strand breakage studies have been reported where a variety of sensitizers have been used but usually in the presence of oxygen (28,41,42). The contribution of the photoinduced electron transfer reaction and singlet oxygen in the strand breakage pathway is not easily evaluated.…”

Simultaneous Photoconjugation of Methylene Blue and cis-Rh(phen)2Cl2+ to DNA via a Synergistic Effect †

“…to form 8-oxo-7, 8-dihydroguanine, is unable to block DNA replication (52). It is also worth noting that the quantum efficiency for MB-sensitized DNA breaks (1-3 ϫ 10 Ϫ7 ) (27,28) is several orders of magnitude lower than the values in Table 3. However, these values for strand cleavage are comparable to the covalent binding data of the dye to calf thymus DNA, which we estimate to be ca 1 ϫ 10 -6 .…”

“…In particular, dye incorporation was evidenced by a permanent blue coloration of the DNA. Though MB-photosensitized nicking of DNA is well documented in the literature (25)(26)(27)(28), covalent binding of MB to DNA is unprecedented. No such blue coloration was observed for DNA that was mixed with dye and complexed and kept in the dark or in DNA isolated from the irradiation of MB in the absence of BISPHEN.…”

“…MB has recently been investigated for its photovirucidal activity in blood (22)(23)(24) where it operates by, predominantly if not exclusively, targeting viral nucleic acid (25,26). DNA inactivation proceeds primarily through strand cleavage (27,28); there are no reports in the literature of the formation of MB-DNA covalent linkages.…”

LASER‐INDUCED STRAND BREAK FORMATION IN DNA and POLYNUCLEOTIDES

“…The latter result indicates that peroxyl radicals of the sugar are precursors of strand breakage. Kelly and coworkers (Blau et al, 1987;Croke et al, 1988) have proposed that the guanine radical cation is the reactive species leading to ssb of DNA.…”