The rare base 4-thiouridine (s4U), present in various transfer RNA (tRNA) molecules from Escherichia coli, occupies usually the strategically important 8 th position between the double helices of the acceptor and the dihydrouridine stems of the cloverleaf. This unusual base is largely used as an intrinsic build-in probe for RNA conformational and RNA(DNA)–protein interaction studies through triplet excited state photochemistry related to covalent adducts formation. Here, by applying laser transient absorption saturation spectroscopy, we measured the intersystem crossing yield ϕST and the excited triplet state absorption εT−T of s4U within tRNA. While the incorporation of s4U in tRNA induced appreciable changes in the latter, no important variation of the intersystem crossing yield was observed, which is in contrasts with the published data.Rijetka baza 4-thiouridine (s4U), koju nalazimo u raznim prijenosnim molekulama RNA (tRNA) Escherichie coli, obično uzima strateški važan 8. položaj između dviju zavojnica primatelja i dihidrouridinskih stapki lista djeteline. Ta se neobična baza najviše rabi kao unutarnja ugradbena proba za proučavanje interakcija konformalne RNA s RNA(DNA)–proteinima preko fotokemije tripletnog uzbuđenog stanja, povezanog sa stvaranjem kovalentnih adukata. U ovom smo radu primjenom laserske prijelazne apsorpcijske spektroskopije sa zasićenjem mjerili prinos međusustavnih prijelaza ϕST i apsorpciju tripletnog stanja εT−T s 4U u tRNA. Dok ugrađivanje s4U u tRNA uzrokuje prilične promjene u tRNA, nismo opazili promjene prinosa međusustavnih prijelaza, što nije u skladu s objavljenim rezultatima

Angelov, D.

Shaqiri, Z.

Vidolova-Angelova, E.

English

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Printed ISSN 1330–0008Online ISSN 1333–9125CD ISSN 1333–8390CODEN FIZAE4MEASUREMENT OF THE INTERSYSTEM CROSSING YIELD OF s4UWITHIN tRNA BY TIME-RESOLVED ABSORPTION BLEACHINGZ. SHAQIRI1, D. ANGELOV and E. VIDOLOVA-ANGELOVAaInstitute of Solid State Physics, Bulgarian Academy of Sciences,72, Tsarigradsko Shaussee blvd., 1784 Sofia, BulgariaaE-mail address: evidol@issp.bas.bgReceived 6 March 2008; Accepted 19 November 2008Online 28 January 2009The rare base 4-thiouridine (s4U), present in various transfer RNA (tRNA)molecules from Escherichia coli, occupies usually the strategically important 8thposition between the double helices of the acceptor and the dihydrouridine stemsof the cloverleaf. This unusual base is largely used as an intrinsic build-in probe forRNA conformational and RNA(DNA)–protein interaction studies through tripletexcited state photochemistry related to covalent adducts formation. Here, by apply-ing laser transient absorption saturation spectroscopy, we measured the intersys-tem crossing yield ϕST and the excited triplet state absorption εT−T of s4U withintRNA. While the incorporation of s4U in tRNA induced appreciable changes in thelatter, no important variation of the intersystem crossing yield was observed, whichis in contrasts with the published data.PACS numbers: 87.14.Gg, 87.15.Kg UDC 539.199, 539.196Keywords: 4-thiouridine (s4U), RNA conformational and RNA(DNA)–protein interac-tion, laser transient absorption saturation spectroscopy, intersystem crossing yield, excitedtriplet state absorption1. IntroductionAbout 70% of the different transfer RNA (tRNA) molecules content in the 8thposition the rare nucleotide 4-thiouridine (s4U). The substitution of the heavy sulfurfor oxygen in position 4 of s4U result in drastic changes in the spectral properties,namely the long wavelength absorption band maximum is shifted from 260 to 3301Present address: Faculty of Natural and Mathematical Sciences, 38000, Prishtina, Kosovo,e-mail address: zekiinst@yahoo.comFIZIKA A (Zagreb) 17 (2008) 3, 109–116 109shaqiri et al.: measurement of the intersystem crossing yield of s4U . . .nm [1, 2]. While the free s4U or tRNA incorporated is not fluorescent, it exhibitsat room temperature an unusual emission peak at 550 nm attributed to phospho-rescence from the long lived triplet state T1 [1 – 4]. The values of the intersystemcrossing yield ϕST and other spectroscopic constants of monomer s4U have beendetermined by using different methods. In early works, the value ϕST ∼ 2 10−2 [3]was found by actinometry, using anthracene as standard, and the energy transferdetermined triplet-triplet extinction value εTT520 = 5104 M−1cm−1 in acetonitrile[3, 4]. However, the latter value turned out, to be an order of magnitude overesti-mated, thus leading to equivalent underestimation of ϕST. By contrast, the valueϕST ∼ 1 was found in Ref. [5] by transient absorption spectroscopy, which is ob-viously overestimated due to neglecting the triplet-triplet absorption. Finally, themost reliable value of ϕST = 0.67 was determined [6, 7] by using several differenttechniques, as the singlet oxygen phosphorescence, laser optogalvanic spectroscopyand time resolution thermal lens.NMR data suggest the formation of non Watson-Crick base pairs between s4Uand adenine at position 13 in tRNA [8]. This has been directly confirmed by pho-tochemistry showing the formation of covalent photocrosslinks between s4U andcytosine at position 13 [9, 10]. Therefore, the spectroscopic properties of s4U mightbe expected to vary by changes in the secondary and tertiary structures of tRNAinduced by salt and/or temperature. Indeed, T1 state lifetime increases from 200 nsto 6.6 µs, depending on mono- and di-valent cation concentration [7, 8, 11], leadingto an increase of the phosphorescence yield in a temperature-dependent way.While the shifted spectral maximim enables selective excitation of s4U withintRNA, the higher intersystem crossing yield is useful for efficient population ofits triplet state. Although, the value of ϕST for free s4U was firmly established,there is little information whether this value varies upon incorporation in tRNAand its structural transitions. The only data [12] provided show an importantdecrease of the intersystem crossing yield for s4U incorporated in tRNA. Here wehave measured the intersystem crossing yield of s4U within tRNA by transientabsorption saturation spectroscopy and found its value similar to that for free s4Uand independent on salt and temperature-induced conformation changes.2. Experimental section2.1. Productss4U and total tRNA used in the measurements are from Sigma and BoeringerMannheim, respectively. All other chemicals were obtained from Merck. All solu-tions were prepared using bi-distilled de-ionized water.To completely remove divalent cations present in the commercial sample, tRNAwas dissolved in water at 10 mg/ml and successively dialyzed three times at 4◦Cagainst 250 ml of 25 mM phosphate buffer, pH 7.2 - 20 mM EDTA - 0.15 M NaCl.This was followed by three 2h dialyses against 25 mM phosphate buffer, pH 7.2 - 5mM NaCl. The dialysed stock solution was aliquoted and stored at -20◦C.110 FIZIKA A (Zagreb) 17 (2008) 3, 109–116shaqiri et al.: measurement of the intersystem crossing yield of s4U . . .The solution concentration was adjusted by optical density readings using thefollowing values of the molar extinctions at the maximum of the 330 – 335 nmabsorption band: 22000 M−1cm−1 and 14660 M−1cm−1 for the free s4U and tRNA,respectively [7].2.2. Laser flash-photolysisThe laser flash photolysis set-up used (Fig. 1) was based on the third harmonic(λ = 355 nm, pulse duration τp = 35 ns) of a Nd:YAG laser (JK) as an excitationsource. The relative laser pulse energy was measured by large area UV photodiode.The transient absorption was monitored at the perpendicular direction by usingthe collimated beam of a pulsed Xe flash-lamp. The dimensions of the laser andthe lamp beams at the entrance of the 0.5 cm× 2 cm quartz cell were adjusted to0.3 cm× 0.3 cm and 0.25 cm× 0.1 cm, respectively, by using diaphragms. The tem-perature was set up at 20◦C by means of water circulating pump monitored by athermocouple. The probing beam was focussed on the slit of a Spex single-gratingmonochromator and the signal recorded by the model R 928 Hamamatsu photo-multiplier connected to a Hewlett Packard 1 Gb/s digital oscilloscope interfaced toa PC via the IEEE 488 protocol.Fig. 1. Scheme of the laser flash-photolysis set-up. M - monochromator, PM -photomultiplier, DO - digital oscilloscope, PC - personal computer.3. Results and discussionAn overview of the literature shows that for free s4U the value ϕST = 0.67± 15% is the most reliable. By contrasts, the only published value ϕST = 0.35 fors4U within tRNA is questionable [12] since excited state absorption was neglected.We have measured this value applying a direct method of ground state absorptionsaturation. The method consists in measuring the optical density at the end of laserpulse as a function of exciting laser intensity. For the purpose, the above describedtechnique of nanosecond laser flash photolysis has been used.FIZIKA A (Zagreb) 17 (2008) 3, 109–116 111shaqiri et al.: measurement of the intersystem crossing yield of s4U . . .The optical density change at the maximum of the absorption band (λ = 330nm) was calculated from the experimentally measured steady state photomultipliercurrent before (A) and at the end the laser pulse (∆A) using the formula∆D = − log(AA + ∆A)(1)Thus, taking into account the 0.3 cm irradiated pathlength in the direction of theprobing beam (see the irradiation geometry in Fig. 1), the transient optical densityin a 1 cm pathlength is given byD = D0 −∆D0.3= D0 − 3.33 log(AA + ∆A)(2)where D0 is the initial optical density of the solution measured by a conventionalspectrophotometer for the 1 cm path length.Let us analyze the reasons for the optical density change in the maximum ofabsorption band λ = 330 − 340 nm (see Fig. 2). Since the excited S1 populationis negligible due to its short lifetime of about 10−12 s, the experimentally observedbleaching of the first absorption band around 330 nm is due uniquely to the de-pletion of the ground S0 state at the expense of the population of the triplet T1state. The lifetime of the latter is considerably longer than the laser pulse duration.By contrast to Ref. [12], we do not neglect the triplet state absorption and con-sider it to play a role in the ground state bleaching. In addition, our observation ofthe lack of significant transient absorption from hydrated electrons at 700 nm (notshown) demonstrates that the biphotonic ionization is negligibly small. Probablythe excitation energy of about ∼ 6 eV by two 355 nm photons absorption is belowthe ionization limit of s4U. Therefore, the contribution of radicals generated bytwo-quantum mechanism is negligible in the 330 – 340 nm region of absorption.Under these conditions for the 1 cm pathlengthD = ε1C1 + ε2C2 (3)where D is the transient optical density at the maximum of the absorption band, ε1and ε2 are the molar extinctions of the ground S0 and the excited triplet T1 state,and C1 and C2 are the population concentrations of these states, respectively.The rate equations for the molecule concentration in approximation of thin layerexcitation are as follows:dC1dt= −ϕSTσC1I(t), t ≤ 0, I(t) = 0, C1 = C0, C2 = 0,C1 + C2 = C0, 0 < t ≤ τp, I(t) = I, (4)where σ is the ground state absorption at 355 nm, φST = τ−1ST/(τ−1ST+ τ−11) is theintersystem crossing yield, I(t) is the laser intensity (photons/(cm2s)), C0 is the112 FIZIKA A (Zagreb) 17 (2008) 3, 109–116shaqiri et al.: measurement of the intersystem crossing yield of s4U . . .initial ground state concentration, τp is the laser pulse duration (35 ns), τ1 and τTare the singlet and triplet state lifetimes respectively (τ1 ≪ τp ≪ τT ) [1, 12].Fig. 2. Simplified energy diagram used to describe the experiment. S0 - the groundstate, S1, T1 - the excited singlet and triplet states, respectively. ε1 and ε2 are themolar extinctions of S1 and T1, respectivey, σ - the absorption cross-section of theS0 → S1 transition (λ = 355 nm).The solutions of the rate equations at the end of the laser pulse t = τp are:[S0]= C0 exp(−φSTσE)[T1]= C0[1 − exp(−φSTσE)](5)where E is the laser pulse dose (E = I τp) (photons/cm−2). By replacing (5) into(3) we obtainD = D0[(1 −ε2ε1)exp(−φST σE) +ε2ε1](6)The experimental curves D = f(E) for s4U free and within tRNA are presentedin Fig. 3. Taking into account that values of ε1(334) = 22000 (14660) M−1cm−1and σ(355) = 2.88 10−17 (3.44 10−17) cm2 for free s4U and tRNA-incorporated areFIZIKA A (Zagreb) 17 (2008) 3, 109–116 113shaqiri et al.: measurement of the intersystem crossing yield of s4U . . .known, the transient optical density depends on two unknown parameters ϕST andε2 only. Their values have been determined by least squared fitting to experimen-tally determined values of D as a function of the laser pulse dose E (D = f(E)),using Eq. (6). The experimental curves D = f(E) at zero delay time from thebleaching pulse for s4U, free and within tRNA, are presented in Fig. 3.Fig. 3. Transient absorbance D(334) at the end of the laser pulse versus the laserfluence E in the absence (A) or presence (B) of 1.5 mM MgCl2.It should be noted that the laser pulse energy has been measured in relativeunits by using non-calibrated photodiode. Therefore, we used the known valueϕST = 0.67 ± 15 % for free s4U for calibration of the laser pulse dose. In this waywe obtain ϕtRNAST= k ϕs4UST, where k = 0.85±10 %. Therefore, ϕtRNAST= 0.57±30 %.The obtained value of ϕtRNASTis close to that of free s4U. By contrasts, the authors ofRef. [12] obtained k = 0.3 and ϕtRNAST= 0.33. This discrepancy with Ref. [12] is dueto the neglecting of different extinction values of free and tRNA-incorporated s4Uin the calculation of ϕtRNASTfrom experimental data in Ref. [12] and importantlyalso in neglecting the excited triplet state absorption which is considerable fortRNA. The method of the ground state absorption saturation allowed not only todetermine ϕST, but also to estimate the T-T excited state absorption. We obtainedεT−T340= 1700 and 4100 M−1cm−1 for s4U free and within tRNA, respectively, theerror being about 10 – 15 %. It is to be noted that experimental data on this valuefor s4U within tRNA are lacking in the literature. Therefore, the value of εT−T2/ε1in the maximum of absorption band (330-340 nm) is 0.086 for free s4U and 0.30 fors4U within tRNA.Finally, the saturation curves of tRNA do not show any changes with the ad-dition of divalent ions that is known to induce structural changes. Therefore, thevalue of ϕST for s4U not only shows little dependence on its incorporation in tRNA,but is independent also on its secondary and tertiary structure.114 FIZIKA A (Zagreb) 17 (2008) 3, 109–116shaqiri et al.: measurement of the intersystem crossing yield of s4U . . .4. ConclusionUsing laser transient absorption spectroscopy, we have shown that the inter-system crossing yield of the rare nucleoside s4U remains unchanged when it isincorporated in tRNA as well as under divalent ion induced folding of the latter.This finding is useful in designing experiments that use the s4U as a built-in probefor 3D analysis of RNA and ribonucleic complexes.AcknowledgementsThis investigation was partly supported by the Ministry of Education and Sci-ence, Bulgaria - the National Fund for Science (grant N 1402/2004).References[1] A. Favre, Photochem. Photobiol. 18 (1973) 135.[2] N. Shalitin and J. Feitelton, J. Chem. Phys. 59 (1973) 1045.[3] C. Salet, R. V. Bensasson and A. Favre, Photochem. Photobiol. 38 (1983) 521.[4] R. V. Bensasson and E. J. Land, Trans. Faraday Soc. II (1971) 1904.[5] S. J. Milder and D. S. Kliger, J. Am. Chem. Soc. 107 (1985) 7365.[6] K. Heihoff, R. W. Redmond, S. E. Braslavsky, M. Rougee, C. Salet, A. Favre andR. V. Benssason Photochem. Photobiol. 51 (1990) 635.[7] A. Favre, 4-Thiouridine as intrinsic photoaffinity probe of nucleic acid structure andnucleic acid-protein interactions, in Bioorganic Photochemistry, Vol. I, ed. H. Morrison,J. Wiley & Sons, New York (1990) pp. 379-425.[8] N. Shalitin and J. Feitelson, Biochemistry 15 (1976) 2092.[9] E. I. Hyde and B. R. Reid, Biochemistry 24 (1984) 4315.[10] Y. L. Dubreuil, L. Kaba, E. Hajnsrorf and A. Favre, Biochemistry 25 (1985) 5726.[11] J. L. Leroy, M. Gueron, G. Thomas and A. Favre, Eur. J. Biochem. 74 (1977) 567.[12] S. J. Milder, P. S. Weiss and D. S. Kliger, Biochemistry 28 (1989) 2258.FIZIKA A (Zagreb) 17 (2008) 3, 109–116 115shaqiri et al.: measurement of the intersystem crossing yield of s4U . . .MJERENJE PRINOSA MED– USUSTAVNOG PRIJELAZA s4U U tRNAVREMENSKI-RAZLUČENIM IZBJELJIVANJEMRijetka baza 4-thiouridine (s4U), koju nalazimo u raznim prijenosnim molekulamaRNA (tRNA) Escherichie coli, obično uzima strateški važan 8. položaj izmed–udviju zavojnica primatelja i dihidrouridinskih stapki lista djeteline. Ta se neobičnabaza najvǐse rabi kao unutarnja ugradbena proba za proučavanje interakcija kon-formalne RNA s RNA(DNA)–proteinima preko fotokemije tripletnog uzbud–enogstanja, povezanog sa stvaranjem kovalentnih adukata. U ovom smo radu prim-jenom laserske prijelazne apsorpcijske spektroskopije sa zasićenjem mjerili prinosmed–usustavnih prijelaza ϕST i apsorpciju tripletnog stanja εT−T s4U u tRNA. Dokugrad–ivanje s4U u tRNA uzrokuje prilične promjene u tRNA, nismo opazili prom-jene prinosa med–usustavnih prijelaza, što nije u skladu s objavljenim rezultatima.116 FIZIKA A (Zagreb) 17 (2008) 3, 109–116

Mjerenje prinosa međusustavnog prijelaza s4U u tRNA vremenski-razlučenim izbjeljivanjem

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