0022-202X/ 8 1/ 770 1-00J9$02.00/ 0 THE JOURNAL OF I NV r, STIGATIVE DEIlMATOLOGY, 77:39-44, IH8 1 Vol. 77, No. I Copy righ t © 198 1 by The Williams & Wilkins Co. Printed ill U.S.A. Psoralen Photochemistry and Nucleic Acid Structure

JOHN E. H EARST, PH.D. Departm.ent of Chemistry, University of California, B erkeley, California, U.S.A .

Many new psorale n derivatives have b een synthesized nated in the production of many new psoralens, several of which in an effort to enhance their water solubility and their have superior photoreactivity with both DNA and RNA as binding to nucleic acids. Availability of the very soluble compared to TMP and 8-MOP. The 5 new derivatives shown strongly binding compounds has improved our abilities here are 4' adducts of TMP and their structW"es ru-e shown (3 - to follow the optical changes associated with the photo­ 7). T he complete characterization of methoxymethyltrioxsalen chemistry of psoralens with DNA. Changes in both ab­ (MMT, 4) , hydroxymethyltrioxsalen (HMT, 5) and aminome­ sorbance and fluoresce nce are prese nted in this r eview. thyltrioxsalen (AMT, 7) wi th respect to their reactivity with A kinetic model for the photochemistry concludes that nucleic acids, including a theoretical treatment, is described in the detailed kinetics is dominated by the equilibrium a recent paper by Isaacs et al [6]. A related set of soluble 8- constant for intercalation of the psoralen in the DNA, methoxypsoralen derivatives have been synthesized by Isaacs, the quantum yield for photoaddition to DNA once inter­ Chun, a nd Hearst [7] a nd ru'e listed as compounds 8-11. Ad­ calated and the quantum yield for photodestruction of ditional advances in psora len synthetic chemistry have been the drug in water. With these 3 parameter s the kinetics presented by Bender, Hearst, and Rapoport [8] a nd in 3 asso­ of photochemistry is predictable. The values of these ciated patents [9-11]. parameters for numerous derivatives of 8-me thoxypso­ Briefly, th e advantages of the new compounds MMT (4), ralen and 4,5',8 trimethylpsoralen are presented. Appli­ HMT (5), AMT (7), MMX (9), and HMX (10) are higher cation of this photoche mistry to a study of nucleic acid aqueous solubility and, in the case of the aminomethyl com­ secondary structure in chromatin, fd bacteriophage, and pound (7), a positively charged side chain. T hese factors greatly in ribosomes is reviewed. enhance the binding of the compound to nucleic acids and thus allow more dl'U g to be covalently bound to DNA or RNA in the photoaddition reaction for a given dose of drug and light. One At present there exist over 100 psoralen derivatives in the example of the advantage gained here is found in viral inacti­ chemical literature of which approximately half are natW"ally vation, as discussed below. occurring and the remainder synthetically prepared. Of this The current position on the therapeutic potential of these large number, however, only 2 derivatives al'e cUJTently in new derivatives falls mainly into 2 areas which should not be widespread scientific and clinical use in the United States. interpreted as limiting. A method enabling selective control of These 2 compounds ar e known by their trade names as Tri­ nucleic acid replication, hence viral and cellulru' reproduction, oxsalen (4, 5',8-trimethylpsoralen or TMP, 1) and Methoxsalen obviously lends itself to a broad range of application. (8-methoxypsoralen or 8-MOP, 2). ANTIVIRAL VACCINE PRODUCTION Current methods for inactivating viJ"U ses for the purpose of ~3 vaccine production consist primarily of fo rmaldehyde or heat ~ denatW"ation of the intact virus. The disadvantage of such c~o~o "O~~O proceduTes is the concomitant denaturation or ch emical modi­ CH 3 OCH 3 fi cation of vU'al protein, ch anging the antigenic structures to 4,5',B-trimethylpsoralen (TMP) 8- methoxypsoralen (B-MOP) which a ntibodies are formed. With psoralens, which have very weak reactivity with protein, I 2 it has been possible to completely inactivate both DNA and ~ RNA viruses with an efficiency several orders of magnitude My laboratory has become involved in many fundamental greater than the denaturation methods. In the case of the RNA aspects of psoralen chemistry, including probing the secondary vU'us, VesiculaT Stomatitis vU'us (Hearst and Thu'y [12], Tri­ structW"es of Escherichia coli 16 S ribosomal RNA in vitro and oxsalen was found capable of reducing the number of survivors in th e 30 S ribosomal subunit [1-3], of Drosophila melanogas­ to 10% of the original vU'al titer for a given dose; an equivalent ter 5 S ribosomal RNA in vitro [4], and of the circular single­ dose of the new derivative AMT was found to leave a fraction stranded DNA genome of fd bacteriophage virus in the virus of 10- 4 sW"viving plaques after treatment. In both cases the [5]. In the coW"se of our investigations it became clear that required time of uTadiation for inactivation was on the order of modification of the psoralen nucleus in various ways would minutes. Even more effective AMT inactivation has been dem­ enable different types of experiments to be carried out which onstrated by H anson, Riggs, and Lennete [13] using Western are not possible to do with either TMP or 8-MOP. With this in Equine Encephalitis vU·us. mind, an organic synthesis project was initiated which culm i- In theory, a psoralen inactivated vU'us should produce a superior vaccine, as the protein a ntigenic component of the This work was supported in part by Grants GM 11180 and GM 25 151 virus is, in aUprobability, unmodified by the inactivation pro­ fro m the United States Public Health Service, and Grant NP 185 fro m cedure. the America n Ca ncer Society. A final point on vu"a l inactivation deserves comment. Recent Reprint requests to: John E. Hearst, Ph.D., Department of Chemis­ a ttention has been given to the oncogene theory. If this theory try, University of California, Berkeley, Cali fornia 94720. is correct, an inactivated vU'us may contain a n oncogene which Abbreviations: could be excised and reinserted into the host genome, giving AMT: aminomethyltrioxsalen HMT: hydroxmethyltriosalen rise to transformation. With psoralen inactivation, however, it MMT: methoxymethyltrioxsalen is possible to chemically alter lout of every 4 base pau's, cleru'ly 8-MOP: 8-methoxypsoralen eliminating the possibility of the repair or recombination of TMP: 4,5',8-trimethylpsoralen viral information.

39 40 HEARST Vol. 77, No.1

CH 3 H CH3 ' CH3 CH3 " , I' , 1 I" )!. 1 /. 1 /. CH 0 /. 0 0 CH 0 0 CH 0 0 M3 CH lm3 CH lm3 CH 3 3 3 4'-ChloromethYltrioxsolen 4'-Methoxymethyltrioxsolen 4'- Hydroxymethyltrioxsolen 3 4 o CH3 1/ N2:¢"",: ~ 1 t /. o CH 0 0 0 o , 3 CH 3

4' -N - phtha limidomethyltrioxsolen 4' - Ami nomethy Itrioxsolen hydrochloride 1

5 -Chloromethyl- 8 -methoxypsorolen 5- Methoxymethyl-8 -methoxypsorolen 5-Hydrox ymethyl-8-met hoxypsorolen 8 9 10

Pj1 N~

QO¥O~O~ OCH 3 N-(5-Me thylene-8-methoxypsorolen) -pyridinium chloride

TUMOR CHEMOTHERAPY oadduct and that only this monoadduct will be fluorescent One possible approach to tumor chemotherapy employing when excited at 365 nm [14-16]. These conclusions have re­ psoralens and light combines oral or locai administration of the cently been verified in our laboratory. drug followed by local irradiation of solid tumor mass via needle Figure 2 shows the excitation (left) and emission spectra guided light pipes. Such an approach is essentially the same in (right) of AMT alone, where the excitation spectrum was re­ principle as the photochemotherapy of psoriasis, but it does not corded at the fluorescence wavelength of 450 nm while the limit itself to an external surface. If found to be workable, the emission spectrum was recorded utilizing exciting radiation at procedure would provide a means. to affect the controlled killing 330 nm [17]. The fluorescence peaks at 457 nm independent of of neoplastic cells without the trauma of surgery or high energy the wavelength of the exciting light between 240 nm and 380 ionizing radiation. nm. There are 3 obvious bands in the excitation spectrum corresponding to the 3 absorption bands seen in Fig 1. These bands lead to fluoresence with an efficiency which is the reverse OPTicAL PROPERTIES OF THE PSORALENS AND of their absorption efficiencies, the shortest wavelength band THEIR REACTION PRODUCTS WITH DNA leading to the least fluorescence. The absorption spectrum of AMT is shown in Fig 1. Typi­ In the presence of poly d(A-T) (Fig 3), there is a 23% cally, the photochemistry is activated at wavelengths between depression of fluorescence intensity of AMT at 457 nm. Under 340 and 380 nm, well away from the absorption bands of nucleic the conditions of this experim'ent, essentially all the AMT was acids alone, which absorb below 300 nm. The absorption spec­ intercalated into the alternating poly d(A-T) before photoreac­ trum of the psoralens is similar to that of the corresponding tion because of its strong binding constant to DNA. coumarins, suggesting that conjugation in the furan ring is As this reaction mixture is irradiated at 365 nm, Fig 3 shows rather weak. In fact, benzofuran does not absorb significantly that the 457 nm fluorescent peak is depressed and a new peak above 320 nm. For this reason one would predict that the furan at 380 nm appears, corresponding to the buildup of furan ring monoadduct to pyrimidine would still absorb between 320 monoadduct. Further irradiation leads to the loss of the 380 run to 380 while the pyrone monoadduct should not. This also fluorescence, leaving a nonfluorescent crosslinked product suggests that the precursor to crosslink is the furan ring mon- [17]. July 1981 PSORALEN PHOTOCHEMISTRY 41 I .4 ,...--,--r-r-,---,--,---.--,--,---,-r-,--,-'-,---.--,--,---,-, amount of intercalation prior to photoreaction shown as [PS]/ [S] for the saturated solution. -2 is the quantum yield for monoaddition assuming the extinction coefficient of the psoralen at 365 nm intercalated in the DNA is not different from the free solution extinction coefficient, an approximation which has been experimentally verified for AMT. ct>3 is the quantum yield for photo breakdown by a first order reaction in psoralen concentration. We find at the very dilute conditions of this chemistry that photodimeri­ zation of free drug is not an important process. >- From Table II [7], we see a range of quantum yields for addition to DNA of 8% down to 0.06%, the highest value being -

~e. ~ 330 nm " "' ...... K = [P][S] = ~ (1) 0- [PST kJ 220 260 300 34CJ 420 460 500 540 580 Typically, this constant is measured by equilibrium dialysis A (nm) utilizing radiolabeled psoralen. Dissociation constants, extinc­ FIG 2 , Corrected excitation (left) and em ission (right) spectra of tion coefficients, and solubilities can be found in Table I [7]. aminomethyltrioxsalen (AMT) alone at a conce ntration of 27,8 j.tM and The solubility of the compound in water at room temperature of AMT [25.9 11M] with polyd d(A· T) [0,15 mM phosphate] in the Tris! as shown in Table I allows for the calculation of the maximum EDTA buffer. 42 HEARST Vol. 77, No.1

750 sec

on /~-.... No poly d (A - T) C I \ Q)

340 380 420 460 500 540 580 380 420 460 500 540 580 A (nm) A (nm)

FIG 3. Fluorescence emission spectra in the course of the photoreaction AMT with poly d(A- T). Note that fluorescence is depressed when AMT intercalates in the poly d(A- T). T he numbers associated with each curve in the figure on the left represent the time in seconds of photoreaction at constant intensity of 365 nm light. The buildup of the flu orescence peak at 380 nm is associated with monoaddition of AMT to thymine residues in the polymer. Loss of the' same peak is associated with crosslink formation and is shown in the fi gure on the right where the numbers associated with each curve represent minutes of photoreaction.

TABLE 1. Dissociation constants, extinction coefficients and solu.bilities

Exlin Cl io ll Solubili ties [PS]![S] Compo und Abbrev. Molec. coefficient. K"DNA Saturated Name Weight molll solu tion .250nm O/ mol - ' em- ') I'g/ml mol /I 4,5',8-Trimetbylpsoralen TMP 228 1.5 X ]0" 0.6 2.6 X 10-'; 1.3 X 10- " .05 (Trioxsalen) 4' Aminomethyltrioxsalen AMT 257 2.5 X 10" >10" 3.4 X 1O - ~ 6.6 X 10-" 5000 4'Hydroxymethyltrioxsalen HMT 258 2.1 X 10" 41 1.6 X lO- " 2.9 X 10- " .55 4' Methoxymethyltrioxsalen MMT 272 2.5 X 10" 10 3.7 X 10- [' 5.4 X 10-' .39 N -(4' methylenetrioxsalen)- PMT 356 2.5 X 10' > 10" 2.8 X lO- " 2.6 X 10- [' 1080 pyridinium chloride Psoralen PSOR 186 35 1.9 X 10- ' 4.0 X 10-" .05 Xanthotoxin 8-MOP 216 2. 1 X 10" 38 1.8 X 10-' 1.3 x 10- " .14 (8-Methoxypsoralen) 5-Hydroxymeth ylxanthotoxin HMX 246 1.8 x 10" 46 1.8 x 10-' 5.0 x 10-" .04 2 5-Methoxymethylxanthotoxin MMX 260 1.7 X 10' 55 2.1 x 10-" 1.0 X 10- .02 N -{5-methylene-8-methoxy- PMX 344 2.3 x lO" > 10" 2.9 x 10- 2 5.2 x 10- " 560 psoralen) -pyridinium- chloride

Table II. Kinetic constants (or photochem.islI y-quantum yields for DNA addition and breakdown Equilibriu m Plateau bound Platea u of ' 2 ,!, bound per photoaddition 1000 b.p. per 1000 b. p. eq uilibrium bound 2 TMP 8.4 x 10 2 4.7x10 1.8 15 28 1.9 AMT 2.2 x 10-" 2.2 X 10- 2 1.0 61 67 1.1 HMT 1.6 X 10-' 4.9 x 10- " 3.2 14 38 2.7 MMT 4.7 X lO- ;] 1.6 X 10-" 2.9 13 23 1.8 PMT 1.8 X 10-" 3.] X 10- ' 5.8 49 55 1.1 P SOR 3.0 X 10- ' 8.3 X 10-" 3.6 1.2 9 7.5 8-MOP 1.3 X lO- 2 5.5 X 10-' 24 3.7 (42) (11.4) HMX 1.4 X 10-" 1.4 X 10-" 10 1.0 (4) (4.0) MMX 1.2 X lO-" 8.9 X lO- " 1.3 0.5 (2) (4.0) PMX 6.2 X lO- ' 8.3 X 10-' 0.74 39 (67) (1.7) July 1981 PSORAL E N P HOTOCHEMIST RY 43

A Specifi c DNA Orientation in the Filamen tous Bacteriophage fd as P robed by Psoralen Crosslink ing and __ ------~------~AMT E lectron M icroscopy Shen a nd Hearst [5] have stabilized the moleculru' structure of single-stranded fd DNA inside its fIl amen tous virion by the ___ ~------~~T photochemical crosslinking with psoralens in solutions of dif­ fe rent concentrations of NaCl, short (100-300 base pairs) hair­ pins can be stabilized on denatured Simia n virus 40 (SV40) __--~8MOP DNA for electron microscope visualization. Six major hairpins were mapped. This study was extended by S hen & H eal"st [26] __~------~~~ __~~------~--~HMT by crosslinking single-stranded SV40 linear molecules, EcoRI­ SV40 a nd Bg1 I-SV40, in the presence of magnesium ions. After __----~~------~TMP the reaction, in addition to the hairpins detected before, 7 DNA hairpins, and hairpin loops with sizes from 300 to neal"l y 2,000 __ ------~MMT nucleotides, are observed. T hese loops are located at specific regions on the SV 40 genome. At least 3 of these loops ru'e fo und in regions though t to be involved in splicing of the eal'ly a nd late SV40 transcripts. These loop structures ru'e most likely to ~_------~PSOR arise fro m base pail"ing between distant regions of the single­ stra nded DNA. RNA transcribed from th e double-stranded t~~~~~~~~~~~;=~~~~~c=:E~~j . HMMMXX DNA template is expected to behave in a similar way, possibly 10 20 30 40 50 60 70 80 90 100 110 120 130 140 providing recognition sites fo r processing (splicing) enzymes. MINUTES OF IRRADIATION FIG 4. Kinetics of photoaddition of deri vatives of Trioxsalen, deriv­ B ase-P airing B etween Distant Regions of th e Escherichia atives of 8-M OP and psoralen to calf thymus D NA. Abbreviations w'e coli 16 S Ribosomal RNA in Solution as fo llows: AMT, aminomethyltrioxsalen; xanthotoxin or 8- methoxy­ Wollenzien et al [3] have introduced intramolecular cross­ psoralen; H M T , hydroxymethyltrioxsalen; TMP, Trioxsalen; MMT, methoxymethyltrioxsalen; and PSOR, psoralen. links into Escherichia coli 16 S ribosomal R NA in aqueous solution by irradiation in the presence of h ydroxymethyltri­ methylpsoralen. When the crosslinked R NA is denatured and examined in the electron microscope, the most striking features APPLICATIONS OF PSORALEN PHOT OCHEMISTRY are a variety of large open loops. In addition, because the TO T HE SOLUTION OF SIGNIFICANT crosslinked molecules are shortened compared to noncros­ STRUCTURAL P ROBLE MS slinked molecules, there ru"e likely to be s mall hairpins not Chromatin S tructure resolved by the present technique. The sizes and positions of 11 The nucleosome structure of chromatin im parts a specificity loop classes have been determined and oriented on the mole­ to the photochemical reaction of the psoralens wi th DNA. The cule. T he frequency of occurrence of the d ifferent classes of ph otoaddition occurs primarily in the interbead regions of this loops depends on the crosslinking conditions. When the cross­ structul"e. This has been demonstrated by denaturation electron linking is done in solutions containing Mg2-+- , at least 4 of the microscopy on high molecular weight DNA isolated from Dro­ loop classes appear with greater frequency th an they do in 3.5 sophila m elanogaster nuclei after photoreaction of the nuclei mM -NaCI. T he loops presumably ru'ise because complementary with Trioxsalen, and also by dena tul"ation electron microscopy sequences separated by long in tervening regions ru'e being cross­ on short pieces of DNA obtained by micrococcal nuclease linked. These base-pairing interactions between residues distant digestion of these same nuclei (Wiese ha hn et al [21, 22], Hyde in the primru'y structure apperu" to be prominent features of the secondary structure of rRNA in solution. & H eal"st [23]). Tritium labeled Trioxsalen is removed prefer­ entially from these nuclei by micrococcal cleavage, demonstrat­ ing that the sites of nuclease attack in nuclei and the sites of Crosslinking Studies on the Organization of the ]6 S photoaddition of Trioxsalen to chl"omatin are in close proximity R ibosomal R NA within the 30 S E coli R ibosomal Subunit on the DNA. Thammana et al [1] have examined the structure of 16 S RNA in th e 30 S subunit, T he location and frequency of RNA Secondary Structure in S ing le-Stranded fd DNA crosslinks induced by photoreaction of hymoxymethyltrime­ The photochemical crosslinking of DN A by 4,5' ,8-trimethylp­ thylpsoralen with 30 S E coli ribosomal subunits have been soralen (Trioxsalen) has been used by S hen and Heal"st [24] to determined by electron microscopy. At least 7 distinct crosslinks freeze the secondru'y structUl"es of single-stra nded DNA mole­ between regions distant in the 16 S rRNA primary structure cules of bacteriophage fd at different ionic strengths. These ru'e seen in the inactive conformation of the 30 S pal"ticle. All secondary structures (hairpins or looped hai.rpins) have been correspond to crosslinked features seen wh en the free 16 S visualized in the electron microscope. M ost of the single rRNA is treated with hydroxymethyltrimethylpsoralen. T he str anded circular fd DNA molecules show only one hairpin most frequently observed crosslink occurs between residues after irradiation at 15° in 20 mM NaCI in the presence of near one end of the molecule and residues about 600 nucleotides T rioxsalen. As the ionic strength is increased, more hairpins away to generate a loop of 570 bases. T he size and orientation a ppeal" on the DNA molecules. T o map these secondary struc­ of this feature indicate it corresponds to the crosslinked feature tilles, double-stranded supercoiled fd DNA (RFI) was cleaved located at the 3' end of free 16 S rRNA. with the restriction enzyme Hind II, which makes only one cut When active 30 S pru,ticles aTe crosslinked in 5 mM Mg2+ , 6 on each RFI molecule. After denatul"ation and crosslin king of of the 7 featw'es seen in the inactive 30 S pal" ticle can still be th e lineal" single-stranded fd DNA (a mixture of viral and detected. H owever, the frequency of several of the features, and complementru'y strands), all the hairpins have been mapped on particularly the 570-base loop feature, is dTa matically de­ th e DNA molecule with respect to this Hind II site. The results creased. T his suggests that the long-range contacts that lead to show that these hairpins occur at specific sites. The most stable these crosslinks are either a bsent or inaccessible in the active h airpin has been assigned to the position where the initiation conformation. Crosslin king results in some loss of fu nctional site for the conversion of single-stra nded fd DNA to the double­ activities of the 30 S pal"ticle. T his is consistent with the notion stranded covalently closed form has been mapped. that the presence of the crossli nk that generates the 570-base 44 HEARST Vol. 77, No.1 loop traps t he subunit in an inactive form, which cannot asso­ ber 25, 1979 ciate with 50 S particles. 11. H earst JE, Rapoport H, Isaacs S, Shen C- KJ: Psoralens. U.S. Patent No. 4,196,28 1. University of California, Berkeley, April 1, The arrangement of the interacting regions crosslinked by 1980 hydroxym ethyltrimethylpsoralen suggests that the RNA may 12. Hearst JE, Thiry L: The photoinactivation of an RNA animal viTUs, be organized into 3 general domains. A striking feature of the vesicul ar stomatitis virus, with the aid of newly synthesized crosslinking pattern is that 3 of t he 7 products involve regions psoralen derivatives. Nucl Acids Res 4:1 339-1347, 1977 13. Hanson CV, Riggs JL, Lennette EH: Photochemical in activation of near the 3' end of the 16 S rR NA. These serve to tie together DNA and RNA viruses by psoralen derivatives. J Gen Virol 40: large sections of rRNA. Thus structural changes at the 3' end 345-358, 1978 could, in principle, be felt through the entire 30 S particle. 14. Kra uch CH, Kramer DM, Wacker A: Zum wirkungsmechanismus photodynamischer furocumarine photoreaktion von psoralen­ I must acknowledge the many scientists wi th whom I have colla bo­ (4 - "'C) mit DNS, RNS, homopolynucleotiden und nucleosid en. rated in these last 5 yr which I have devoted to psora len chemistry. In Photochem Photobiol 6:341-354 , 1967 particular, I acknowledge the assistance and friendship of J ean-Pierre 15. Musajo L, Bordin F, Bevilacqua R: Photoreactions at 3655 A linking the 3-4 double bond of furocoumarins with pyrimidine bases. Bachellerie, Stephen Isaacs, Henry Rapoport and Gary Wiesehahn. Photochem Photobiol 6:927-93 1, 1967 16. Musajo L, Bordin F, Caporale G, Marciani S, Rigatti G: Photoreac­ REFERENCES tions at 3655 A between pyrimidine bases and skin-photosensitiz­ 1. Thammana P, Cantor CR , Wo!J enzien P L, Hearst JE: Crosslinking ing furocourmarins. Photochem Photobiol 6:711-719, 1967 studies on the organization of the 16 S ribosomal RNA wi thin 17. Johnston BH: Dynamics of the psoraien-DNA photoreaction. Doc­ the E scherichia co li ribosomal subunit. J Mol Bioi 135:271-283, toral dissertation, University of California, Berkeley, 1980 1979 18. Johnston BH, Hearst JE: Low level psoralen-DNA crosslinks in­ 2. WoLl enzien PL, Hearst JE, Squires C, Squires C: Determining the duced by single laster pulses. Biochemistry 20:739-745, 198 1 polan ty of the map of crosslinked in teractions in Escherichia 19. Johnston GH, J ohnson MA, Moore CB, Hearst JE: Psoralen-DNA co li 16 S ribosomal RNA. J Mol Bioi 135:285- 291 , 1979 photoreaction: Controlled production of mono- and diadducts 3. Woll enzien P, Hearst J E, Thammana P, Cantor CR: Base-pairing with nanosecond ultraviolet laser pulses. Science 197:906- 908, between distant regions of the E scherichia coli 16 S ribosomal 1977 RNA in solution. J Mol Bioi 135:255- 269, 1979 20. J ohnston BH, Kung A, Moore CB, Hearst JE: Kinetics of forma tion 4. 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Biochemistry 16:925-932, 1977 toreactivity with DNA and RNA. Biochemistry 16:1058-1064, 23. Hyde JE, Hearst JE: Binding of psoralen derivatives to DNA and 1977 chromatin: Influence of the ionic environment on dark binding 7. Isaacs ST , Chun C' . Hearst J E: Trends in Photobiology. Plenum and photoreactivity. Biochemistry 17:1251-1257, 1978 Press, New York, 111 press, 1981 24. Shen C-KJ, Hearst JE: Psoralen-crosslinked secondary structure 8. Bender DR, Hearst J E, Rapoport H: Psoralen synthesis. Improve­ map of single-stranded virus DNA. Proc Nat! Acad Sci USA 73: ments in furano ring formation. Application to the synthesis of 2649- 2653, 1976 4,5',8-trimethylpsoralen. J Org Chern 44 :2 176- 2180, 1979 25. Shen C-KJ, Hearst JE: Mapping sequences wit h 2-fold symmetry 9. Hearst J E, Rapoport H , Isaacs S, Shen . C-KJ: Psoralens. U.S. on the simian vi.rus 40 genome: A photochemical crosslinking Patent N o. 4,1 24,598. University of California, Berkeley, Novem­ approach. Proc Nat! 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