PSORALEN PHOTOCHEMISTRY #9159 · The adducts of 4’-hydroxymethyl 4,5’,8-trimethylpsoralen to both DNA and RNA have been isolated by enzymatic degradation of the photore-acted

An~ Re~. Biophys. Bioeng. 1981. 10:69-86Copyright © 1981 by Annual Re~iews Inc. All rights reserved

PSORALEN PHOTOCHEMISTRY #9159

John E. HearstDepartment of Chemistry, University of California, Berkeley, California 94720

INTRODUCTION

Psoralen or furocoumarin is a linear three ring heterocyclic compound(Structure 1).

I Psoralen

The activity of this family of compounds was apparently described inold Arabic literature (1400 BC), where reference was made to the use fruits of Ammi Majus as remedy for leukoderma. Derivatives of psoralenhave been isolated from many fruits of tropical plants as well as from anumber of fungi. An excellent review of the history, toxicity, and use ofthe furocoumarins has been written by Perone (36) in Microbial Toxins.Not until 1959 was it realized that vesicular and bulbous dermatitis,along with de- and hyperpigmentation in celery workers, was caused by4,5’,8-trimethylpsoralen (trioxsalen) and by 8-methoxypsoralen(xanthotoxin), products of celery pink rot (a fungus). After carefulscrutiny, it was realized that only those workers who handled the rottingcelery in the sunlight were inflicted with skin lesions.

Synthetic chemistry associated with the psoralens has been an activearea of research; the pioneering work was done by Sp/ith et al (44).Recent activity prior to our work was dominated by Kaufman (26-29).Thin layer chromatography of the furoeoumarins is discussed by Egger(13) and Pathak & Kr/imer (35). The furocoumarins have also characterized by 1H NMR to some extent (29)--a fact that has provenhelpful in establishing the structure of the adducts.

69

0084-6589/81/0615-0069501.00

Annual Reviewswww.annualreviews.org/aronline

Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.

http://www.annualreviews.org/aronline

70 HEARST

The chemistry associated with the photoreaction of the psoralens withDNA has also been studied, although the information at the structurallevel with respect to DNA photoproducts has been largely suggestive. Aseries of papers has been presented by Dali’Acqua et al (10-12), wherephotoproducts from mixtures of pyrimidine bases and the furoc-oumarins, as well as hydrolysis products of photoreacted DNA, showthat a C4 cyclo-addition reaction occurs between the 5,6 double bond ofthe pyrimidine bases and the 3,4 or the 4’,5’ double bonds of thefurocoumarins. Both Dall’Acqua et al (11) and Cole (7) have pothesized the mechanism of DNA cross-linkage to be the formation ofthe double adduct at both the 3,4 and 4’,5’ double bonds of thepsoralens. It appears that the order of reaction may be importantbecause the 3,4 photoadducts no longer absorb at 360 nm. The 4’,5’adducts do, and therefore they are the primary candidates for furtherreaction. Dali’Acqua’s papers also provide valuable solubility, spectral,and binding information for many derivatives of psoralen. He hasshown (6) that alternating poly d(A-T) is cross-linked by the psoralenphoto-reaction and that the polypurine-polypyrimidme (A.’I) is not.This observation has been confirmed by Shen & Hearst with a Drosophilamelanogaster satellite containing long runs of polypurine and poly-pyrimidine (40, 41).

Cole has studied recA excision and repair in Escherichia coli cells (8)after treatment with 0.17 #g/ml of trioxsalen and irradiation for 30rain. Under these conditions 1 cross-link/33,000 base pairs is formedand the frequency of monoadduct is three times that of cross-link. Morerecently, Cole et al (9) have implicated uvrA, uvrB, uvrC, and polA(5’-3’ exonuclease) in the initial steps of cross-link repair in E. coll. Thelaboratory of Howard-Flanders (5, 31) has actively studied the relativeeffects of monoadduct and cross-link in the induction of genetic ex-change between E. coli K-12 (k) lysogens and photosubstituted k phage.They concluded that cross-links were 20 times more effective thanmonoadducts for inducing recombination under repressed conditions.They implicated the uvrA, uvrB, and recA genes in these recombinationevents.

THE COMPOUNDS

At present there exists over one hundred psoralen derivatives in thechemical literature, of which approximately half are naturally occurringand the remainder synthetically prepared. Of this large number, how-ever, only two derivatives are currently in widespread scientific andclinical use in the United States. These two compounds are known as


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


PSORALEN PHOTOCHEMISTRY 71

trioxsalen [4,5’,8-trimethylpsoralen or TMP (Structure 2)] andmethoxsalen [8-methoxypsoralen or 8-MOP, (Structure 3)].

CH3

4,5~,8-trimet hylpsorolen (TMP)

2

8-methoxypsorolen (8-MOP)

3

My laboratory has become involved in many fundamental aspects ofpsoralen chemistry and has used this chemistry to probe the secondarystructures of E. coli 16S ribosomal RNA [in vitro and in the 30Sribosomal subunit (46, 50, 51)], of D. melanogaster 5S ribosomal RNA invitro (47), and of the circular single-stranded DNA genome of bacteriophage virus in the virus (40). In the course of our investigationsit became clear that modification of the psoralen nucleus (Structure 1)in various ways would enable different types of experiments to becarried out that are not possible to do with either TMP or 8-MOP. Withthis in mind an organic synthesis project was initiated which culminatedin the production of many new psoralens, several of which have super-ior photoreactivity with both DNA and RNA as compared to TMP and8-MOP. The five new derivatives shown here are all 4’ adducts of TMP.The complete characterization of methoxymethyltrioxsalen [MMT(Structure 5)], hydroxymethyltrioxsalen [HMT (Structure 6)], aminomethyltrioxsalen [AMT (Structure 8)] with respect to their reactiv-ity with nucleic acids, including a theoretical treatment, is described in arecent paper by Isaacs et al (21). Additional advances in psoralensynthetic chemistry have been presented by Bender, Hearst, & Rapoport(4) and in two associated patents (15,16).

Briefly, the advantages of the new compounds MMT (Structure 5),HMT (Structure 6), and AMT (Structure 8) are higher aqueous solu-bility and, in the case of the aminomethyl compound (Structure 8), positively charged side chain. These factors greatly enhance the bindingof the compounds to nucleic acids and thus allow more drug to becovalently bound to DNA or RNA in the photoaddition reaction for agiven dose of drug and light. One example of the advantage gained hereis found in viral inactivation, as discussed below.


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


72 HEARST

The current position on the therapeutic potential of these new deriva-tives falls mainly into two areas that should not be interpreted aslimiting. A method enabling selective control of nucleic acid replication,hence viral and cellular, reproduction, obviously lends itself to a broadrange of application.

CI CH~ CH~

CH~ CH~

4~- Chloromelhylt rioxsolen 4/- Met hoxymet hylt rioxs(~len

4 5

CH5

°C~o

CH3

4/- Hydroxymethyltrioxs(]len

6

Antiviral Vaccine Production

Current methods for inactivating viruses for the purpose of vaccineproduction consist primarily of formaldehyde or heat denaturation ofthe intact virus. The disadvantage of such procedures is the concomitantdenaturation or chemical modification of viral protein, changing theantigenic structures to which antibodies are formed.


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.



With psoralens, which have very weak reactivity with protein, it hasbeen possible to completely inactivate both DNA and RNA viruses withan efficiency several orders of magnitude greater than the denaturationmethods. In the case of the RNA virus, vesicular stomatitis virus [Hearst& Thiry (17)], trioxsalen was found capable of reducing the number survivors to 10% of the original viral titer for a given dose; an equiva-lent dose of the new derivative AMT was found to leave a fraction of10-4 surviving plaques after treatment. In both cases the required timeof irradiation for inactivation was on the order of minutes. Even moreeffective AMT inactivation has been demonstrated by Hanson et al (14)using western equine encephalitis virus.

In theory, a psoralen-inactivated virus should produce a superiorvaccine, as the protein antigenic component of the virus is in allprobability unmodified by the inactivation procedure.

A final point on viral inactivation deserves comment. Recent atten-tion has been given to the oncogene theory. If this theory is correct, aninactivated virus may contain an oncogene that could be excised andreinserted into the host genome, giving rise to transformation. Withpsoralen inactivation, however, it is possible to chemically alter one outof every four base pairs, clearly eliminating the possibility of the repairor recombination of viral information.

Tumor Chemotherapy

One possible approach to tumor chemotherapy employing psoralensand light combines oral or local administration of the drug followed bylocal irradiation of solid tumor mass via needie-guided light pipes. Suchan approach is essentially the same in principle as the photo-chemotherapy of psoriasis, but it does not limit itself to an externalsurface. If found to be workable, the procedure would provide a meansto affect the controlled killing of neoplastic ceils without the trauma ofsurgery or high energy ionizing radiation.

CHEMICAL SPECIFICITY OF HMT

PHOTOADDITION

The adducts of 4’-hydroxymethyl 4,5’,8-trimethylpsoralen to both DNAand RNA have been isolated by enzymatic degradation of the photore-acted polymers. In the case of DNA, calf thymus DNA is typicallyreacted with 1 HMT molecule per 100 basepairs of DNA and with asaturating dose of 365 am light. Drosophila melanogaster 5S ribosomalRNA, E. coli phe tRNA, and E. coli, 16S ribosomal RNA have beenreacted with HMT.


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


74 HEARST

The Reaction With DNA

The irradiated .HMT-DNA sample has been hydrolyzed at pH 5 usingDNAase II (E.C. 3.1.4.6) and spleen phosphodiesterase (E.C. 3.1.4.18).After 48 h the hydrolysis mixture was adjusted to pH 8.6 and treatedwith alkaline phosphatase (E.C. 3.1.3.1) for 12

Large scale preparations (200 rag) of HMT-modified DNA were.chromatographed by Straub et al (45) on a Biogel P6 column afterhydrolysis. High pressure liquid chromatography (HPLC) was thendone utilizing either a Whatman M90DS eolurrm or an Altex 5 inUltrasphere ODS column.

Those peaks from the HPLC, which are cyclobutane adducts on thefuran end of the psoralen molecule and the pyrone end of the psoralenmolecule, have been distinguished by permethyl derivatization and massspectrometry. The dinucleoside-psoralen cross-link peak from the Biogelcolumn has been analyzed by mass spectroscopy; although this study is

’still in a preliminary stage, the requirement for field desorption tech-niques has been demonstrated. The more conventional electron-impactprobe for mass spectroscopy results in the immediate breakdown of thecross-linked dinucleoside to a monoadduct plus a free nucleoside. Thecytosine-psoralen cyclobutane adduct is unstable and typicallydeaminates to form a uracil-psoralen adduct that is readily distinguishedfrom the thymidine adducts by mass spectroscopy.

High resolution (360 MHz) NMR .has proven extraordinarily valuablein identifying monoadduct products of the photochemistry in DNA, afeat that has not been previously possible. In a formal sense, 32stereoisomers are possible for the furan adduct. Of these 32 potential

~.stereoisomers, 24 involve a trans cyclobutane ring, which would consistof highly strained and unlikely products from photochemical reactions.Of the remaining eight cis stereoisomers, most of which are found bydirect reaction of HMT with thymidine, only two are formed in theDNA reaction. Thus,-the ’DNA helix has a major influence on thestereochemistry of the products.

The same potential diversity of products is possible at the pyronedouble bond. This bond also reacts differently in DNA and free insolution. The stereochemical possibilities for the cross-link are vast innumber and the products of the actual reaction have not yet beenworked out.

The Reaction With RNA

A more biochemical approach has been taken to the characterization ofthe RNA-HMT adducts by Bachellerie et al (3) in my laboratory.Complete degradation to mononueleotides is possible with ribonuclease


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.



T2 (E.C. 3.1.4.23). Digestions with ribonu¢lease A (E.C. 3.1.4.22) ribonuclease T1 (E.C. 3.1.4.8) are also possible, with the rates cleavage often dependent upon the position of sites of monoadditionrelative to cleavage sites. All mono- and dinucleotide products of thedegradation of RNA after reaction with HMT can be separated bypaper electrophoresis. (See Figure 1 for an impression of relative ratesof migration.) In the case of RNA, 90% or more of the monoadditionoccurs on the uracil residues. Utilizing these methods of analysis as wellas gel electrophoresis, there have been remarkable achievements inmapping cross-links in D. melanogaster 5S ribosomal RNA (47), proving

1.30,

1.20

1.10

1.00

0.90

0.80

0.70

0.60

0.50

0.40

0.30

0.20

----" UpGp

-- Up --"-- (U~UP)A

-- -- UpA~’--- (U~UP)B

-- Gp

-- U~ A -- UpU

_-- U~B

-- U~ c -- GpU

-- ApU

-- CpU---- Cp

--UpCp

-- (U~U)A-- (U~U)B-- (U~G)A"--- (U$~)B

-- (UI~A)B-- (Ui~A)c

-- UpC

Figure 1 The rates of electrophoretic migration of mono- and dinucleotides on Whatmanpaper. Only monoaddition of uracil is observed with high efficiency. The positions of thehydroxymethyltrioxsalen (HMT) substitution on the uracil is indicated by an asterisk (*).The differing stereochemical products of each are arbitrarily designated A, B, and C inorder of decreasing rates of migration. The Rv’s are presented on the ordinate relative tothat of 7-methyl-guanine, which migrates in the opposite direction under these conditions.


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


76 HEARST

its secondary structure. The reactive positions in E. coli phe tRNA havealso been mapped (2). The long-range prospects for psoralen photo-chemistry as a probe to RNA structure in vitro and in vivo are brightindeed.

As is the case with almost all cyclobutane photoaddition products, thepsoralen adducts to pyrimidines are photoreversed by 250 nm radiation(37). Photoreversal makes it very much easier to identify the oligo-nucleotides from T1 digestion that are involved in either monoadditionor cross-linkage (47). The problem of cytosine adduct deamination more severe in RNA than it is in DNA. Eventually, the same massspectroscopic and high resolution NMR characterization as has beenaccomplished with DNA must also be available for RNA. The possibil-ity that the stereochemical structure of the product adduct will bediagnostic for RNA structure at the position of the addition is very real.

Typically, we have found that the most reactive sites in RNA are atthe ends of helical runs and at G-U mismatches in helices. There is aremarkable correlation between RNA reactivity and secondary struc-ture, which is indicated by the temperature dependence of the photore-action and by the correlation between photoreactivity and circulardichroism of the reactive RNA (1). This strongly suggests the need for stacking interaction between the psoralen and the nucleic acid basesprior to reaction.

Recently, some photoreactions with purines have been reported. Ou& Song (34) observed photoaddition of a psoralen to adenine in tRNA.Hochkeppel & Gordon (18) have evidence for the cross-linking polyinosinic acid. polycytidylic acid by AMT photochemistry. Bachel-lerie et al (3) have paper electrophoretic evidence for a guanine adductto AMT (the structure of which has not yet been established). Thephotoefficiency of this reaction is orders of magnitude below thatobserved with uracil.

OPTICAL PROPERTIES OF THE PSORALENS AND

THEIR REACTION PRODUCTS WITH DNA

The absorption spectrum of AMT is shown in Figure 2. Typically, thephotochemistry is activated at wavelengths between 340 and 380 nm,well away from the absorption bands of nucleic acids alone, whichabsorb below 300 rim. The absorption spectrum of the psoralens issimilar to that of the corresp,onding coumarins, suggesting that conjuga-tion in the furan ring is rather weak. In fact, benzofuran does notabsorb significantly above 320 nan. For this reason one would predictthat the furan ring monoadduct to pyfimidine would still absorb be-tween 320 to 380 am while the pyrone monoadduct should not. This


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.



I’itl I I I I I ’ I I I I I I I I I I II.

~0.8

0.6

O.

?__2_..0 2_40 2_60 2_80 ~00 .’.’.’5’;ZO ~40 ~ =080 400X (nm)

Figure 2 Absorption spectrum of aminomethyltrioxsalen (AMT) at a concentration 46.6 pg/ml in 10 mM Tris/l mM EDTA pH 7.0.

also suggests that the precursor to cross-link is the furan ring monoad-duct and that only this monoadduct will be fluorescent when excited at365 nm (30, 32, 33). These conclusions have recently been verified in ourlaboratory.

Figure 3 shows the excitation (left) and emission spectra (right) ofAMT alone, where the excitation spectrum was recorded at the fluores-cence wavelength of 450 nm while the emission spectrum was recordedutilizing excitating radiation at 330 nm (23). These spectra are correctedfor source and detector response. The fluorescence peaks at 457 rim,independent of the wavelength of the exciting light between 240 and 380nm. There are three obvious bands in the excitation spectrum corre-sponding to the three absorption bands seen in Figure 2. These bandslead to fluorescence with an efficiency that is the reverse of theirabsorption efficiencies; the shortest wavelength band leads to the leastfluorescence.

In the presence of poly d(A-T) (Figure 4), there is a 23% depressionof fluorescence intensity of AMT at 457 nm. Under the conditions ofthis experiment, essentially all the AMT was intercalated into thealternating poly d(A-T) before photoreaction, because of the strongbinding constant of AMT to DNA.


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


78 HEARST

:303 AMTolone / ~

220 260 300 340 380 420 460 500 540 580X

Figure 3 Excitation (left) and emission (right) spectra of aminomethyltrioxsalen (AMT)at a concentration of 27.8 ~M in the Tris/EDTA buffer (corrected).

700

~ iIl-~’\Nko poly d(A-T)

/ \ / o \

~ / \//~--~ \\

340 ~0 420 460 500 540X (nm)

~T ~ ~I~ d(~-~. Note ~at ~uotes~ is d~t~d ~ ~T ~tet~ates~ly d(A-~. ~e n~bers ~ciated ~ ~ch c~e repre~nt ~e ~e ~ s~n~ ofphotoreacfion at ~mmt ~te~ity of 365 m fi#t. ~e b~dup of ~e fluor~at 380 m is ~ted ~ monoad~fion of ~ to ~y~e residues ~ ~e ~l~er.~ss of ~e ~me ~ is ~so~at~ ~ ~oss-~ rotation.


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.



As this reaction mixture is irradiated at 365 nm, Figure 4 shows thatthe 457 nm fluorescent peak is depressed and a new peak at 380 nmappears, corresponding to the buildup of furan monoadduct. Furtherirradiation leads to the loss of the 380 nm fluorescence, leaving anonfluorescent cross-linked product (23).

In a similar experiment using the helix of homopolymers poly d(A-T)[(poly dA-poly dT)], Johnston & Hearst (23) have shown that buildup of the 380 nm fluorescent peak still occurs with the formationof furan monoadduct, but the loss of this fluorescence is slower andleads to a thus far undefined photobreakdown product that is fluo-rescent at 460 nm.

KINETICS OF PHOTOADDITION TO DNA

Isaaes et al (21) have presented a tentative kinetic model for psoralenaddition to DNA involving three steps: (a) drug intercalation, (b)photoaddition, (c) photobreakdown of unbound drug,

k~P+s ~ Ps (a)

PS + hv--> A ( b

P +hv--~ B (c)

where P is the psoralen, S is an intercalation site, PS is an intercalateddrug, A is the photoadduct, and B is the breakdown product. Theadduct A may absorb a second photon to form a cross-link, but it is stillconsidered adduct A, because the experiment only measures 3H psora-len covalently added to the DNA.

We have characterized the psoralen derivatives that we handle interms of four parameters relating to this scheme. First, the dissociationconstant associated with the equilibrium represented by Reaction 1:

[ PSI kl "

Typically, this constant is measured by equilibrium dialysis utilizingradiolabeled psoralen. ~2 is the quantum yield for monoaddifion, assum-ing the extinction coefficient of the psoralen at 365 nm intercalated inthe DNA is not different from the free solution extinction coefficient,an approximation that has been experimentally verified for AMT. #3 isthe quantum yield for photobreakdown by a first order reaction inpsoralen concentration. We find at the very dilute conditions of this


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


80 HEARST

Table 1 Kinetic and equilibrium data for five psoralens

Compound Kv ~ ~ ~/~’3 Solubifity"

Trioxsalen 1.3×10-4 8.4x 10-2 4.7×10-2 1.8 2.6x 10-6

AMT 6.6 × 10-6 2.2 × 10-2 2.2× 10-2 1.0 3.4× 10-2HMT 2.9× I0-4 1.6× 10-2 4.9× 10-3 3.2 1.6× 10-48-MOP 1.3×10-3 1.3×10-2 5.5x 10-4 24.0 1.8×10-4

Psoralen 4.0)< 10-3 3.05< 10-2 8.3× 10 -3 3.6 1.95< lO-4

aSolubility expressed in moles/liter.

chemistry that photodimerization of free drug is not an importantprocess. FinaLly, the solubility of the compound in water at roomtemperature is shown in Table 1, so that the maximum amount ofintercalation prior to photoreaction can be calculated.

From Table 1 (20) we see a range of quantum yields for addition DNA of 8.4% down to 1.3%; the highest value is associated withtrioxsalen, the lowest with 8-MOP. The quantum yields for drug break-down in solution range from 4.7% down to 0.06%. Typically the quan-tum yield for addition is one to five times larger than the quantum yieldfor photobreakdown. Oaly 8-methoxypsoralen is a variant from thispattern and appears to resist photobreakdown about ten times betterthan the other compounds in comparison to addition. Figure 5 showsthe kinetics of addition of these compounds to DNA and one can seethe qualitatively different behavior of 8-MOP in that the addition of thecompound to DNA continues for times about ten times longer than forthe remaining compounds. These long times are associated with thediffusion and intercalation of unbound 8-MOP into the DNA, a processwhich is less likely in the other derivatives because of photobreakdown.At present the relationships between these quantum yields and thestructures of the compounds are not explicable, but with a sufficientlylarge sampling such understanding should be realizable.

Johnston et al (22,24,25) have studied the kinetics of cross-linkformation in DNA with AMT using laser flash photolysis. There arethree major conclusions in these references. First, a single laser pulse of10 ns duration at modest energy densities forms only monoadducts.Second, the delay required between the first and second of such laserpulses for efficient cross-link formation is 1/ts, suggesting the need for aDNA conformational change in the course of cross-link formation.Finally, at higher energy densities a small number of single pulsecross-links do form. Although the mechanism for formation of thesesingle pulse cross-links is uncertain, one possibility is that a small


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.



70 !

5¢

IlJillllll]

t i ~ I I I I ~ 1 L i I I

AMT

XT

HMT

TMP

MMT

PSOIR

0 I0 20 50 ~ 50 60 70 80 90 I00 I10 12_0 1:~)014.0Minutes of Irrodiation

PHOTOADDITION OF TRIMETHYLPSORALEN,XANTHOTOXIN AND PSORALEN

Figure 5 I~netics of photoaddition of derivatives of trioxsalen, derivatives of 8-MOP,and psoralen to calf thymus DNA. Abbreviations are as follows: AMT,aminomethyltrioxsalen; XT, xanthotoxin or 8-methoxypsoralen; HMT, hydroxymethyltri-oxsalen; TMP, trioxsalen; MMT, methoxymethyltrioxsalen; and PSOR, psoralen.

fraction of intercalated molecules are at sites that are already conforma-tionally modified at the time of the pulse, so two photons can beabsorbed over a very short time, both leading to reaction. A secondattractive possibility is that a small number of excited molecules inter-system cross to a triplet biradical that is capable of reaction at bothends.

APPLICATIONS OF PSORALENPHOTOCHEMISTRY TO THE SOLUTION OFSIGNIFICANT STRUCTURAL PROBLEMS

Chromatin Structure

The nucleosome structure of chromatin imparts a specificity to thephotochemical reaction of the psoralens with DNA. The photoadditionoccurs primarily in the interbead regions of this structure. This has been


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


82 HEARST

demonstrated by denaturation electron microscopy on high molecularweight DNA isolated from Drosophila melanogaster nuclei after photore-action of the nuclei with trioxsalen [Hanson et al (14)] and also denaturation electron microscopy on short pieces of DNA obtained bymicrococcal nuclease digestion of these same nuclei [Wiesehahn et al(48,49), Hyde & Hearst (19)]. Tritium-labeled trioxsalen is removedpreferentially from these nuclei by micrococcal cleavage, demonstratingthat the sites of nuclease attack in nuclei and the sites of photoadditionof trioxsalen to chromatin are in dose proximity on the DNA.

Secondary Structure in Single-Stranded fd DNA

The photochemical cross-linking of DNA by 4,5’,8-trimethylpsoralen(tdoxsalen) has been used by Shen & Hearst (38) to freeze the ondary structures of single-stranded DNA molecules of bacteriophagefd at different ionic strengths. These secondary structures (hairpins orlooped hairpins) have been visualized in the electron microscope. Mostof the single-stranded circular fd DNA molecules show only one hairpinafter irradiation at 15° in 20 mM NaCI in the presence of trioxsalen. Asthe ionic strength is increased, more hairpins appear on the DNAmolecules. To map these secondary structures, double-stranded super-coiled fd DNA (RFI) was cleaved with the restriction enzyme HindII,which makes only one cut on each RFI molecule. After denaturationand cross-linking of the linear single-stranded fd DNA (a mixture ofviral and complementary strands), all the hairpins have been mapped onthe DNA molecule with respect to this HindlI site. The results showthat these hairpins occur at specific sites. The most stable hairpin hasbeen assigned to the position where the initiation site for the conversionof single-stranded fd DNA to the double-stranded covalently closedform has been mapped.

A Specific DNA Orientation in the Filamentous

Bacteriophage fd as Probed by Psoralen

Cross-linking and Electron Microscopy

Shen et al (43) have stabilized the molecular structure of single-strandedfd DNA inside its filamentous virion by the photochemical reactionwith a psoralen derivative and have examined it in the electron micro-scope. The results support the notion that the 6408 nucleotide-longDNA molecule is folded back on itself inside the 1 #m-long proteincoat. At one end of the virion, there exists a DNA hairpin region200___50 base-pairs long. This "end hairpin" is mapped on the fdgenome at the site of the replication origin. The most stable in vitro


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


PSORALEN PHOTOCHEIVffSTRY 83

hairpin of fd DNA has been shown previously to be at this same site.This unique duplex region of fd DNA may play an important role in theformation of specific protein-DNA complexes that are crucial to stagesof the fd life cycle: the adsorption of the phage to the bacteria, theinitiation of replication of the single-stranded DNA, and the assemblyof newly synthesized DNA strands into the filamentous virions.

A Technique for Relating Long-Range Base Pairing onSingle-Stranded DNA and Eukaryotic RNA Processing

Shen & Hearst (39) have shown that after photochemical cross-linkingwith psoralens in solutions of different concentrations of NaC1, short(100-300 base pairs) hairpins can be stabilized on denatured simianvirus 40 (SV40) DNA for electron microscope visualization. Six majorhairpins were mapped. This study was extended by Shen & Hearst (42)by cross-linking single-stranded SV40 linear molecules, EcoRI-SV40and BglI-SV40, in the presence of magnesium ions. After the reaction,in addition to the hairpins detected before, seven DNA hairpins, andhairpin loops with sizes from 300 to nearly 2000 nucleotides are ob-served. These loops are located~at specific regions on the SV40 genome.At least three of these loops are found in regions thought to be involvedin splicing of the early and late SV40 transcripts. These loop structuresare most likely to arise from base pairing between distant regions of thesingle-stranded DNA. RNA transcribed from the double-stranded DNAtemplate is expected to behave in a similar way, possibly providingrecognition sites for processing (splicing) enzymes.

Base Pairing Between Distant Regions of the Escherichiacoli 16S Ribosomal RNA in SolutionWollenzien et al (51) have introduced intermolecular cross-links idtoEscherichia coli 16S ribosomal RNA in aqueous solution by irradiationin the presence of hydroxymethyltrimethylpsoralen. When the cross-linked RNA is denatured and examined in the electron microscope, themost striking features are a variety of large open loops. In addition,because the cross-linked molecules are shortened compared to noncross-linked molecules, there are likely to be small hairpins that are notresolved by the present technique. The sizes and positions of 11 loopclasses have been determined and oriented on the molecule. Thefrequency of occurrence of the different classes of loops depends on thecross-linking conditions. When the cross-linking is done in solutionscontaining Mg2+, at least four of the loop classes appear with greaterfrequency than they do in 3-5 mM-NaC1. The loops presumably arise


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


8,4 HEARST

because complementary sequences separated by long intervening re-gions are being cross-linked. These base-pairing interactions betweenresidues distant in the primary structure appear to be prominent fea-tures of the secondary structure of rRNA in solution.

Cross-linking Studies on the Organization of the 16SRibosomal RNA Within the 30S Escherichia coliRibosomal Subunit

Thammana et al (46) have examined the structure of 16S R_NA in the30S subunit. The location and frequency of RNA cross-links induced byphotoreaction of hydroxymethyltrimethylpsoralen with 30S Escherichiacoli ribosomal subunits have been determined by electron microscopy.At least seven distinct cross-links between regions distant in the 16SrRNA primary structure are seen in the inactive conformation of the30S particle. All correspond to cross-linked features seen when the free16S rRNA is treated with hydroxymethyltrimethylpsoralen. The mostfrequently observed cross-link occurs between residues near one end ofthe molecule and residues about 600 nucleotides away, to generate aloop of 570 bases. The size and orientation of this feature indicate thatit corresponds to the cross-linked feature located at the 3" end of free16S rRNA.

When active 30S particles are crosslinked in 5 mM-Mg2+, six of theseven features seen in the inactive 30S particle can still be detected.However, the frequency of several of the features, and particularly the570-base loop feature, is dramatically decreased. This suggests that thelong-range contacts that lead to these cross-links are either absent orinaccessible in the active conformation. Cross-linking results in someloss of functional activities of the 30S particle. This is consistent withthe notion that the presence of the cross-link that generates the 570-baseloop traps the subunit in an inactive form, which cannot associate with50S particles.

The arrangement of the interacting regions cross-linked by hy-droxymethyltrimethylpsoralen suggests that the RNA may be organizedinto three general domains. A striking feature of the cross-linkingpattern is that three of the seven products involve regions near the 3’end of the 16S rRNA. These serve to tie together large sections ofrRNA. Thus, structural changes at the 3’ end could, in principle, be feltthroughout the entire 30S particle.


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


PSORALEN PHOTOCHEMISTRY

ACKNOWLEDGMENTS

I wish to acknowledge the assistance of the many scientists with whom Ihave collaborated in these last five years that I have devoted to psoralenchemistry. In particular, I acknowledge the assistance and friendship ofJean-Pierre Bachellerie, Stephen Isaaes, Henry Rapoport, and GaryWiesehahn.

This review has been supported in part by Grants GM 11180 andGM25151 from the United States Public Health Service, and GrantNP 185 from the American Cancer Society.

Literature Cited

1. BaeheIlerie, J. P., Hall, K., Hearst, J.E. 1980. Biochemistry. In press

2. Bachellerie, J. P., Hearst, J. E. 1980.Biochemistry. In press

3. Bachellerie, J. P., Thompson,Hearst, J. E. 1980. Biochemistry. Inpress

4. Bender, D. R., Hearst, J. E., Rapo-port, H. 1980. J. Org. Chem. 44:2176-80

5. Cassuto, E., Gross, N., Bardwell, E.,Howard-Flanders, P. 1977. Biochim.Biophys. Acta 475:589-600

6. Chandra, P., Marciani, S., Dall’Acqua,F., Vedaldi, D., Rodighier6, G., Bi-swa, R. K. 1973. FEBS Lett. 35:243-

7. Cole, R. S. 1971. Biochim. Biophys.Acta 254:30-39

8. Cole, R. S. 1973. Proc. Natl. Acad.Sci. USA 70:1064-68

9. Cole, R. S., Levitan, D., Sinden, R. R.1976. J. Mol. Biol. 103:39-59

10. Dall’Acqua, F., Marciani, S., Ciavatta,L., Rodighiero, G. 1971. Z. Natur-forsch. Teil B 26:561-69

11. Dall’Acqua, F., Marciani, S., Rodi-ghiero, G. 1970. FEBS Lett. 9:121-23

12. Dall’Acqua, F., Terboje~ch, M., Ben-venuto, F. 1968. Z. Naturforsch. TetlB 23:943-45

13. Egger, K. 1969. Thin Layer Chro-matography, ed. E. Stahl, pp. 687-706.New York: Springer-Verlag. 1041 pp.

14. Hanson, C. V., Riggs, J. L., Lennete,E. H. 1978. J. Gen. Virol. 40:345-58

15. Hearst, J. E., Rapoport, H., Isaac*, S.,Shen, C. -K. J. 1978. Psoralens. USPatent No. 4,124,598. Univ. Calif.,Berkeley, Nov. 7, 1978

16. Hearst, J. E., Rapoport, H., Isaacs, S.,Shen, C.-K. J. 1979. Psoralens. USPatent No. 4,169,204. Univ. Calif.,

Berkeley, Sept. 25, 197917. Hearst, J. E., Thiry, L. 1977. Nucleic

Acids Res. 4:1339-4718. Hochkeppel, H. -K., Gordon, J. 1979.

Biochemistry 18:2905-1019. Hyde, J. E., HearsL J. E. 1978. Bio-

chemistry 17:1251-5720. Isaac,, S. T., Chua, C., Hearst, J. E.

1981. Trends in Photoblology. NewYork: Plenum. In press

21. Isaac,, S. T., Shen, C. -K. J., HearsLJ. E., Rapoport, H. 1977. Biochemistry16:1058-64

22. Johnston, B. H., Hearst, J. E. 1980.Biochemistry. In press

23. Johnston, B. H. 1980. Thesis. Univ.Calif., Berkeley

24. Johnston, B. H., Johnson, M. A.,Moore, C. B., Hearst, J. E. 1977. Sci-ence 197:906-8

25. Johnston, B. H., Kung, A., Moore, C.B., Hearst, J. E. 1980. Biochemistry. Inpress

26. Kaufman, K. D. 1961. J. Org. Chem.26:117-21

27. Kaufman, K. D., Russey, W. E.,Worden, L. R. 1962. J. Org. Chem.27:875-79

28. Kaufman, K. D., Worden, L. R. 1961.J. Or8. Chem. 26:4721-22

29. Kaufman, K. D., Worden, L, R.,Lode, E. T., Strong, M. K., Reitz,C. 1970. J. Org. Chem. 35:157-60

30. Krauch, C. H., Kr~hner, D. M.,Wacker, A. 1967. Photochem. Photo-biol. 6:341-54

31. Lin, P. -F., Bradwell, E., Howard-Flanders, P. 1977. Proc. Natl. Acad.Sci. USA 74:291-95

32. Musajo, L., Bordin, F., Bevilacqua, R.1967. Photochem. Photobiol. 6:927-31

33. Musajo, L., Bordin, F., Caporale,Marciani, S., Rig~tti, G. 1967. Photo-


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


chem. Photobiol. 6:711-1934. Ou, C. -N., Song, P. -S. 1978. Bio-

chemistry 17:1054-5935. Pathak, M. A., Kriimer, D. M. 1969.

Biochim. Biophys. Acta 195:197- 20636. .Perone, V. B. 1972. Microbial Toxin~,

ed. S. Kadis, A. Ciegler, S. J. Ajl,8:71-80. New York: Academic. 400PP.

37. Rabin, D., Crothers, D. M. 1979.Nucleic Acids Res. 7:689-703

38. Shen, C. -K. J., Hearst, J. E. 1976.Proc. Natl. Acad. Sci. USA 73:26~9-53

39. Shen, C. -K. J., Hearst, J. E. 1977.Proc. Natl. Acad. Sci. USA 74:1363-67

40. Shen, C. -K. J., Hearst, J. E. 1977. J.Mol. Biol. 112:495-507

41. Shen, C. -K. J., Hearst, J. E. 1978.Cold Spring Harbor Symp. 42:179-89

42. Shen, C. -K. J., Hearst, J. E. 1979.Anal. Biochem. 95:108-16

43. Shen, C. -K. J., Ikoku, A., Hearst, J.E. 1979. J. Mol. Biol. 127:163-75

44. Sp/ith, E., Majunath, II. L., Pailer, M.,Jois, H. S. 1936. Ber. Dtsch. Chem.Ges. 69:1087-90

45. Straub, K., Kanne, D., Hearst, J. E.,Rapoport, H. 1980. J. Am. Chem. Soc.In press

46. Thammana, P., Cantor, C. R., Wol-lenzien, P. L., Hearst, J. E. 1980. J.MoL Biol. 135:271-83

47. Thompson, J., Wegnez, M., Hearst, J.E. 1980. J. Mol. Biol. In press

48. Wies~hahn, G., Hearst, J. E. 1976. InMolecular Mechanisms in the Controlof Gene Expression, ed. D. P. Nierlich,W. J. Rutter, C. F. Fox. 5:27-32.New York: Academic. 655 pp.

49. Wiesehahn, 13., Hyde, J. E., Hearst, J.E. 1977. Biochemistry 16:925-32

50. Wollenzien, P. L,, Hearst, J. E.,Squires, Catherine, Squires, Craig.1979. J. Mol. Biol. 135:285-91

51. Wollenzien, P., Hearst, J. E., Tham-maria, P., Cantor, C. R. 1979. J. Mol.Biol, 135:255-69


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.


Ann

u. R

ev. B

ioph

ys. B

ioen

g. 1

981.

10:6

9-86

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

UC

Ber

kele

y on

08/

16/0

5. F

or p

erso

nal u

se o

nly.

Documents

PSORALEN PHOTOCHEMISTRY #9159 · The adducts of 4’-hydroxymethyl 4,5’,8-trimethylpsoralen to both DNA and RNA have been isolated by enzymatic degradation of the photore-acted