J. theor. Biol. (1979) 78, 61-75

Sequence-Specific Crosslinking Agents for Nucleic Acids: Design and Functional Group Testing JAMES SUMMERTON?

Department of Molecular Biology, University of California, Berkeley, California 94720, U.S.A. (Received 18 September 1978) The genetic material of most organisms is composed of two complementary strands of DNA fitting together much like a long zipper. For this double helical DNA to function the two strands must first separate and then one or both of the separated strands can code for RNA or new copies of DNA. Herein a general method is outlined for sequence-specific inactivation of DNA and RNA. This involves attachment of a new class of crosslinking agent of the form NH,NHCO(CH,),C(OR),CHR’X at multiple sites on a single-stranded polynucleotide (the carrier). These compounds are designed to form covalent interstrand crosslinks when the derivatized carrier strand is annealed with a complementary polynucleonde (the target). Such crosslinks between a carrier and its target should irreversibly block that specific sequence with respect to all information transfer processes (replication, transcription, translation). Experimental studies are reported concerning optimal conditions for transamination of cytidine by acylhydrazides (attachment step), acid catalyzed conversion of a-haloketals to a-haloketones (activation step), and alkylation of guanosine by a-haloketones (crosslinking step). The requirements of these three reactions are discussed with regard to the constraints they place on the parameters of the crosslinking agent (n, R, R', and 2’). Nucleosides and model compounds corresponding to the two halves of the crosslinking agent were used to facilitate study of the reactions and characterization of the products.

1. Introduction I propose that inactivation of specific genetic sequences can be accomplished with a specially designed bifunctional agent bound to a single-stranded carrier polynucleotide such that upon annealing of the carrier with its complementary target sequence the carrier-bound agent crossiinks the two sequences. As a consequence of the specificity residing in the carrier sequence rather than in the crosslinking agent a single compound should suffice for inactivation of all desired target sequences. 7 Current address: Biochemistry-Biophysics Department, Oregon State University, Corvalfis, Oregon 97331 U.S.A. 61 0 1979 Academic Press Inc. (London) Ltd. 0022-5193/79/090061+15 $02.00/o

62

J.

SUMMERTON

A compound with the above characteristics should be useful as a tool for in repair, transcription, and translation. A particularly interesting application is the systematic mapping and characterization of viral genomes through the introduction of specific blocks using agent-derivatized restriction fragments. Since cells have natural mechanisms for taking up single-stranded DNA and pairing this newly-taken-up DNA with complementary sequences within the cell (Lewin, 1977) it may be possible to exploit these natural mechanisms as a means for delivering agent-derivatized carrier polynucleotides to their target sequences within living cells. If such in viva delivery proves feasible, the agent would have wide application due to its potential as a sequence-specific mutagen. In bacteria a technique for blocking specific sequences might be applied to answering questions about direction and control of replication, in vivo mechanisms of DNA repair, and the effects of specific blockage on transcription of polycistronic genes. If sequence-specific inactivation can be carried out in eucaryotic cells then the agent would have, among others, applications revolving around the function and control of the “oncogene” (src gene in Rous transformed chicken cells, etc.). In addition to its experimental applications, the compound could conceivably enable a fundamental approach to treating viral diseases and virally-mediated cancers by providing a means for intracellular inactivation of the pathogens’ genetic material. A variety of compounds that crosslink DNA have been described. These include bifunctional alkylating agents such as diepoxides and the nitrogen and sulfur mustards, nitrous acid, platinum compounds, mitomycin C, and fucocoumarins (psoralen being the best known). However, none of these compounds can be used to block a particular nucleotide sequence to the exclusion of other similar sequences. In 1967 Belikova, Zarytova & Grineva reported the first compound which could be attached directly to an oligonucleotide such that when the derivatized oligonucleotide was annealed with a complementary nucleotide sequence the bound agent crosslinked the paired sequences, thereby blocking the target sequence at a specific site. More recently several additional compounds have been reported which can be attached directly to oligonucleotides for the purpose of crosslinking to complementary structures (Budker et al., 1974; Pongs & Lanka, 1975; Schwartz & Ofengand, 1974; Wagner & Gassen, 1975). However, none of these compounds are generally adaptable for crosslinking any desired polynucleotide to its complementary sequence because they require unusual functional groups on the carrier strand that are specific for each system. Furthermore, a common feature of these compounds is that only one molecule can be attached per oligonucleotide chain. Although this is an acceptable limitation in the applications for which vitro studies of essentially all genetic mechanisms including replication,

SEQUENCE-SPECIFIC

CROSSLINKING

AGENTS

63

these agents were designed it is unlikely such compounds could irreversibly block a long nucieotide sequence in a complex in vitro system or in vivo. Irreversible blockage in a complex system will likely require many crosslinks because of two factors: first, alkylation at the most commonly alkylated site in DNA-the N7 of guanine residues-leads to a somewhat unstable crosslink which can be broken via depurination of the alkylated guanine; second, cells contain complex repair systems capable of repairing low levels of interstrand crosslinks in DNA (Cole, Levitan & Sinden, 1976). Described below are the design criteria for a class of crosslinking agents which I believe will prove suitable for general application in complex biological systems. The first step in the design of potential sequence-specific crosslinking agents was to carry out a survey of the literature on compounds known to react at specific sites on nucleic acids and to make a thorough study of a CPK space filling molecular model of a segment of double-helical DNA. By that means the following sites and functional groups were selected. The C4 position of cytosine was picked as the attachment site for the crosslinking agent because it allows attachment of many molecules of agent to essentially all nucleic acids; it faces the major groove in a double-helix configuration ; and it is proximal via the major groove to the N7 position of a base-paired guanine. An acylhydrazide was chosen as the attachment moiety of the agent because it specifically transaminates the C4 position of cytosine under conditions where nucleic acids are not damaged (Hayatsu & Ukita, 1964). The N7 position of guanine was chosen as the site for the crosslinking reaction both because it is accessible to an agent bound at the C4 of a base-paired cytosine, and because it is, in the case of most alkylating agents, the most readily alkylated site on the bases (Price et al., 1969). An a-haloketal was selected as the crosslinking moiety of the agent. This group is compatible with acylhydrazides; it can be converted by mild acid hydrolysis to an a-haloketone; and the reactivity of the activated form (a-haloketone) can be varied by changing the halogen and/or changing a modifying group on the cc-carbon. At the time this moiety was selected the reactivity of a-haloketones with guanine residues had not been tested but c+haloketones had been reported to react with the imidazole ring of histidine residues at the active sites of several proteolytic enzymes (Schoellman & Shaw, 1963). It thus seemed reasonable to expect a-haloketones to react with the imidazole ring of a guanine residue-presumably at the N7 position. Figure 1(a) illustrates the expected structures and reactions in the formation of a crosslink by the prospective agents. Note that the parameters n, R, R', and 2’ are unspecified in the crosslinking agent (structure fa of Fig. 1). The experimental portion of this paper deals with specifying these parameters, finding optimal reaction conditions, and characterizing the products in the

resdue of carr,er 0 ‘\\ ,C--Wdn UN \ NH / 2a

0

? R

I

0 --L-L

of target

H-

R’ x

cytldine

H

(b)

u,

)-(CH HN

\ HNH

HN’ lb

0 2 )3-CH3

‘L(CH*)3-CH3

0

SEQUENCE-SPECIFIC

CROSSLINKING

65

AGENTS

crosslinking sequence. Model compounds as illustrated in Fig. l(b) were used in place of crosslinking agents and polynucleotides to facilitate characterization of these conditions and products. In a separate paper (Summerton & Bartlett, 1978) results from the model studies illustrated in Fig. l(b) have been applied in the synthesis and use of a crosslinking agent of structure la. Applications of that compound include linking cytidine to guanosine as illustrated in Fig. l(a) and crosslinking RNA to its complementary DNA. 2. Experimental (A)

TRANSAMINATION

OF CYTIDINE

BY PENTANOHYDRAZIDE

: PREPARATION

OF

2b

A mixture of 2.0 g pentanohydrazide (m.p. 63” ; Beilstein El 11, 2 p. 677) 0.5 g cytidine, 0.6 ml water, and 0.25 ml 91 “/;, formic acid was incubated 14 h at 37°C (final pH = 4.1). The reaction mixture was added to 100 ml of ether and the ether insoluble residue fractionated by descending paper chromatography (developing solvent 1: n-butanol/water/ethanol ; 16/5/2 by vol.). The product (R, = 064) was eluted with water, dried, and recrystallized from a mixture of acetone and ethylacetate; giving a compound (m.p. 153”) which chromatographed as a single spot in solvent 1 and solvent 2 (isopropanol/ 1% aqueous ammonium formate; 2/l by vol.) (Rf = O-78). Elemental analysis for C1,H,,N,06 (342.35): calculated: found:

C, 49.11; H, 6.48; N, 16.37; C, 48.96; H, 6.39; N, 16.19.

The pK, values, measured spectrophotometrically, were 2.1 and 10.2. U.V. spectra showed Aa, at pH 1, 5.5, and 13 of 287 nm, 255 nm, and 299 nm respectively with an .sZg9at pH 13 of 205 x 103. In a separate experiment the rate of formation of this product was found to be maximal at pH 3.9. FIG. 1. Crosslink formation. (a) Attachment entails bonding between the attachment site (C4 of cytosine) of the carrier strand and the attaching moiety of the crosslinking agent (NH,NHCOof structure la). Activation of the bound agent (structure 2~) entails acid catalyzed conversion of the ketal to a ketone to give structure 3~. Annealing between the activated carrier strand and a complementary target strand pairs a guanine residue with a derivatized cytosine residue. thereby placing an activated crosslinking moiety in proximity to the crosslinking site (N7 of guanine) of the target strand. Subsequent alkylation of the N7 should give the desired interstrand crosslink (structure 4~). (b) Attachment was studied using cytidine and compound lb. Nine ketals were used in studying the activation step. These included the dimethoxy (R = CH,), diethoxy (R = CH,CHZ), and ethylenedioxy (R = CH,) ketals of chloroacetone (R’ = H, X = Cl). bromoacetone (R’ = H, X = Br), and 3-bromo-2-butanone (R’ = CH,, X = Br). The crosslinking reaction was studied using guanosine and the three a-haloketones, chloroacetone, bromoacetone, and 3-bromo-2-butanone. Finally, stabilities of compounds Z’b, Zb, 36. and 46 were characterized.

66

J.

SUMMERTON

Based on a comparison of the properties of this compound (pK.‘s, U.V. spectra, .cbmax in alkali, and pH optimum for formation) with properties of a variety of 4-deamino-4-acylhydrazidocytidines reported in the literature (Hayatsu & Ukita, 1964; Kikugawa, Hayatsu & Ukita, 1967; Gal-Or, Mellema, Moudrianakis & Beer, 1967 ; Kikugawa, Hayatsu & Ukita, 1969) I conclude this new product is 4-deamino-4pentanohydrazidocytidine (structure 2b of Fig. 1). The attachment of pentanohydrazide at the C4 of cytidine was further characterized with respect to its rate as a function of water concentration in the reaction mixture. When water concentration was decreased from 75 y0 of the reaction mixture (v/u) to 25 7; (ethanol replacing the water), the reaction rate was increased 22 %. This suggests that decreasing water concentration to stabilize ketal moieties of the agent during the attachment step will not adversely affect the rate of attachment. The stability of this cytidine derivative (2b) in HzO, 0.1~ HCl, and 0.1~ NaOH was determined spectrophotometrically and verified by paper chromatography. After 67 h at 25°C no more that 2 ‘4 of the material was degraded in either Hz0 or NaOH, while in the same time period 40 7; of the compound was degraded in 0.1~ HCl. (B)

STABILITIESOF

a-HALOKETALS

UNDER

CONDITIONS:CONVERSIONOF

ATTACHMENTAND

2’b

TO

ACTIVATION

3b

Three a-haloketals (dimethoxy, R = CH,; diethoxy, R = CH,CH,; and ethylenedioxy, R = CHJ were prepared from each of the following ahaloketones: bromoacetone (R’ = H, X= Br), 3-bromo-2-butanone (R = CH,, X = Br), and chloroacetone (R’ = H, X = Cl). R, R’, and Xrefer to structural parameters of the compunds shown in Fig. 1. Since the crosslinking moiety of the prospective agent must remain in the protected state (ketal) during the attachment step [Fig. l(a)], stabilities of the four least-stable ketals were measured under attachment conditions (pH 41, 50% water by vol., 2YC), as shown in Fig. 2(a). In Fig. 2(b) the stability of a representative ketal under attachment conditions (pH 4*1,25X) was determined as a function of water concentration. Finally, the hydrolysis rates of all nine ketals were measured under standard activation conditions (pH 2.5, > 99 % H,O, ZYC), as shown in Fig. 2(c). (c)

STABILITY•

FSINGLE-STRANDEDDNAUNDERACTIVATI~NCONDITI~NS

DNA is known to undergo depurination in acidic conditions (Tamm, Hodes & Chargaff, 1962) and thus it was of interest to determine the rate at which a carrier DNA strand will sustain depurination damage under

SEQUENCE-SPECIFIC

CROSSLINKING

AGENTS

):

i-

I-

me ,_

! -

(a) I 2

I 4

I 6

Time(h)

III 0

30

I 45

I 60

% H,O

10

20 Time

30

4( 1

(min)

FIG. 2. Haloketal hydrolysis. The ketals in these hydrolysis experiments are the 3-bromo-2,2dialkoxybutanes (m), the I-bromo-2,2-dialkoxypropanes (a), and the I-chloro-2,2dialkoxypropanes (A). The alkoxy radicals include methoxy (me), ethoxy (et), and ethylenedioxy (ed). (a) Hydrolysis of the 4 least-stable ketals at pH 4.1, 50% H,O, and 25°C (standard attachment conditions). (b) Hydrolysis of 3-bromo-2,2-diethoxybutane as a function of water concentration. (c) Hydrolysis of ketals at pH 2.5 in > 99 7; Hz0 at 25°C (standard activation conditions).

conditions used for activation of the carrier-bound agent (Fig. l(a)). For this purpose a solution of salmon sperm DNA in the single-stranded state was incubated under standard activation conditions (pH 25, > 99 ‘T!! H20, 25T). At 0, 1, 2, 4, and 8 h samples were neutralized ; DNA was removed by precipitation with ethanol; supernatants were passed through an anion exchange column to remove any nucleotides or oligonucleotides; and, after adjustment to pH 2, the AZs2 of the solutions were measured. Absorbance at 252 nm was used because at this wavelength adenine and guanine (the alcoholsoluble products of depurination) have equivalent molar absorption coefficients (cts2 = 11.0 x 103) at pH 2. Under the conditions of this experiment single-stranded DNA was found to lose 065 7; of its purines per hour. (D)

STABILITIES

OF Q-HALOKETONES:

STRUCTURE

3b

Activated crosslinking moieties must be stable both during the activation step and during the time required for the carrier polynucleotide to find and anneal with its complementary target sequence. Since nucleic acids can be

68

J.

SUMMERTON

annealed in neutral conditions at 37°C in the presence of appropriate concentrations of such denaturing agents as dimethylsulfoxide, formamide, and sodium perchlorate, and since in vivo applications would most likely entail conditions of neutral pH and 37°C the stabilities of a-haloketones were measured under these conditions (pH 7, 37°C) as well as in activation conditions (pH 2.5,25”(Z) and alkaline conditions (pH 13,25”(Z). Stabilities of chloroacetone, bromoacetone, and 3-bromo-2-butanone in dilute aqueous solution (~0.5’4 haloketone by vol.) at pH 25 were assessed spectrophotometrically at their respective i,,, ~~~~~ = 21 for chloroacetone; s2s4 = 45 for bromoacetone; .s2s9= 101 for 3-bromo-2-butanone). The three compounds were found to undergo no detectable degradation at this pH over a period of 1 h at 25°C. For verification it was shown that no bromide or chloride ion was released under these same conditions. The stabilities of the three haloketones in neutral (0.2~ phosphate to pH 7.0 with NaOH) and alkaline (0.3~ NaOH) solutions (1.25 9; haloketone by vol.) were assessed by measuring released halide ion. All three haloketones were completely degraded within 5 min in alkali at 25°C. The times for 50 *jO degradation in neutral solution at 37°C were found to be 6 h for 3-bromo-2butanone, 13 h for bromoacetone, and 7 days for chloroacetone. (E)

ALKYLATION

OF GUANOSINE

BY Q-HALOKETONES:

PREPARATION

OF

4b

A mixture of 7 ml formamide, I .25 ml H,O, 0.5 g guanosine, and 0.5 ml bromoacetone (freshly distilled) was incubated for 21 h at 37°C. The mixture was extracted with 100 ml ether to remove residual bromoacetone, and then fractionated by paper electrophoresis using 0.05~ ammonium acetate adjusted to pH 5.5 with acetic acid. The high-electrophoretic-mobility material (positively charged), showing a deep blue fluorescence under 254 nm light, was eluted and further resolved by descending chromatography (solvent 1) into a major component (Rf = 0.20) and several minor components (Rf’s > 0.25). The major component was eluted and recrystallized from a mixture of methanol and ether. The product (structure 4b) chromatographed as a single spot in solvent 1 and solvent 2 (Rf = O-52) and moved as a single spot during electrophoresis at pH 5.5. It had a pK, of 7.1 (determined spectrophotometrically) and its U.V. spectra at both pH 1 and 5.5 showed a i,,, at 258 nm. The compound was somewhat unstable below pH 2 and above pH 8 and the ionized form tended to lose its countervion when dried under vacuum. Elemental analysis of 46 gave variable results-presumably because of these instabilities. Alkali treatment of 4b converted it to a single new product (Rf = 0.30 in solvent 1 and 0.46 in solvent 2) which was no longer fluorescent under 254 nm

SEQUENCE-SPECIFIC

CROSSIJNKING

AGENTS

69

light ; carried no charge at pH 5.5 ; and had A,,, at pH 1 and 13 of 27 1 nm and 264 nm respectively-all characteristics common to imidazole-ring-opened 7alkylguanosines (Haines, Reese & Todd, 1962; Brookes, Dipple & Lawley, 1968; Singer, 1972; Lawley & Jarman, 1972). Acid treatment of 4b (0.1~ HCl for 90 min at 100°C) converted it to a new product (dec 380’) with U.V. spectra at pH 1, 5.5, and 13 (A,,,,, of 250 nm, 284 nm, and 280 nm respectively) characteristic for 7-alkylguanines (Haines et al., 1962; Fasman, 1975). Elemental analysis for this aglycone, C,H,N,02 (207.19): calculated: found:

C, 46.37; H, 4.38; N, 33.80; C, 46.09; H, 4.51; N, 32.93.

On the basis of a comparison of the properties of 4b (fluorescence, pK,, U.V. spectra, acid and alkali lability) and the properties of its products arising from acid and alkali treatment with the properties of a variety of previously reported 7-alkylguanosines (Haines et al., 1962; Brookes et al., 1968; Singer, 1972; Lawley & Jarman, 1972; Fasman, 1975) I conclude the major product from the reaction of bromoacetone with guanosine is 7-acetonylguanosine (structure 4b where R’ = H). Chloroacetone and 3-bromo-2-butanone also react with guanosine to give products with pK,, spectra, and alkali labilities identical to that of 7-acetonylguanosine. Relative rates were determined for the alkylation of guanosine by bromoacetone, chloroacetone, and 3-bromo2-butanone, as shown in Fig. 3. 3. Discussion

The objective of this project is the development of a general method for crosslinking specific nucleotide sequences. The three reactions in this crosslinking scheme are transamination of cytosine residues by an acylhydrazide moiety (attachment); acid hydrolysis of c+haloketals (activation); and alkylation of guanine residues by a-haloketones (crosslinking). In the experimental section all three reactions were shown to occur in the desired manner under conditions in which nucleic acids are both soluble and relatively stable. The parameters which remain to be specified in the crosslinking agent (structure la of Fig. 1) include: X, the halogen; R’, a modifying group on the halogen-bearing carbon; R, the alkyl group of the ketal; and n, the number of methylenes linking the attachment and crosslinking moieties. These parameters will now be considered in the context of the constraints arising from the requirements of the attachment, activation, and crosslinking reactions.

70

J. SUMMERTON

l-

I-

l-

20,

,

Time(h)

FIG. 3. Rate of reaction between guanosine and a-haloketones. A 1: 1 (by vol.) solution of ethanol and phosphate buffer (0.4~ H,POI to pH 5 with trizma base) was saturated with guanosine. One volume of this guanosine solution (A *s2 = 80) was mixed with 0.18 vol. ahaloketone (chloroacetone, A; 3-bromo-2-butanone, H; bromoacetone, 0) and incubated at 37’C. At various times, 0.2-ml samples were extracted with ether, treated with alkali, adjusted to pH 1 with HCl and the ALSOand AzT2 measured (at pH 1 the& 2s0of guanosine is equal to the s2s0 of alkali-treated 7-acetonylguanosine). The AzT2/A 2s,, is O-756 for guanosine, and 2.70 for the high-electrophoretic-mobility fraction. The percent reaction at time t was calculated as:

&,~/Am)r--0~756 2.70 - 0.756 These values for per cent guanosine derivatized correlate well with values determined by fractionating reaction mixtures by electrophoresis and quantitating the zero-electrophoreticmobility fraction (guanosine) and the high-electrophoretic-mobility fraction (7-alkylated guanosine derivatives).

(A)

OPTIMAL

REACTIVITY

FOR

THE

CROSSLINKING

MOIETY:

R’

AND

x

Since an insufficiently reactive crosslinking moiety will not give the desired crosslink in an acceptable length of time and an overly reactive moiety will lack the desired specificity (i.e. it will react with structures other than the

SEQUENCE-SPECIFIC

CROSSLINKING

71

AGENTS

target strand) it is of value to determine an optimal reactivity for the prospective crosslinking agent. Although final determination of the best reactivity will likely require studies carried out at the polynucleotide level, information on the relative rates of alkylation by the three a-haloketones described in section 2(e) should considerably simplify these future optimization studies. As illustrated in Fig. 3, the reactivity of the crosslinking moiety with guanosine is a function of both R’ and X. Relative reactivities available include: 1 (R’ = H, X = Cl); 6.3 (R’ = CH3, X = Br); and 46 (R’ = H, X = Br). Finally, as noted in section 2(d), the above three crosslinking moieties in the activated state have acceptable stabilities under both activation and annealing conditions. (B)

OPTIMAL

STABILITY

FOR

THE

PROT’BCTIVE MOIETY:

GROUP

OF THE

CROSSLINKING

R

The attachment step requires conditions in which single-stranded nucleic acids are soluble and the ketal of the agent is not significantly hydrolyzed. These requirements are satisfied by a pH of 4.1 (the attachment rate decreases substantially at higher pH values) and the presence of the minimum concentration of water compatible with solubility of the nucleic acid being derivatized (Fig. 2(b) demonstrates the relationship between water concentration and rate of ketal hydrolysis). It should be noted that some water seems to be required for the attachment reaction since I find little or no attachment occurs in nonaqueous solvents such as formamide and dimethylsulfoxide. Under reasonable attachment conditions (0.05~ cytidine, 2~ acylhydrazide, 50 “/:, Hz0 by vol., pH 4.1) about 2.5 % of the cytidine is derivatized per hour. The activation step requires a pH sufficiently low that the ketal will be hydrolyzed within a reasonable period of time. However, since nucleic acids are precipitated by acid, care must be taken in choosing the conditions for activation. Zimmerman (1973) has reported on the solubility of polynucleotides as a function of pH and ionic strength. His results indicate that essentially all nucleic acids (except polyC) are soluble in aqueous solution at pH 2.5 and less than 0.9~ salt. In view of these solubility considerations, and because the ketals under consideration have reasonable hydrolysis rates at pH 2.5 and 25”C, these conditions were chosen for the activation conditions. Additional points to consider are: under these conditions the cytidine-agent linkage is quite stable [section 2(a)] ; activated crosslinking moieties (a-haloketones) are stable [section 2(d)] ; and DNA loses only 0.65 “/;, of its purines per hour [section 2(c)].

72

.I.

SUMMERTON

Since rx-haloketones react readily with acylhydrazides (forming hydrazones and a-aminoketones) the crosslinking moiety is initially in a protected state (ketal) and it must remain so throughout the attachment step. In contrast to its required stability during attachment, the protective group should be sufficiently labile that the carrier-bound agent can be activated without excessive damage to the carrier polynucleotide. In Fig. 4 I have tabulated the estimated fraction of cytosine residues which can be derivatized by each of the prospective agents under standard attachment conditions during the period of

FIG. 4. Ketal stabilities under attachment and activation conditions. The times for 5 7; hydrolysis of the various ketal moieties under standard aftachment conditions were estimated from the results shown in Fig. 2(a). The corresponding percents of cytosine residues which could be derivatized by agents containing the respective ketals (S) were then calculated on the basis of an attachment of 2.5 7; of cytosines derivatized per hour. The times for 90 “/I hydrolysis of the various ketal moieties under activation conditions were estimated from the results shown in Fig. 2(c). The corresponding percents of purines which would be lost during these activation times (0) were calculated on the basis of a depurination rate of 0.65 7; per hour.

time in which only 5 % of their respective ketal moieties would be hydrolyzed. Also shown is the fraction of purines expected to be lost from a DNA carrier strand during activation of 90% of each of the prospective agents. Agents containing ethylenedioxy ketals are not included in the figure because carrier strands would be excessively depurinated long before the agents were even marginally activated. Values in Fig. 4 suggest that the most suitable group for most applications will be the dimethoxy ketal (R = CH,).

SEQUENCE-SPECIFIC (C)

OPTIMAL

NUMBER

OF

CROSSLINKING

METHYLENES LINKING

FOR

AGENTS

LINKING

MOIETIES:

ATTACHMENT

73 AND

CROSS-

!‘I

The number of methylenes needed to link the attachment and crosslinking groups (n) was estimated in the following way: CPK atomic models (Eahng Corp.) were used to construct a guanine-cytosine base-pair with attaching and crosslinking groups bound at appropriate sites. Methylenes were then added to bridge the gap between the bound groups. Results with these models suggest that IZshould be 3. A model of the crosslinking agent containing three methylenes was attached at the C4 of cytosine (structure 3a of Fig. 1) in a segment of DNA double helix constructed from CPK atomic models. The structure fit easily into the major groove of the helix, suggesting that the agent will not seriously interfere with base-pairing between a derivatized cytosine residue and its complementary guanine residue. Finally, a model of the crosslink structure (4~ of Fig. 1) containing three methylenes was attached at the appropriate positions on a G-C base-pair of the DNA model. The structure bridged from the C4 of cytosine to the N7 of a paired guanine and required no displacement of structures bounding the major groove. This suggests that in the double-helix configuration, the N7 of guanine is readily accessible to an agent bound at the C4 of a paired cytosine.

(D)

PREDICTED

STABILITY

OF THE

INTERSTRAND

CROSSLINK

The cytidine-agent linkage [section 2(a)] was found to be quite stable in conditions ranging from strong acid (half-time of cleavage = 80 h in O,~M HCl) to strong alkali (no significant cleavage after 1 week in 0.1~ NaOH). Thus the stability of the interstrand crosslink (structure 4a of Fig. 1) is expected to be primarily a function of the sugar moiety of the crosslinked guanine residue. For the case of a crosslinked guanosine (sugar = ribose) one would predict the crosslink should be stable in conditions ranging from mild acid to strong alkali (Haines et al., 1962)-and indeed, for the case of cytidine linked to guanosine by the compound 6-bromo-5,5-dimethoxyhexanohydrazide, this has been found to be true (Summerton & Bartlett, 1978). It should be noted that although alkali opens the imidazole ring of the crosslinked guanosine this does not break the interstrand crosslink since the glycosidic bond of guanosine remains intact. For the case of a crosslinked deoxyguanosine (sugar = deoxyribose) its glycosidic bond is expected to undergo cleavage at pH 7 and below, thereby breaking the interstrand crosslink. Estimates of the half-time of this cleavage at pH 7 and 37°C in 7-methyldeoxyguanosine range from 17 h (Lawley &

74

J.

SUMMERTON

Brookes, 1963) to 36 h (Haines et al., 1962). However, I contend that an interstrand crosslink to a guanine residue in a target DNA strand will be somewhat more stable than would be predicted on the basis of the glycosidic bond stability of 7-methyldeoxyguanosine. I base this contention on work by Hendler, Furer & Srinivasan (1970) who have reported that depurination of 7alkylated deoxyguanosine is significantly slower when it is a part of a polynucleotide. It should be noted that depurination of DNA leads to opening of the deoxyribose ring, rendering the adjoining 3’-phosphate ester sensitive to hydrolysis by alkali or heat (Tamm et al., 1953). Consequently, if a DNA target strand is crosslinked and the crosslinked deoxyguanosine subsequently undergoes depurination, the target strand will become susceptible to strand scission at the apurinic site. Above pH 9 crosslinked deoxyguanosine should undergo opening of the imidazole ring in the same manner as crosslinked guanosine. This in turn should stabilize the glycosidic bond (and hence the interstrand crosslink) toward conditions ranging from mild acid to strong alkali (Haines et al., 1962). (E)

EXPERIMENTAL

EVIDENCE

FOR THE

FEASIBILITY

OF THE

PROPOSED

CROSSLINKING

SCHEME

A separate paper (Summerton & Bartlett, 1978) describes the synthesis of 6-bromo-5,5-dimethoxyhexanohydrazide, its attachment to cytidine, its activation, and its subsequent linking to guanosine. The resulting product is shown to have electrophoretic and spectral properties consistant with structure 4a of Fig. 1. Also reported is the attachment of this crosslinking agent to T7-RNA, activation of this derivatized RNA, and annealing to complementary T7-DNA. Evidence is presented indicating the formation of covalent interstrand crosslinks between the complementary sequences. I would like to thank C. K. Mathews, Marshall Dinowitz, and V. Hruby, all of the University of Arizona, for their encouragement and helpful criticism during the planning of this project; Robert Shapiro of New York University for suggesting a protected crosslinking moiety; F. R. Green for help with practical chemical matters; and J. Kusmierek for helpful discussions of experimental techniques. I am particularly indebted to Paul Bar&t for bringing to my attention a-haloketones as alkylating agents and for his continual advice and assistance during the course of this work. Critical review of this manuscript by Kathleen Hercules is gratefully acknowledged. Finally, I would like to express my appreciation to H. Fraenkel-Conrat and B. Singer for jointly sponsoring this project, and for their advice, help, and constructive criticism throughout my two years in their laboratory. Financial support for this work included National Institutes of Health Postdoctoral Fellowships CA-04039 and CA-05883, Research Grant CA-12316 from the National

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