Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion

Psoralen intercalates into double-stranded DNA and photoreacts mainly with thymines to form monoadducts and interstrand cross-links. We used an oligonucleotide model to demonstrate a novel mechanism: the reversal of psoralen cross-links by base-catalyzed rearrangement at 90 degrees C (BCR). The BCR reaction is more efficient than the photoreversal reaction. We show that the BCR occurs predominantly on the furan side of a psoralen cross-link. The cleavage does not result in the breaking of the DNA backbone, and the thymine base freed from the cross-link by the cleavage reaction appears to be unmodified. Similarly, BCR of the furan-side monoadduct of psoralen removed the psoralen molecule and regenerated the unaltered native oligonucleotide. The pyrone-side psoralen monoadduct is relatively resistant to BCR. One can use BCR to perform efficient oligonucleotide-directed, site-specific delivery of a psoralen monoadduct. As a demonstration of this approach, we have hybridized a 19 base long oligonucleotide vehicle containing a furan-side psoralen monoadduct to a 56 base long complementary oligonucleotide target strand and formed a specific cross-link at the target site with 365-nm UV. Subsequent BCR released the oligonucleotide vehicle and deposited the psoralen at the target site.     

Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae.

Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer). We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins. The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides. A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN. When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends. The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used. Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro. We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer. The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing.     

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.     

The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers.

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.     

Long-range guanine oxidation in DNA restriction fragments by a triplex-directed naphthalene diimide intercalator

Naphthalene diimide (NDI), a powerful oxidant that binds avidly to DNA by intercalation, is seen to damage the 5' guanine of 5'-GG-3' sites by photoactivated charge transport through DNA. When covalently tethered to the center of a triplex-forming oligonucleotide and delivered by triplex formation within a pyrimidine.purine-pyrimidine motif to a specific site on a restriction fragment, NDI can photooxidize guanine over at least 25-38 bp in each direction from the site of binding. Charge migration occurs in both directions from the NDI intercalator and on both DNA strands of the target, but the oxidation is significantly more efficient to the 3' side of the triplex. NDI and octahedral rhodium intercalators, when tethered directly to the 5' terminus of the triplex-forming strand as opposed to the center, generate significant amounts of oxidative damage only in the immediate vicinity of the intercalation site. Given that long-range charge transport depends on DNA stacking, these results suggest that the base stack is distorted at the 5' end of the triplex region in the duplex-triplex junction. Targeting of photooxidative damage by triplex formation extends our previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed. Moreover, triplex targeting of oxidative damage provides for the first time a typical distance distribution for genomic charge transport of approximately 200 A around the oxidant.     

Photoreactivities and thermal properties of psoralen cross-links

We have studied the photoreaction of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP), and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) with a pair of 18-base-long oligonucleotides in which a 14-base region is complementary. Only one 5'TpA site, favored for both monoadduct and cross-link formation with psoralen, is present in this oligonucleotide pair. We have used this model system to demonstrate, for the first time, strand specificity in the photoreaction of psoralen with DNA. We found that the two types of cross-links which form at this site have large differences in thermal stabilities. In addition, the denaturation of each cross-link isomer duplex occurred in at least three stages, which can be visualized as three bands in thermal equilibrium under the conditions of a denaturing polyacrylamide gel. This novel observation suggests that there are several domains differing in thermal stability in a psoralen cross-link.     

Thermostability of double-stranded deoxyribonucleic acids: effects of covalent additions of a psoralen

We have carried out a thermodynamic study on the effects of covalent additions of the psoralen derivative HMT, 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, on the stability of double-stranded deoxyoligonucleotides. This was done with two systems. The first was a double-stranded DNA formed by two non-self-complementary oligonucleotides, 5'-GAAGCTACGAGC-3' and 5'-GCTCGTAGCTTC-3', where we site specifically placed an HMT molecule on the thymidine residue in oligonucleotide 5'-GAAGCTACGAGC-3' as either a furan-side monoadduct or a pyrone-side monoadduct. The second was a double-stranded DNA formed by a self-complementary oligonucleotide, 5'-GGGTACCC-3', where we placed an HMT molecule on the thymidine residue of each strand as a furan-side monoadduct or cross-linked the two strands with an HMT molecule linked to the two thymidines. We found that HMT cross-linking of the two strands stabilizes the double helix formed by 5'-GGGTACCC-3', as one might expect. Less predictable results were that the monoaddition of a psoralen stabilizes the double helix formed by the two non-self-complementary oligonucleotides by as much as 1.3 kcal/mol as a furan-side monoadduct and 0.7 kcal/mol as a pyrone-side monoadduct at 25 degrees C in 50 mM NaCl. In contrast, the monoaddition of a psoralen on each of the two thymidines in the double helix formed by 5'-GGGTACCC-3' destabilizes the helix by 1.8 kcal/mol at 25 degrees C in 1 M NaCl. This destabilization arises from an unfavorable enthalpy change (8.6 kcal/mol) and a favorable entropy change (23 cal/K X mol) due to the two HMT molecules at the centers of each strand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Method for cloning single-stranded oligonucleotides in a plasmid vector

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.     

Construction of DNA substrates modified with psoralen at a unique site and study of the action mechanism of ABC excinuclease on these uniformly modified substrates

Psoralens bind to DNA noncovalently and upon exposure to near UV (320-400 nm) light produce covalent adducts. Thymidine residues in DNA, especially those at 5'-TpA-3' sequences, are most susceptible to the photochemical reaction. This property of the reaction and the recent advances in oligonucleotide synthesis and separation has enabled us to construct DNA fragments containing psoralen adducts at a specific site. The octanucleotide 5'-TCGTAGCT-3' was photoreacted (in the presence of the complementary strand) with the synthetic psoralen 4'-hydroxymethyl-4,5',8-trimethylpsoralen to obtain oligonucleotides adducted via the furan or pyrone ring at the internal thymine. These modified octanucleotides were ligated to nonmodified oligonucleotides to obtain a 40-base pair DNA fragment containing a psoralen adduct at a central location. The modified fragment having the thymine-furan side 4'-hydroxymethyl-4,5',8-trimethylpsoralen adduct was irradiated with 360 nm of light to produce an interstrand cross-link, and this cross-linked DNA was purified to homogeneity. These uniquely modified DNAs were used as substrates for Escherichia coli ABC excinuclease to determine its incision mechanism unambiguously and to determine the contact sites of the enzyme. ABC excinuclease mediates the cleavage of the 8th and 5th phosphodiester bonds 5' and 3', respectively, to psoralen monoadducts, and the 9th (5') and 3rd (3') phosphodiester bonds to the furan-side thymine of the cross-link. Preliminary DNaseI footprinting studies show that ABC excinuclease protects the whole 40-base pair fragment from DNaseI, and binding of the A and B subunits to the furan side-monoadducted substrate produces two hypersensitive phosphodiester bonds in the vicinity of the 5' incision site of ABC excinuclease.     

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.     

Characteristics of triplex-directed photoadduct formation by psoralen-linked oligodeoxynucleotides.

A triplex-forming oligopyrimidine has been attached at its 5'-end to a photoreactive psoralen derivative and used to target a sequence which forms part of the coding region of the human aromatase gene. The 20 base pair sequence is not a perfect triplex target since it contains three pyrimidine interruptions within the purine-rich strand. Despite this, we have detected triplex-directed photoadduct formation at pH 7.0 between the psoralen-linked oligonucleotide and a 30mer duplex representing the aromatase target. Photoadduct formation was found to be sensitive to pH, temperature, cation concentration and the base composition of the third strand. By varying the base sequence of the target duplex around the psoralen intercalation site, we have characterised the site and mode of psoralen intercalation. The attached psoralen has been found to intercalate at the triplex-duplex junction with a strong preference for one orientation. We have shown that the psoralen will bind at the junction even when there is a preferred TpA step at an adjacent site. We have also compared the binding affinity and photoreactivity of oligodeoxyribonucleotides linked to two different psoralen derivatives and found differences in the rate of crosslinking and the extent of crosslink formation. Finally, we have examined oligodeoxyribonucleotides which are attached to psoralen by polymethylene linkers of different lengths.