Inhibition of 5'-UTR RNA conformational switching in HIV-1 using antisense PNAs.

BACKGROUND: The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5'-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5'-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization. METHODS AND RESULTS: We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes. CONCLUSIONS: Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.

Efficient sequence-directed psoralen targeting using pseudocomplementary Peptide nucleic acids.

A pair of decameric pseudocomplementary PNAs which bind to their mixed purine-pyrimidine sequence target in duplex DNA by double duplex invasion has been synthesized with a derivative of 8-methoxypsoralen conjugated to one of the PNAs. It is shown that this pair of psoralen-conjugated pseudocomplementary PNA oligomers, which target a site in the pBluescriptKS+ vector, upon irradiation with long-wavelength UV light (UVA) with high efficiency and specificity form photoadducts to an adjacent 5'-TA site, and more than 50% of these adducts are DNA interstrand cross-links. Transcription elongation by T7 or T3 RNA polymerase is specifically arrested at the psoralen cross-linking site, yielding more than 90% arrested product. These results emphasize the potential of pseudocomplementary PNA oligomers for highly specific gene targeting, in particular, with respect to sequence-directed psoralen photomodification of double-stranded DNA. Thus, such psoralen-PNA conjugates could be very useful in a range of biology and drug discovery applications.

DNA triplex stabilization by a delta-carboline derivative tethered to third strand oligonucleotides.

A delta-carboline derivative was covalently coupled to a 7 mer oligonucleotide at its 5'- or 3'-end. The stability of triplexes formed from the conjugates and a double-helical target was studied by UV melting experiments. Compared to the unmodified control triple helices, triplexes with the conjugate exhibit a significantly higher stability. However, the degree of stabilization depends on the particular triplex structure formed.

Interstrand cross-links: a new type of gamma-ray damage in bromodeoxyuridine-substituted DNA.

Interstrand cross-links (ICL) represent one of the most toxic types of DNA damage for dividing cells. They are induced both by natural products (e.g., psoralens + UVA) and by several chemical agents, some of which are used in chemotherapy (e.g., carboplatin and mitomycin C). Although repair mechanisms exist for interstrand cross-links, these lesions can induce mutations, chromosomal rearrangements, and cell death. Here, we report, for the first time, the formation of ICL by gamma-rays in brominated DNA. It is well established that the radiosensitization properties of bromodeoxyuridine (BrdUrd) result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of a uridinyl radical (dUrd(*)) or uridinyl anion (dUrd-) upon addition of an electron. We observe that the radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. We have used a model DNA containing a bulge, formed by five mismatched bases, and have observed a linear dose-response for the formation of strand breaks on the single-stranded regions of both the brominated strand and the opposite nonbrominated strand. Surprisingly, we have observed the formation of interstrand cross-links exclusively in the mismatched region. Thus, we propose that the radiosensitization effects of bromodeoxyuridine in vivo will almost certainly be limited to single strand regions such as found in transcription bubbles, replication forks, mismatched DNA, and possibly the loop region of telomeres. Our results suggest that interstrand cross-links may contribute to the radiosensitization effects of BrdUrd. These findings may have profound implications for the clinical use of bromodeoxyuridine as a radiosensitizer, as well as for the development of targeted radiosensitizers.

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion.

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats.

Mechanism of site-specific psoralen photoadducts formation in triplex DNA directed by psoralen-conjugated oligonucleotides.

Triplex-formation oligonucleotides attached with a photoreactive psoralen molecule (psoTFO) can be used to induce site-specific DNA damage and to control gene expression. Inhibition of transcription by psoralen-cross-linked triplexes results in both arrest and termination of RNA Pol II transcriptional complexes during elongation. To understand the relationship between triplex psoralen cross-linking products and the fate of RNA Pol II elongation complexes, it is important to delineate the mechanism for creating site-specific psoralen photoadducts in a target duplex DNA. To investigate the mechanism of photoadduct-formation by psoralen photo-cross-linking, triplex structures were generated by targeting a DNA duplex with psoTFOs of different lengths. The psoralen photoadducts were then analyzed after UV irradiation, which initiates the psoralen cross-linking reaction. Our results demonstrated that UV irradiation of triplexes formed between a psoTFO and a DNA duplex generated two distinct groups of psoralen photoadducts: monoadducts and psoralen interstrand cross-link products. The formation of these psoralen photoadducts was also photoreversible through exposure to short wavelength UV irradiation. The length of a psoTFO was shown to establish the position at which psoralen was added to the target DNA duplex and determined which photoadducts products formed predominantly. Kinetic experiments that monitored the formation of the psoralen photoadducts also suggested that the length of the psoTFO influenced which photoadducts were preferentially formed at faster rates. Taken together, these studies provide new insight into the mechanism associated with the formation of psoralen photoadducts that are directed by psoTFO during triplex formation.

Alpha-anomeric deoxynucleotides, anoxic products of ionizing radiation, are substrates for the endonuclease IV-type AP endonucleases.

Alpha-anomeric 2'-deoxynucleosides (alphadN) are one of the products formed by ionizing radiation (IR) in DNA under anoxic conditions. Alpha-2'-deoxyadenosine (alphadA) and alpha-thymidine (alphaT) are not recognized by DNA glycosylases, and are likely removed by the alternative nucleotide incision repair (NIR) pathway. Indeed, it has been shown that alphadA is a substrate for the Escherichia coli Nfo and human Ape1 proteins. However, the repair pathway for removal of alphadA and other alphadN in yeast is unknown. Here we report that alphadA when present in DNA is recognized by the Saccharomyces cerevisiae Apn1 protein, a homologue of Nfo. Furthermore, alphaT is a substrate for Nfo and Apn1. Kinetic constants indicate that alphadA and alphaT are equally good substrates, as a tetrahydrofuranyl (THF) residue, for Nfo and Apn1. Using E. coli and S. cerevisiae cell-free extracts, we have further substantiated the role of the nfo and apn1 gene products in the repair of alphadN. Surprisingly, we found that bacteria and yeast NIR-deficient mutants are not sensitive to IR, suggesting that DNA strand breaks with terminal 3'-blocking groups rather than alphadN might contribute to cell survival. We propose that the novel substrate specificities of Nfo and Apn1 play an important role in counteracting oxidative DNA base damage.

Effects of the C4'-oxidized abasic site on replication in Escherichia coli. An unusually large deletion is induced by a small lesion.

The C4'-oxidized abasic site (C4-AP) is produced in DNA as a result of oxidative stress by a variety of agents. For instance, the lesion accounts for approximately 40% of the DNA damage produced by the antitumor antibiotic bleomycin. The effect of C4-AP on DNA replication in Escherichia coli was determined using the restriction endonuclease and postlabeling (REAP) method. Three-nucleotide deletion products are the sole products observed following replication of plasmids containing C4-AP under SOS conditions in wild-type cells. Full-length products are formed in varying amounts depending upon the local sequence in wild-type cells under non-SOS-induced conditions. The "A-rule" is followed for the formation of substitution products. C4-AP is the first example of a DNA lesion that produces significant levels of three-nucleotide deletions in a variety of sequence contexts. Experiments carried out in cells lacking specific polymerases reveal that formation of three-nucleotide deletion products results from a coordinated effort involving pol II and pol IV. This is the first example in which these SOS inducible polymerases are shown to work in concert during lesion bypass. Three-nucleotide deletions are not observed during the replication of other abasic lesions, and are rarely produced by bulky adducts. The effect of C4-AP on DNA replication suggests a significant role for this lesion in the cytotoxicity of bleomycin. Formation of the C4-AP lesion may also be responsible for the formation of mutant proteins containing single-amino acid deletions that exhibit altered phenotypes.

LC-MS/MS identification and yeast polymerase eta bypass of a novel gamma-irradiation-induced intrastrand cross-link lesion G[8-5]C.

Reactive oxygen species can give rise to intrastrand cross-link lesions, where two neighboring nucleobases are covalently bonded. Here, we employed LC-MS/MS and demonstrated for the first time that gamma irradiation of a synthetic duplex oligodeoxyribonucleotide can give rise to an intrastrand cross-link lesion G[8-5]C, where the C8 carbon atom of guanine and the C5 carbon atom of its 3'-neighboring cytosine are covalently bonded. We also carried out in vitro replication studies of a substrate containing a site-specifically incorporated G[8-5]C, and our results showed that yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion. Steady-state kinetic analyses for nucleotide incorporation by pol eta showed that the 3'-cytosine moiety of the cross-link did not significantly affect either the efficiency or the fidelity of nucleotide incorporation. The 5' guanine portion of the cross-link lesion, however, markedly reduced both the efficiency and the fidelity of nucleotide incorporation; the insertion of dGMP or dAMP was slightly favored over the insertion of the correct nucleotide, dCMP, which was in turn favored over the insertion of dTMP. The above results support that the oxidative cross-link lesion, if not repaired, can be mutagenic.

Synthesis and properties of DNA analogs consisting of the benzene-phosphate backbone.

This paper describes DNA analog synthesis consisting of the benzene-phosphate backbone, of which the monomer units are built from benzene-core units connected via a biaryl-like axis to the nucleobases. It was found that the duplex comprised of the analogs had a non-helical structure and was thermally more stable than the corresponding natural DNA duplex.

Efficient generation of 2'-deoxyuridin-5-yl at 5'-(G/C)AA(X)U(X)U-3' (X = Br, I) sequences in duplex DNA under UV-irradiation.

A highly sequence-specific photoreaction of 5'-(G/C)AA(X)U(X)U-3' was found by using 450-base pair DNA fragments containing (X)U under UV-irradiation (X = Br, I). The molecular basis of this high photoreactivity was investigated in detail using synthetic octanucleotides. Based on the experimental data, the mechanism for the efficient photoreaction was proposed.

Electrochemical behavior of gold electrodes modified with photosensitizer-tethered DNA.

We investigated electrochemical characteristics of gold electrodes modified with photosensitizer-tethered DNA. We synthesized 3'-thiol-modified DNA strands (ODN-SH) and 3'-anthraquinone-modified DNA strands (AQ-ODN). We prepared an AQ-ODN/ODN-SH duplex-modified electrode through hybridization of AQ-ODN with ODN-SH on the gold electrode. We used an amperometric technique for its photocurrent measurement. The large photocurrent was observed by irradiation of a DNA modified electrode at 365 nm. We also measured photocurrent with different DNA sequences on gold electrodes.

Silver (I) cation specifically stabilizes heteroduplex with C:C mismatch base pair: toward the efficient detection of single nucleotide polymorphism (2).

We examined the effect of silver (I) cation on the thermal stability of heteroduplex and homoduplex. Addition of silver (I) cation increased the melting temperature of heteroduplex containing C:C mismatch base pair by about 3-4 degrees C. The thermal stability of homoduplex and heteroduplexes containing other kinds of mismatch base pairs was not significantly changed by the addition of silver (I) cation. We conclude that silver (I) cation specifically stabilizes heteroduplex containing C:C mismatch base pair. Our results certainly support the idea that the addition of silver (I) cation to C:C mismatch base pair in heteroduplex could be a convenient strategy for heteroduplex analysis and may eventually lead to progress in single nucleotide polymorphism genotyping.

Photo-oxidation of duplex DNA with the stable trioxatriangulenium ion.

We show that 5'-Gs in 5'-GG-3' duplex DNA dinucleotide steps are preferentially oxidized by the trioxatriangulenium ion (TOTA( plus sign in circle )) upon photo-activation, an oxidation pattern characteristic of guanine radical cation formation. Some photo-oxidation of the 3'G in 5'GG3' steps and of isolated guanines is also observed but reactions carried out in D(2)O reveal only a minor increase in oxidation damage at these sites, indicating that electron transfer is the primary mechanism of guanine oxidation.

Site-specific strand breaks in RNA produced by (125)I radiodecay.

Decay of (125)I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of (125)I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3'-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when (125)I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that (125)I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules.

Long-range oxidative damage in DNA/RNA duplexes.

Oxidative damage as a result of DNA-mediated long-range charge transport occurs readily and at high yield in duplex DNA, and it is of interest whether similar damage can occur in duplex oligonucleotides that include both ribo- and deoxyribonucleotides. Assemblies containing RNA and mixed RNA.DNA strands were constructed containing tethered ethidium as a photooxidant. In photooxidation experiments, long-range oxidative damage to the ribose-containing strand of the oligonucleotide duplexes was examined. Hole injection by photoexcited ethidium followed by radical migration to oxidatively susceptible guanines afforded significant damage on ribose-containing strands at long range ( approximately 35 A). This damage does not differ substantially in yield and location from that found in B-DNA duplexes. No oxidative damage was found upon photooxidation of DNA/RNA duplexes containing tethered metallointercalator, despite the ability of the rhodium complex to promote oxidative damage at a distance in DNA duplexes. This result is attributed to the poor coupling of the rhodium complex into the A-like RNA/DNA duplex. The ability for long-range charge transport to occur in double-stranded nucleic acids of different comformations is considered in light of modeling studies that show interstrand base-base overlap between the opposing, complementary strands that make up RNA/DNA hybrid duplexes. Thus, the possibility of long-range radical migration to effect oxidative damage or signaling may be considered also in the context of transcriptional events.

Polarity of human replication protein A binding to DNA.

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3'-end of the upstream oligonucleotide or at the 5'-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5'-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3'-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer-template systems containing 5'- or 3'-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.

Radioprobing of a RecA-three-stranded DNA complex with iodine 125: evidence for recognition of homology in the major groove of the target duplex.

A fundamental problem in homologous recombination is how homology between DNAs is recognized. In all current models, a recombination protein loads onto a single strand of DNA and scans another duplex for homology. When homology is found, a synaptic complex is formed, leading to strand exchange and a heteroduplex. A novel technique based on strand cleavage by the Auger radiodecay of iodine 125, allows us to determine the distances between (125)I on the incoming strand and the target sugars of the duplex DNA strands in an Escherichia coli RecA protein-mediated synaptic complex. Analysis of these distances shows that the complex represents a post-strand exchange intermediate in which the heteroduplex is located in the center, while the outgoing strand forms a relatively wide helix intertwined with the heteroduplex and located in its minor groove. The structure implies that homology is recognized in the major groove of the duplex.

Enthalpy and heat capacity changes for formation of an oligomeric DNA duplex: interpretation in terms of coupled processes of formation and association of single-stranded helices.

The thermodynamics of self-assembly of a 14 base pair DNA double helix from complementary strands have been investigated by titration (ITC) and differential scanning (DSC) calorimetry, in conjunction with van't Hoff analysis of UV thermal scans of individual strands. These studies demonstrate that thermodynamic characterization of the temperature-dependent contributions of coupled conformational equilibria in the individual "denatured" strands and in the duplex is essential to understand the origins of duplex stability and to derive stability prediction schemes of general applicability. ITC studies of strand association at 293 K and 120 mM Na+ yield an enthalpy change of -73 +/- 2 kcal (mol of duplex)-1. ITC studies between 282 and 312 K at 20, 50, and 120 mM Na+ show that the enthalpy of duplex formation is only weakly salt concentration-dependent but is very strongly temperature-dependent, decreasing approximately linearly with increasing temperature with a heat capacity change (282-312 K) of -1.3 +/- 0.1 kcal K-1 (mol of duplex)-1. From DSC denaturation studies in 120 mM Na+, we obtain an enthalpy of duplex formation of -120 +/- 5 kcal (mol of duplex)-1 and an estimate of the corresponding heat capacity change of -0.8 +/- 0.4 kcal K-1 (mol of duplex)-1 at the Tm of 339 K. van't Hoff analysis of UV thermal scans on the individual strands indicates that single helix formation is noncooperative with a temperature-independent enthalpy change of -5.5 +/- 0.5 kcal at 120 mM Na+. From these observed enthalpy and heat capacity changes, we obtain the corresponding thermodynamic quantities for two fundamental processes: (i) formation of single helices from disordered strands, involving only intrastrand (vertical) interactions between neighboring bases; and (ii) formation of double helices by association (docking) of single helical strands, involving interstrand (horizontal and vertical) interactions. At 293 K and 120 mM Na+, we calculate that the enthalpy change for association of single helical strands is approximately -64 kcal (mol of duplex)-1 as compared to -210 kcal (mol of duplex)-1 calculated for duplex formation from completely unstructured single strands and to the experimental ITC value of -73 kcal (mol of duplex)-1. The intrinsic heat capacity change for association of single helical strands to form the duplex is found to be small and positive [ approximately 0.1 kcal K-1 (mol of duplex)-1], in agreement with the result of a surface area analysis, which also predicts an undetectably small heat capacity change for single helix formation.

UV light-induced crosslinking of short DNA duplex strands: nucleotide sequence preferences and a prominent role of the duplex ends.

By various doses of UV light, we irradiated 32 short DNA duplexes having between 12 and 40 nucleotide residues in length, and monitored the induced crosslinks between the complementary DNA strands by denaturing polyacrylamide gel electrophoresis. The experiments revealed that the crosslinking was strongest with the alternating sequence of T and A and weaker with the alternating sequence of T and G (C and A in the complementary strand). On the other hand, GC blocks of any sequence provided undetectable amounts of interstrand crosslinks even at the highest doses of UV irradiation. The amount of crosslinked strands logarithmically increased with the UV dose but it did not depend on the oligonucleotide concentration, ionic strength, divalent magnesium or manganese cations and pH at least within the examined regions of the experimental conditions, unless the oligonucleotide denatured or isomerized into a unimolecular foldback. The extent of crosslinking also did not depend on the (dT-dA)n duplex length to indicate that the crosslink was predominantly localized at a specific duplex locus. Experiments with (dT-dA)8 "mutants" in which AT pairs were systematically replaced by GC pairs at various molecule positions, revealed that the crosslinking predominantly occurred at the oligo(dT-dA) duplex ends. The crosslinking is a direct method to detect duplexes of DNA, which is here, for example, demonstrated with the heteroduplex of (dT-dA)12 and (dT-dA)16.