Modulation of psoralen DNA crosslinking kinetics associated with a triplex-forming oligonucleotide

A triplex-forming oligonucleotide (TFO), HPRT3, conjugated to a psoralen derivative, was designed to target a psoralen reaction site within the HPRT gene. HPRT3 bound with high affinity to a synthetic duplex target sequence. At a uniform UVA radiation dose, the ratio of psoralen monoadducts (MA) to interstrand crosslinks decreased and inverted with increasing TFO concentration. As the TFO concentration increased from 10 nm to 10 microm, the efficiency of psoralen MA formation remained relatively constant but the efficiency of interstrand crosslink formation increased several-fold. Neither shortening the TFO to reduce its dissociation constant nor altering the DNA sequences flanking the TFO binding site altered the concentration dependence of MA and crosslink yields. The psoralen photokinetics associated with 10 nm HPRT3 converted to those associated with 10 microm HPRT3 with the addition of other unrelated TFOs at 10 microm that do not specifically interact with the HPRT3 target sequence. Glycerol at concentrations of 0.5% (vol/vol) or higher also mimicked high TFO concentrations in enhancing crosslink formation. These results demonstrate that while psoralen may be targeted to react at a particular sequence by TFOs, photoreactivity associated with triplex formation is also modulated by sequence-independent factors that may affect the local macromolecular environment.     

Rearrangement of the (6S,8R,11S) and (6R,8S,11R) exocyclic 1,N2-deoxyguanosine adducts of trans-4-hydroxynonenal to N2-deoxyguanosine cyclic hemiacetal adducts when placed complementary to cytosine in duplex DNA

trans-4-Hydroxynonenal (HNE) is a peroxidation product of omega-6 polyunsaturated fatty acids. The Michael addition of deoxyguanosine to HNE yields four diastereomeric exocyclic 1,N(2)-dG adducts. The corresponding acrolein- and crotonaldehyde-derived exocyclic 1,N(2)-dG adducts undergo ring-opening to N(2)-dG aldehydes, placing the aldehyde functionalities into the minor groove of DNA. The acrolein- and the 6R-crotonaldehyde-derived exocyclic 1,N(2)-dG adducts form interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence context. Only the HNE-derived exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry forms interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence context. Moreover, as compared to the exocyclic 1,N(2)-dG adducts of acrolein and crotonaldehyde, the cross-linking reaction is slow (Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc. 2003, 125, 5687-5700). Accordingly, the chemistry of the HNE-derived exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry has been compared with that of the (6R,8S,11R) adduct, when incorporated into 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCGCTAGC)-3', containing the 5'-CpG-3' sequence (X = HNE-dG). When placed complementary to dC in this duplex, both adducts open to the corresponding N(2)-dG aldehydic rearrangement products, suggesting that the formation of the interstrand cross-link by the exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry, and the lack of cross-link formation by the exocyclic 1,N(2)-dG adduct of (6R,8S,11R) stereochemistry, is not attributable to inability to undergo ring-opening to the aldehydes in duplex DNA. Instead, these aldehydic rearrangement products exist in equilibrium with stereoisomeric cyclic hemiacetals. The latter are the predominant species present at equilibrium. The trans configuration of the HNE H6 and H8 protons is preferred. The presence of these cyclic hemiacetals in duplex DNA is significant as they mask the aldehyde species necessary for interstrand cross-link formation.     

DNA interstrand cross-link formation initiated by reaction between singlet oxygen and a modified nucleotide

DNA is the target of many anti-cancer therapies. These agents damage the biopolymer by oxidation or by alkylation. Interstrand DNA cross-links are believed to be the source of cytotoxicity of anti-tumor agents, such as mitomycin C, which alkylate the biopolymer. In contrast, deoxyguanosine oxidation is the result of reaction between DNA and singlet oxygen, which is the damaging species produced in photodynamic therapy. We have shown that, upon oxidation by singlet oxygen, an analogue of thymidine (2) rearranges to a methide, which forms DNA-DNA interstrand cross-links. This novel process suggests that 2 may be a useful adjuvant in photodynamic therapy.     

Fast interstrand cross-linking of cisplatin-DNA monoadducts compared with intrastrand chelation: a kinetic study using hairpin-stabilized duplex oligonucleotides

The antitumor drug cisplatin forms two kinds of guanine-guanine cross-links with DNA: intrastrand, occurring mainly at GG sites, and interstrand, formed at GC sites. The former are generally more abundant than the latter, at least in experiments with linear duplex DNA. The formation of interstrand cross-links requires partial disruption of the Watson-Crick base pairing, and one could therefore expect the cross-linking reaction to be rather slow. In contrast with this expectation, kinetic measurements reported here indicate that interstrand cross-linking is as fast as intrastrand, or even faster. We have investigated the reactions between two hairpin-stabilized DNA duplexes, containing either a d(TGCA)(2) sequence (duplex TGCA) or a d(G(1)G(2)CA)-d(TG(3)CC) sequence (duplex GGCA), and the diaqua form of cisplatin, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), in an unbuffered solution kept at pH 4.5 +/- 0.1 and 20 degrees C. Using HPLC as the analytical method, we have determined the platination (first step) and chelation (second step) rate constants for these reaction systems. Duplex TGCA, in which the two guanines are quasi-equivalent, is found to be platinated very slowly (k=0.5 +/- 0.1M(-1)s(-1)) and to form the final interstrand cross-link very rapidly (k=13 +/- 3 x 10(-3) s(-11)). For GGCA, we find that G(1) is platinated rapidly (k=32 +/- 5M(-1)s(-1)) to form a long-lived monoadduct, which is only slowly chelated (k=0.039 +/- 0.001 x 10(-3) s(-1)) by G(2) (intrastrand), while G(2) is platinated one order of magnitude more slowly than G(1) (k=2.0 +/- 0.5M(-1)s(-1)) and chelated fairly rapidly both by G(1) (intrastrand: k=0.4 +/-0.1 x 10(-3) s(-1)) and G(3) (interstrand: k=0.2 +/- 0.1 x 10(-3) s(-1)); finally, G(3) is platinated at about the same rate as G(2) (k=2.4 +/- 0.5M(-1)s(-1)) and chelated very rapidly by G(2) (interstrand: k=10 +/- 4 x 10(-3) s(-1)). These results suggest that the low occurrence of interstrand cross-links in cisplatinated DNA is due to an extremely slow initial platination of guanines involved in d(GC)(2) sequences, rather than to a slow cross-linking reaction.     

pH‐Independent Recognition of the dG ⋅ dC Base Pair in Triplex DNA: 9‐Deazaguanine N7‐(2′‐Deoxyribonucleoside) and Halogenated Derivatives Replacing Protonated dC

Triplex-forming oligonucleotides (TFOs) containing 9-deazaguanine N⁷-(2′-deoxyribonucleoside) 1a and halogenated derivatives 1b,c were synthesized employing solid-phase oligonucleotide synthesis. For that purpose, the phosphoramidite building blocks 5a – c and 8a – c were synthesized. Multiple incorporations of 1a – c in place of dC were performed within TFOs, which involved the sequence of five consecutive 1a – c ⋅ dG ⋅ dC triplets as well as of three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. These TFOs were designed to bind in a parallel orientation to the target duplex. Triplex forming properties of these oligonucleotides containing 1a – c in the presence of Na⁺ and Mg²⁺ were studied by UV/melting-curve analysis and confirmed by circular-dichroism (CD) spectroscopy. The oligonucleotides containing 1a in the place of dC formed stable triplexes at physiological pH in the case of sequence of five consecutive 1a ⋅ dG ⋅ dC triplets as well as three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. The replacement of 1a by 9-halogenated derivatives 1b,c further enhanced the stability of DNA triplexes. Nucleosides 1a – c also stabilized duplex DNA.     

Efficient DNA interstrand cross-link formation from a nucleotide radical

5-(2'-Deoxyuridinyl)methyl radical (1) is produced during gamma-irradiation of DNA and other methods of oxidative stress. Independent generation of this reactive intermediate in duplex results in significant levels of DNA interstrand cross-links. Cross-link formation does not require O2 and involves reaction between the nucleotide where the radical is originally generated and the opposing deoxyadenosine. This is the first example in which formation of a nucleotide radical ultimately results in DNA-DNA cross-links. Interstrand DNA cross-links are difficult to repair and are believed to be the source of cytotoxicity of antitumor agents, such as mitomycin C. The efficient formation of DNA cross-links through 1 should provide the impetus for the design of DNA damaging agents that exploit this pathway.     

Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.     

Cross-linking of a DNA conjugate tethering a cis-bifunctional platinated complex to a target DNA duplex

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a ligand for complexation with K2PtCl4. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. Cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand or cases of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA; conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.     

Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.     

Sequence specific DNA cross-linking triggered by visible light

A new biocompatible strategy for photoinduced DNA interstrand cross-linking is presented. Methylene blue induced (1)O(2) formation triggers furan oxidation; the resulting aldehyde then rapidly reacts with complementary A or C with formation of stable adducts. Easily accessible furan modified nucleosides, a commercially available photosensitizer, and visible light irradiation constitute the necessary tools to achieve selective duplex interstrand cross-linking.     

DNA duplexes and triplex-forming oligodeoxynucleotides incorporating modified nucleosides forming stable and selective triplexes

We have previously reported DNA triplexes containing the unnatural base triad G-PPI·C3, in which PPI is an indole-fused cytosine derivative incorporated into DNA duplexes and C3 is an abasic site in triplex-forming oligonucleotides (TFOs) introduced by a propylene linker. In this study, we developed a new unnatural base triad A-ψ·C(R1) where ψ and C(R1) are base moieties 2'-deoxypseudouridine and 5-substituted deoxycytidine, respectively. We examined several electron-withdrawing substituents for R1 and found that 5-bromocytosine (C(Br)) could selectively recognize ψ. In addition, we developed a new PPI derivative, PPI(Me), having a methyl group on the indole ring in order to achieve selective triplex formation between DNA duplexes incorporating various Watson-Crick base pairs, such as T-A, C-G, A-ψ, and G-PPI(Me), and TFOs containing T, C, C(Br), and C3. We studied the selective triplex formation between these duplexes and TFOs using UV-melting and gel mobility shift assays.     

Highly efficient sequence-specific DNA interstrand cross-linking by pyrrole/imidazole CPI conjugates

We have developed a novel type of DNA interstrand cross-linking agent by synthesizing dimers of a pyrrole (Py)/imidazole (Im)-diamide-CPI conjugate, ImPyLDu86 (1), connected using seven different linkers. The tetramethylene linker compound, 7b, efficiently produces DNA interstrand cross-links at the nine-base-pair sequence, 5'-PyGGC(T/A)GCCPu-3', only in the presence of a partner triamide, ImImPy. For efficient cross-linking by 7b with ImImPy, one A.T base pair between two recognition sites was required to accommodate the linker region. Elimination of the A.T base pair and insertion of an additional A.T base pair and substitution with a G.C base pair significantly reduced the degree of cross-linking. The sequence specificity of the interstrand cross-linking by 7b was also examined in the presence of various triamides. The presence of ImImIm slightly reduced the formation of a cross-linked product compared to ImImPy. The mismatch partners, ImPyPy and PyImPy, did not produce an interstrand cross-link product with 7b, whereas ImPyPy and PyImPy induced efficient alkylation at their matching site with 7b. The interstrand cross-linking abilities of 7b were further examined using denaturing polyacrylamide gel electrophoresis with 5'-Texas Red-labeled 400- and 67-bp DNA fragments. The sequencing gel analysis of the 400-bp DNA fragment with ImImPy demonstrated that 7b alkylates several sites on the top and bottom strands, including one interstrand cross-linking match site, 5'-PyGGC(T/A)GCCPu-3'. To obtain direct evidence of interstrand cross-linkages on longer DNA fragments, a simple method using biotin-labeled complementary strands was developed, which produced a band corresponding to the interstrand cross-linked site on both top and bottom strands. Densitometric analysis indicated that the contribution of the interstrand cross-link in the observed alkylation bands was approximately 40%. This compound efficiently cross-linked both strands at the target sequence. The present system consisted of a 1:2 complex of the alkylating agent and its partner ImImPy and caused an interstrand cross-linking in a sequence-specific fashion according to the base-pair recognition rule of Py-Im polyamides.     

Thermodynamic versus kinetic products of DNA alkylation as modeled by reaction of deoxyadenosine.

Alkylating agents that react through highly electrophilic quinone methide intermediates often express a specificity for the weakly nucleophilic exocyclic amines of deoxyguanosine (dG N(2)) and deoxyadenosine (dA N(6)) in DNA. Investigations now indicate that the most nucleophilic site of dA (N1) preferentially, but reversibly, conjugates to a model ortho-quinone methide. Ultimately, the thermodynamically stable dA N(6) isomer accumulates by trapping the quinone methide that is transiently regenerated from collapse of the dA N1 adduct. Alternative conversions of the dA N1 to the dA N(6) derivative by a Dimroth rearrangement or other intramolecular processes are not competitive under neutral conditions, as demonstrated by studies with [6-(15)N]-dA. Both a model quinone methide precursor and its dA N1 adduct yield a similar profile of deoxynucleoside products when treated with an equimolar mixture of dC, dA, dG, and T. Consequently, the most readily observed products of DNA modification resulting from reversible reactions may reflect thermodynamic rather than kinetic selectivity.     

Chemical properties of oxopropenyl adducts of purine and pyrimidine nucleosides and their reactivity toward amino acid cross-link formation

N2-oxopropenyldeoxyguanosine (2) forms in duplex DNA by modification of dG residues with base propenal or malondialdehyde. The pKa of 2 was estimated to be 6.9 from the pH dependence of its ring-closing to the pyrimidopurinone derivative 1. The acidity of 2 may be an important determinant of its miscoding properties and its reactivity to nucleophiles in DNA or protein. To test this hypothesis, analogous N-oxopropenyl derivatives of dA (4), dC (5), and N1-methyl-dG (6) were synthesized and their pKa's were determined by optical titration. The N-oxopropenyl derivatives of dA and dC both exhibited pKa's of 10.5, whereas the N-oxopropenyl derivative of N1-methyldG exhibited a pKa of 8.2. Cross-linking of 2, 4, 5, and 6 to N(alpha)-acetyl-lysine was explored at neutral pH. Adduct 2 did not react with N(alpha)-acetyl-lysine, whereas 4-6 readily formed cross-links. The structures of the cross-links were elucidated, and their stabilities were investigated. The results define the acidity of oxopropenyl deoxynucleosides and highlight its importance to their reactivity toward nucleophiles. This study also identifies the structures of a potential novel class of DNA-protein cross-links.     

Effects of geometric isomerism and anions on the kinetics and mechanism of the stepwise formation of long-range DNA interstrand cross-links by dinuclear platinum antitumor complexes

Reported herein is a detailed study of the kinetics and mechanism of formation of a 1,4-GG interstrand cross-link by the dinuclear platinum anticancer compound [15N][{cis-PtCl(NH3)2}2{mu-NH2(CH2)6NH2}]2+ (1,1/c,c (1)). The reaction of [15N]1 with 5'-{d(ATATGTACATAT)2} (I) has been studied by [1H,15N] HSQC NMR spectroscopy in the presence of different concentrations of phosphate. In contrast with the geometric trans isomer (1,1/t,t), there was no evidence for an electrostatic preassociation of 1,1/c,c with the polyanionic DNA surface, and the pseudo-first-order rate constant for the aquation of [(15)N]1 was actually slightly higher (rather than lower) than that in the absence of DNA. When phosphate is absent, the overall rate of formation of the cross-link is quite similar for the two geometric isomers, occurring slightly faster for 1,1/t,t. A major difference in the DNA binding pathways is the observation of phosphate-bound intermediates only in the case of 1,1/c,c. 15 mM phosphate causes a dramatic slowing in the overall rate of formation of DNA interstrand cross-links due to both the slow formation and slow closure of the phosphate-bound monofunctional adduct. A comparison of the molecular models of the bifunctional adducts of the two isomers shows that helical distortion is minimal and globally the structures of the 1,4 interstrand cross-links are quite similar. The effect of carrier ligand was investigated by similar studies of the ethylenediamine derivative [15N]1-en. A pKa value of 5.43 was determined for the [15N]1,1/c,c-en diaquated species. The rate of reaction of [15N]1-en with duplex I is similar to that of 1,1/c,c and the overall conformation of the final adduct appears to be similar. The significance of these results to the development of "second-generation" polynuclear platinum clinical candidates based on the 1,1/c,c chelate (dach) series is discussed.     

Metal-salen-base-pair complexes inside DNA: complexation overrides sequence information

Two isomeric salicylic aldehyde nucleobases have been prepared and incorporated into various DNA duplexes. Reaction with ethylenediamine leads to formation of the well-known salen ligand inside the DNA double helix. Addition of transition-metal ions such as Cu(2+), Mn(2+), Ni(2+), Fe(2+), or VO(2+) results in the formation of metal-salen-base-pair complexes, which were studied by using UV and circular dichroism (CD) spectroscopy. HPLC and ESI mass spectrometric measurements reveal an unusually high stability of the DNA-metal system. These metal-salen complexes act as interstrand cross-links and thereby lead to a strong stabilization of the DNA duplexes, as studied by thermal de- and renaturing experiments. Complex formation is strong enough to override sequence information even when the preorganization of the ligand precursors is unfavorable and the DNA duplex is distorted by the metal complexation. Furthermore, melting-point studies show that the salen complex derived from ligand 2 fits better into the DNA duplex, in accordance with results obtained from the crystal structure of the corresponding copper-salen complex 8.     

A cationic dye triplet as a unique "glue" that can connect fully matched termini of DNA duplexes

In this study, we propose that three consecutive cationic p‐methylstilbazoles tethered on D ‐threoninols ( Z residues) at 5′ termini act as a unique “glue” connecting DNA duplexes by their interstrand cluster formation. Interstrand clustering of p‐methylstilbazoles ( ZZZ triplets) induces narrowing and hypsochromic shift of bands at 350 nm, which can be assigned to the absorption of p‐methylstilbazole. However, single‐stranded DNA conjugates involving a ZZZ triplet at the 5′ terminus of 8‐mer native nucleotides is found not to induce such large spectral changes, which implies that the intrinsic self‐assembling property of ZZZ triplets is weak. Interestingly, when this conjugate is hybridized with a complementary 8‐mer native oligonucleotide, a remarkable spectral change is observed, indicating the dimerization of a duplex through the interstrand clustering of ZZZ triplets. Dimerization of the duplex is also evidenced by cold‐spray ionization mass spectrometry. This interstrand clustering is observed only when a ZZZ triplet is tethered to a 5′ rather than 3′ terminus. Furthermore, the stability of the interstrand cluster increases by increasing the number of nucleobases of the DNA portion, and when mismatched base pairs are incorporated or when a base next to the Z residue is deleted, the stability substantially drops. When we apply the ZZZ triplet to the formation of a nanowire using two complementary DNA conjugates, each of which has a ZZZ triplet at the 5′ termini as overhang, we demonstrate the successful formation of a nanowire by native PAGE analysis. Since native sticky ends that have three nucleotides do not serve as “glue”, ZZZ triplets with their unique glue‐like properties are prime candidates for constructing DNA‐based nanoarchitectures.     

Conformationally restricted triplex-forming oligonucleotides (TFOs). Binding properties of α-l-LNA and introduction of the N-glycosylated LNA-guanosine

The method for scaled-up production of α-l-LNA phosphoramidite building blocks containing thymine and 5-methylcytosine nucleobases is described. Binding properties of pyrimidine TFOs modified with α-l-LNA are reported. In contrast to LNA TFOs, the fully modified α-l-LNA forms a stable triplex with a model DNA duplex. Pyrimidine DNA/LNA/α-l-LNA chimeras also efficiently hybridize with a model DNA duplex in the parallel mode. LNA nucleoside containing unnatural N⁷-glycosylated guanine (LNA-⁷G) was synthesized by a convergent method and incorporated into LNA oligonucleotides. The triplex-forming alternating DNA/LNA oligonucleotides containing a single LNA-⁷G modification instead of internal LNA-mC demonstrate improved pH-dependent properties. The single LNA-⁷G modification can also discriminatively reduce competitive binding of TFOs to natural nucleic acids in the antiparallel duplex mode.     

Site-specific synthesis and reactivity of oligonucleotides containing stereochemically defined 1,N2-deoxyguanosine adducts of the lipid peroxidation product trans-4-hydroxynonenal

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of omega-6 polyunsaturated fatty acids. The reaction of HNE with DNA gives four diastereomeric 1,N(2)-gamma-hydroxypropano adducts of deoxyguanosine; background levels of these adducts have been detected in animal tissue. Stereospecific syntheses of these four adducts at the nucleoside level have been accomplished. In addition, a versatile strategy for their site-specific incorporation into oligonucleotides has been developed. These adducts are destabilizing as measured by melting temperature when compared to an unadducted strand. The thermal destablization of the adducted 12-mers ranged from 5 to 16 degrees C and is dependent on the absolute stereochemistry of the adduct. The HNE adducts were also examined for their ability to form interstrand DNA-DNA cross-links when incorporated into a CpG sequence. We find that only one of the HNE stereoisomers formed interstrand DNA-DNA cross-links.