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The X-ray structure of a DNA duplex containing a synthetic [N.sup.4]C-ethyl-[N.sup.4]C interstrand cross-link between the cytosines of the central CpG step is determined at 1.65 Angstrom resolution. This structure reveals that the ethyl cross-link in the CpG major grooves does not significantly disrupt the B-form DNA helix.

The structural properties underpinning the differences in the immediate environment of a N(super 3)T-ethyl-N(super 3)T interstrand cross link that is inserted into a scaffold wherein the cross link is mispair-aligned is described. Results demonstrate that the lesion defined by mismatch-aligned minor groove N(super 3)T-ethyl-N(super 3)T cross-linked thymine bases produces very localized distortions in a DNA stem that may be difficult to recognize by repair mechanisms that are not transcription-or-replication-coupled.

Results show that the cross-link N(sup)4C-Ethyl-N(sup)4C interstrand in the undecamer deoxyoligonucleotide imitates the BCNU-induced G-C cross-link and the C-C cross-link, rendering this DNA duplex recognizable by different DNA repair mechanisms.

Results indicate that bending deformations in the helix involving the -CNG- sequence cross-linked DNA duplexes may be controlled by varying the cross-link length. Data show that the cross-link containing 2,4,or 7 methylene groups joins the C residues of a -CNG- sequence in the DnA duplex.

A threshold level of the triple helix forming oligonucleotides (TFOs), consisting of three or four 2'-AE residues, are 10-fold more active than those with just 2 2'-AE residues in a hypoxanthine phosphoribosyltransferase (HPRT) activity monitored gene knockout assays. Further, the frequencies of HPRT activity with the active TFOs are 300-400 fold higher than the background activity.

This articles describes the synthesis of N(sup)4C--ethyl--N(sup)4C interstrand cross-linked DNA duplexes employing N(sup)4C--ethyl--N(sup)4C phosphoramidite reagent. The ligated oligomers containing staggered cross-links exhibit differences in structure and stability with -GC- staggered cross-links disrupt hydrogen bonding and affecting helix stability by perturbing adjacent base pairs.

Psoralen-conjugated chimeric oligonucleoside methylphosphonates and their triplex formation are discussed. Oligomers that are made up of pyrimidine nucleosides form stable triplexes at neutral pH. Triplex-forming oligonucleotides (TFOs) have been thought to perhaps be DNA-specific ligands that may be used for controlling gene expression. TFO here refers to oligonucleotides designed to bind to purine bases in the major groove of the DNA target.

Oligonucleotides have been synthesized that can bind to TAR RNA and are resistant to nuclease degradation. TAR RNA regulates HIV replication and these oligonucleotides could be used as antisense drugs to block viral replication.

The interaction between the antisense activity and oligomer binding affinity in cell cultures suggests that oligo-2'-O-methylribonucleoside methylphosphonates (mr-OMPs) can be used as effective antisense reagents. The capacity of mr-OMP to bind to its RNA target is higher than that of oligo-2'-deoxyribonucleoside methyphosphonate. The mr-OMP also inhibits herpes simplex virus type 1 (HSV-1) replication in HSV-1 infected Vero cells in culture.

Melting experiments on psoralen-derivatized oligonucleoside methylphosphonates with a random coil or a hairpin in solution reveal that the binding and cross-linking reactions are affected by target structure and sequence. The oligomer binding site position in the target RNA and the nature of the oligomer backbone enhance the binding of oligonucleotides containing phosphodiester or phosphorothiate internucleotide linkages to RNA-hairpins.

Ultraviolet melting experiments were carried out to study the stabilities of oligodeoxyribonucleotide triplexes containing single pyrimidine (Py)-purine (Pu) base pair. The Py-Pu base pair interrupts an otherwise Pu-Py base pair motif. In particular, the formation of X-U-A triads, where U is dexoyuridine, was focused on. Triplex formation in this system was also compared with that involving X-C-G and X-T-A base triads.

The interaction of two oligodeoxyribonucleotides, I(A) and II(A), with the duplex deoxyribonucleotide III-IV(Y-Z) was studied. The results of continuous variation experiments, melting experiments and circular dichroism spectroscopy show that I(A) and II(A) each form triplexes with III-IV(Y-Z) at neutral and basic pH. Moreover, it appears that 8-oxoadenine residue in the two oligodeoxyribonucleotides interacts in a specific manner with G-C base pairs of the duplex.

A study was conducted to understand the cross-link structure of DNA interstrand that affects the repair in nonreplicating reporter plasmids that contain a mispaired [N.sup.4]C-ethyl-[N.sup.4]C (C-C), N3T-ethyl-N3T (T-T), or N1I-ethyl-N3T (I-T) ICL. The insertion of both, C and G residues at the C-C cross-link site without any need for Rev1 for repair indicated that replicative or other translesion polymerases could bypass the C-C remnant.

The effects of the phosphodiester backbone on the reactions of cisplatin with oligodeoxyribonucleotides that lack or contain a GTG sequence are examined. The results have shown that interactions between cisplatin and the negatively charged phosphodiester backbone have played a vital role in facilitating platination of guanine nucleotides in DNA.

Interstrand cross-linked (ICL) linear DNA duplexes are used to examine cross-link processing in mammalian whole-cell extracts. The results have shown that in DNA not undergoing transcription or replication, distortions induced by the presence of an ICL has contributed to initial cross-link recognition and processing.

Triple helix forming oligonucleotides (TFOs) are developed for gene targeting and their influence on bioactivity of the modifications is examined. The enhancement of TFO bioactivity has required chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences.

The synthesis of triple helix forming oligonucleotide (TFOs) designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinpathies, including sickle cell anemia is described. The results validate the modification format described and indicate that 2'-O-methylpseudoisocytidine substitutions are an effective solution to problem of targeting genomic sequences containing adjacent cystosines.

A study characterizes the repair, structure, and flexibility of DNA that contains N4C-ethyl-N4C interstand DNA cross-link in two different orientations. A combination of (super 1)H nuclear magnetic resonance (NMR), restrained molecular dynamics simulations, and atomic force microscopy (AFM) was used to examine the structure and flexibility of DNA containing these cross-links.

The development in living mammalian cells of triple helix-forming oligonucleotides as gene targeting reagents is studied. The results show that for optimal bioactivity, the 2'-0-[2-Aminoethyl] residues should be clustered rather than dispersed.