A synthetic random basic copolymer with promiscuous binding to class II major histocompatibility complex molecules inhibits T-cell proliferative responses to major and minor histocompatibility antigens in vitro and confers the capacity to prevent murine graft-versus-host disease in vivo.

Graft-versus-host disease (GVHD) is a T-cell-mediated disease of transplanted donor T cells recognizing host alloantigens. Data presented in this report show, to our knowledge, for the first time that a synthetic copolymer of the amino acids L-Glu, L-Lys, L-Ala, and L-Tyr (molecular ratio, 1.9:6.0:4.7:1.0; Mr, 6000-8500) [corrected], termed GLAT, with promiscuous binding to multiple major histocompatibility complex class II alleles is capable of preventing lethal GVHD in the B10.D2 --> BALB/c model (both H-2d) across minor histocompatibility barriers. Administration of GLAT over a limited time after transplant significantly reduced the incidence, onset, and severity of disease. GLAT also improved long-term survival from lethal GVHD: 14/25 (56%) of experimental mice survived > 140 days after transplant compared to 2/26 of saline-treated or to 1/10 of hen egg lysozyme-treated control mice (P < 0.01). Long-term survivors were documented to be fully chimeric by PCR analysis of a polymorphic microsatellite region in the interleukin 1beta gene. In vitro, GLAT inhibited the mixed lymphocyte culture in a dose-dependent fashion across a variety of major barriers tested. Furthermore, GLAT inhibited the response of nylon wool-enriched T cells to syngeneic antigen-presenting cells presenting minor histocompatibility antigens. Prepulsing of the antigen-presenting cells with GLAT reduced the proliferative response, suggesting that GLAT inhibits antigen presentation.

Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.

A minor groove binder (MGB) derivative (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate tripeptide; CDPI3) was covalently linked to the 5' or 3' end of several oligodeoxyribonucleotides (ODNs) totally complementary or possessing a single mismatch to M13mp19 single-stranded DNA. Absorption thermal denaturation and slot-blot hybridization studies showed that conjugation of CDPI3 to these ODNs increased both the specificity and the strength with which they hybridized. Primer extension of the same phage DNA by a modified form of phage T7 DNA polymerase (Sequenase) was physically blocked when a complementary 16-mer with a conjugated 5'-CDPI3 moiety was hybridized to a downstream site. Approximately 50% of the replicating complexes were arrested when the blocking ODN was equimolar to the phage DNA. Inhibition was unaffected by 3'-capping of the ODN with a hexanol group or by elimination of a preannealing step. Blockage was abolished when a single mismatch was introduced into the ODN or when the MGB was either removed or replaced by a 5'-acridine group. A 16-mer with a 3'-CDPI3 moiety failed to arrest primer extension, as did an unmodified 32-mer. We attribute the exceptional stability of hybrids formed by ODNs conjugated to a CDPI3 to the tethered tripeptide binding in the minor groove of the hybrid. When that group is linked to the 5' end of a hybridized ODN, it probably blocks DNA synthesis by inhibiting strand displacement. These ODNs conjugated to CDPI3 offer attractive features as diagnostic probes and antigene agents.

Calicheamicin-DNA complexes: warhead alignment and saccharide recognition of the minor groove.

The solution structures of calicheamicin gamma 1I, its cycloaromatized analog (calicheamicin epsilon), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin gamma 1I such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin gamma 1I is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.

Cyclic polyamides for recognition in the minor groove of DNA.

Small molecules that specifically bind with high affinity to any designated DNA sequence in the human genome would be useful tools in molecular biology and potentially in human medicine. Simple rules have been developed to rationally alter the sequence specificity of minor groove-binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Crescent-shaped polyamides bind as antiparallel dimers with each polyamide making specific contacts with each strand on the floor of the minor groove. Cyclic polyamides have now been synthesized that bind designated DNA sequences at subnanomolar concentrations.

RecA.oligonucleotide filaments bind in the minor groove of double-stranded DNA.

Escherichia coli RecA protein, in the presence of ATP or its analog adenosine 5'-[gamma-thio]triphosphate, polymerizes on single-stranded DNA to form nucleoprotein filaments that can then bind to homologous sequences on duplex DNA. The three-stranded joint molecule formed as a result of this binding event is a key intermediate in general recombination. We have used affinity cleavage to examine this three-stranded joint by incorporating a single thymidine-EDTA.Fe (T*) into the oligonucleotide part of the filament. Our analysis of the cleavage patterns from the joint molecule reveals that the nucleoprotein filament binds in the minor groove of an extended Watson-Crick duplex.

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.