Search

The binding of two hairpin polyamide ligands at adjacent sites on DNA is studied using NMR spectroscopy. In all of the double-hairpin complexes the ligand tails are sufficiently close that they could be modified to allow favorable interactions.

In the polyamide-DNA binding process, the large differences in the equilibrium association constants between the unlinked and covalently linked polyamides are ascribed to higher rate constants. However, the polyamide discrimination between matched and mismatched binding sites is due to differences in their dissociation rate constants.

The nonpalindromic recognition site of the Drosophila basic helix-loop-helix (bHLH) Deadpan transcription factor creates asymmetrical DNA binding. These transcription factors bind to hexnucleotide sequences in the major groove.

N-terminal acetylation and C-terminal tail structure effects on binding orientation of imadzaole/pyrrole polyamide DNA ligands were examined.

The affinity of the hydroxypyrrole (Hp)/pyrrole (Py) pair for T.A, A.t, C.G and G.C base pairs is described.

Research concerning the ability of polycytidylate to direct and catalyze the synthesis of oligoguanylates from guanosine 5'-phosphate 2-methylimizadolide is discussed. Results demonstrate that small amounts of isomeric dimers are formed slowly in the absence of poly(C).

Research was conducted to examine whether pyrrole-imidazole polyamides can exert a positive influence on transcription by interfering with the activity of a specific repressor protein. The human cytomegalovirus IE86 repressor protein is appropriate for the study since it is a minor groove DNA-binding protein and transcriptional repression is dependent on IE86 binding to its DNA target site. Results show that IE86 exerts its negative effect on transcription by blocking recruitment of RNA polymerase II to the preinitiation complex.

Three pyrrole-imidazole polyamides of sequence composition ImPyPy-X-PyPyPy-Dp bound to a 13 base pair DNA duplex with a 9 base pair 5'-TGTATATCA-3' match site each adopt an extended antiparallel dimeric conformation with the ligands fully overlapped around a central Py/Py, G/G, or beta/beta pair. Nuclear magnetic resonance characterization data reveal that conformational exchange takes place near the linker for the G-linked complex, but not with the beta or Py linkers. Results provide the first direct structural evidence for formation of the aliphatic beta/beta pairing in the minor groove.

A study was conducted to extend previous investigations of cycle polyamides. The experiment reports the DNA binding affinities and sequence specificities of two eight-ring cycle polyamides that differ by a single amino acid substitution for their respective six base pair match sites, which in turn differ by a single base pair. A pyrrole resin is also described which allows cycle polyamide linear precursors to be synthesized by solid-phase methods.

A hairpin polyamide ImImPy-gamma-ImPyPy-beta-Dp and the corresponding affinity cleaving analogue were synthesized to probe the sequence-specific effects of the gamma-turn and beta-tail amino acids. Three separate techniques, namely, MPE*Fe(II) footprinting, affinity cleaving and DNase I footprinting were used to determine the affinities, binding sites and relative selectivities of these polyamides. Results of the study are discussed.

Polyamides that contain the aromatic amino acids N-methylpyrrole (Py), N-methylimidazole (Im) and N-methyl-3-hydroxy-pyrrole (Hp) are synthetic ligands that follow simple rules when binding with DNA sequences. The DNA-binding properties of one six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp where gamma = gamma-aminobutyric acid, beta = beta-alanine and Dp = dimethylaminopropylamide, are reported. The thermodynamic characterization of the polyamide is also given. Results should prove useful in the design of synthetic polyamides.

The synthesis of oligoguanylate on a polycytidylate template has been undertaken. The oligomerization starts with the dimerization of the first primer (k2) that is followed by the elongation of a preformed primer (ki) and ends with the hydrolysis of the monomer to form the deactivated material (kh). It has been observed that ki' does not depend on i when i is greater than or equal to 4 or less than or equal to 16. The rate constants k3' and ki' for oligoguanylate elongation agree with the template-directed model and enhances next-neighbor assistance.

Synthetic ligand Polyamides containing N-methylpyrrole and N-methyllimidazole amino acids have a specificity for DNA which is comparable to those of naturally occurring DNA binding proteins. More than 100 pyrrole-imidazole polyamides have been found to recognize predetermined sequences, although there are certain difficult Py-Im/DNA base-pair sequences. Polyamides incorporating Py/beta, beta/Py, Im/beta and beta/Im pairings are good examples of aromatic/aliphatic amino acid pairings for minor groove recognition.

The DNA-binding characteristics of two enantiomeric polyamides were examined by footprinting and affinity cleavage on a DNA fragment with two match sites (5'-TGTTA-3' and 5'-ACATT-3') and one 5'-TGTCA-3' mismatch site. Findings revealed that replacement of gamma-aminobutyric acid by (R)-2,4-daiminobutyric acid improves DNA binding affinity for the 5'-TGTTA-3' match site by 13-fold. The improvement is attained without a compromise in sequence selectivity which actually improves and is 100-times higher compared to binding at a single base pair mismatch sequence, 5'-TGTCA-3'.

It has been possible to identify the DNA-binding affinities of three eight-ring hairpin polyamides containing pairings of Im/Py, Py/Im opposite G.C, C.G and either Py/Py, Hp/Py or Py/Hp at a common single point opposite T.A and A.T. A new aromatic acid, 3-hydroxypyrrole (Hp), was added to test for pairings that discriminate A.T from T.A. It was found that replacing a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude.

A study on the orientation of pyrrole was conducted to determine its influence on positive charge, position of positive charge at N- or C-terminus and substitution of the terminal imidazole. The research identified equilibrium association constants for a series of six-ring hairpin polyamides with respect to N and C termini. Results show that second generation design principles on pairing rules optimize sequence specificity of pyrrole-imidazole polyamides for double helical DNA.

The seven base pair sequences in the minor groove of DNA was analyzed by utilizing ten-ring amino acids. The hairpin polyamide motif with gamma-aminobutyric acid served as a turn-specific internal-guide-residue for the synthesis of accessible C-N linkage of polyamide subunits. The process also involved a stepwise synthetic reaction utilizing Boc-beta-alanine Pam resins as starting materials. Furthermore, the resulting binding orientation indicated the presence of gamma-linked five-ring subunits in the 10-ring polyamides.

An eight-ring hairpin polyamides model with varying linear arrangement of pyrrole and imidazole (Im) amino acids was used to identify six base pair DNA sequences having four adjoining G,C base pairs. Notable characteristics of the DNA binding in three polyamides were then analyzed through affinity cleavage and footprinting on DNA fragment with match sites 5'-TGGCCA-3', 5'-TGCGCA-3', and 5'-TGGGGA-3'. Findings revealed that the sequence of Im amino acids has significant effect on the range of binding affinities of pyrrole-imidazole polyamides.

The affinity and binding orientation of the hairpin polyamide ImPyPy-gamma-PyPyPy-beta-Dp were measured for eight possible five base pair 5'-TG(A,T)(sub 3)-3' match sites to test the A.T/T.A degeneracy of pyrrole-imidazole polyamide recognition in the minor groove of DNA. Findings indicated that the Py/Py pair is approximately degenerate for recognition of A,T base pairs, affording generality with regard to targeting sequences of mixed A.T/T.A composition.

The DNA binding properties of of two pyrrole-imidazole polyamides with a hairpin motif were examined to understand how they recognize 5'-GGG-3' sequences in the minor groove of double-stranded DNA. To this end, ImImIm-gamma-PyPyPy-beta-Dp and ImImImPy-gamma-PyPyPyPy-beta-Dp and their EDTA affinity cleaving analogs were prepared using Im = N-methylimidazole-2-carboxamide, Py = N-methylpyrrole-2-carboxamide, beta = beta-alanine, gamma = gamma-aminobutyric acid and Dp = ((dimethylamino)propyl)amide.

The solid phase methodology for the preparation of DNA binding polyamides consisting of N-methylimidazole and N-methylpyrrole amino acids involves a single-step cleavage of the bond between the polyamide and an insoluble matrix. This introduces a positive charge in the polyamide. The technique increases the complexity and number of minor groove binding polyamides that can be prepared and analyzed with respect to DNA binding ability and sequence specificity.

Pyrrole-imidazole polyamides with three-ring subunits linked by beta-alanine have a high sequence-specific recognition and binding affinity for the minor groove of DNA with 9-13 base pairs (bp). The DNA-binding affinities of polyamides with the general sequence ImPyPy-X-PyPyPy-Dp, where X = Py, G, beta, or gamma, for the 9 bp site 5'-TGTTAAACA-3' and the 13 bp sites 5'-AAAAAGACAAAAA-3' and 5'-ATATAGACATATA-3' sequences are examined. The binding affinities are determined using the results of quantitative DNase I footprint titrations.

Quantitative DNase I and MPE.FeII foot printing techniques reveal that four hairpin pyrrole-imidazole polyamides synthesized by the solid phase methodology recognize 5'-TGGTT-3' sequences. The position of the gamma-aminobutyric acid linker has little effect on the binding affinity and sequence specificity of the polyamides. An analysis of the binding affinities and specificities of the designed polyamides is presented.

The addition of a beta-alanine residue at the C-terminus increases the binding ability and sequence specificity of pyrrole-imidazole polyamides that bind to the minor groove of DNA. A glycine residue at the C-terminus however, reduces the DNA binding ability and sequence specificity. The addition of an N-terminal acetyl group regulates the effects of the C-terminal additions. An aliphatic amino acid at the C-terminus makes the hairpin polyamide motif suited for solid phase synthetic and allows the preparation of polyamides with high binding affinity.

The reactivity of the natural polyamines, spermine (spm) and spermidine (spd), with guanosine 5'-phosphate 2-methylimidazolide (2-MeImpG) generates guanosine 5'-phosphate-polyamine derivatives that are very stable for pHs between four and 11. This reaction indicates that the secondary amino groups of the polyamines are unreactive, probably due to a steric effect, leaving only the the primary groups to react with 2-MeImpG. Therefore, only two of the three possible spd products and only one of the two expected spm products are generated. The basicity of the two primary amino groups are identical.

The oligomerization kinetics of guanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpG, in the presence of polycytidylate, poly (C), functioning as a template at 23 degrees Celsius, were analyzed. The presence of additional next-neighbor monomer units aids bond formation between two monomer units or between a primer and a monomer.

Three consecutive amino acid ring Hp/Py pairs can be combined in a hairpin model to specify core sequence 5'-TTA-3' from 5'-TAT-3' in the DNA minor groove. These ring pairs were used in identifying sequences in the minor groove of the DNA. Experiments found that hydroxypyrrole-imidazole-pyrrole polyamides complete the design of the minor groove recognition code. The four-ring pairs Im/Py, Py/Im, Hp/Py and Py/Hp complete the code and complement four Watson-Crick base pairs.

The affinity and specificity of a pyrrole-imidazole polyamide dimer targeting 16 contiguous base pairs in the minor groove of DNA were investigated. An inverse relationship was observed between the length of a polyamide dimer having the general sequence ImPy2-6 and the DNA-binding affinity. Results also show that eight pairs of amide residues form a fully overlapped core as indicated by the strong binding affinity and the affinity cleavage pattern of the 16 base pair polyamide-DNA complex.

A pyrrole-imidazole polyamide oligonucleotide is found to bind cooperatively to a double-stranded DNA template via a sequence specific polyamide dimerization domain. Findings revealed that the unmodified oligonucleotide, 5'-d(TTTTTT(super Me)C-(super Me)CTTT)-3', binds each triple-helix site with an equilibrium association constant of 1.7 X 10(super 6)/M. The untethered polyamide ImPyPy binds to its 5'-TGTCA-3' target site with an equilibrium association constant of 1.5 X 10(super 5)/M.