Stereochemical control of the DNA binding affinity, sequence specificity, and orientation preference of chiral hairpin polyamides in the minor groove

Three-ring polyamides containing pyrrole (Py) and imidazole (Im) amino acids covalently coupled by γ-aminobutyric acid (γ) form six-ring hairpins that recognize five-base-pair sequences in the minor groove of DNA. Selective chiral substitution of the ``gamma-turn'' enhances the properties of polyamide hairpins with regard to DNA affinity and sequence specificity. Polyamides of core sequence composition ImPyPy-γ-PyPyPy which differ by selective stereochemical substitution of the prochiral alpha-position in the γ-turn were prepared. The DNA binding properties of two enantiomeric polyamides were analyzed by footprinting and affinity cleavage on a DNA fragment containing two match sites (5'-TGTTA-3' and 5'-ACATT-3') and one 5'-TGTCA-3' mismatch site. Quantitative footprint titrations demonstrate that replacement of γ-aminobutyric acid by (R)-2,4-diaminobutyric acid enhances DNA binding affinity for the 5'-TGTTA-3' match site 13-fold (K_a = 3.8 x 10^9 M-1). The enhanced affinity is achieved without a compromise in sequence selectivity, which in fact increases and is found to be 100-fold higher relative to binding at a single base pair mismatch sequence, 5'-TGTCA-3'. An (S)-2,4-diaminobutyric acid linked hairpin binds with 170-fold reduced affinity relative to the R-enantiomer and only 5-fold sequence specificity versus a 5'-ACATT-3' reversed orientation site. These effects are modulated by acetylation of the chiral amine substituents. This study identifies structural elements which should facilitate the design of new hairpin polyamides with improved DNA binding affinity, sequence specificity, and orientational selectivity

Answer and Return of Writ

Respondent says that the record in this case affirmatively shows that the petitioner (Sheppard) was awarded a full and fair hearing in the state courts, resulting in reliable findings of fact, and that the state courts applied correct constitutional standards in disposing of the various claims of the petitioner. Respondent denies that the trial court erred in refusing to grant petitioner (Sheppard) a new trial on the ground of newly discovered evidence. Respondent denies that any relevant material or substantial evidence was suppressed by the prosecution or that any unjust tactics were used by the prosecuting authorities in the trial of this case. Respondent denies that the petitioner (Sheppard) was prevented from having a fair and impartial trial by the action of the trial judge as a result of the actions of the bailiffs in permitting the jurors to make telephone calls. Respondent denies that the trial judge coerced the jury into reaching a verdict, and avers that the fact that the jury deliberated for a period of five days merely shows the carefulness and consideration that the jury gave the mass of testimony and over 200 exhibits in the case. Respondent denies each and every other allegation in the petition not herein admitted to be true. Affirmatively, respondent alleges that petitioner was convicted in a court that had jurisdiction of his person and of the crime involved, that petitioner was not deprived of any of his constitutional rights, and that the facts upon which petitioner (Sheppard) relies, even if true, constitute mere error in the trial court, which is not cognizable in an action of habeas corpus. For the foregoing reasons, respondent prays that the petition herein be dismissed

Answer and Return of Writ

Respondent says that the record in this case affirmatively shows that the petitioner (Sheppard) was awarded a full and fair hearing in the state courts, resulting in reliable findings of fact, and that the state courts applied correct constitutional standards in disposing of the various claims of the petitioner. Respondent denies that the trial court erred in refusing to grant petitioner (Sheppard) a new trial on the ground of newly discovered evidence. Respondent denies that any relevant material or substantial evidence was suppressed by the prosecution or that any unjust tactics were used by the prosecuting authorities in the trial of this case. Respondent denies that the petitioner (Sheppard) was prevented from having a fair and impartial trial by the action of the trial judge as a result of the actions of the bailiffs in permitting the jurors to make telephone calls. Respondent denies that the trial judge coerced the jury into reaching a verdict, and avers that the fact that the jury deliberated for a period of five days merely shows the carefulness and consideration that the jury gave the mass of testimony and over 200 exhibits in the case. Respondent denies each and every other allegation in the petition not herein admitted to be true. Affirmatively, respondent alleges that petitioner was convicted in a court that had jurisdiction of his person and of the crime involved, that petitioner was not deprived of any of his constitutional rights, and that the facts upon which petitioner (Sheppard) relies, even if true, constitute mere error in the trial court, which is not cognizable in an action of habeas corpus. For the foregoing reasons, respondent prays that the petition herein be dismissed

Measles vaccination and antibody response in autism spectrum disorder

OBJECTIVE: To test the hypothesis that measles vaccination was involved in the pathogenesis of autism spectrum disorders (ASD) as evidenced by signs of a persistent measles infection or abnormally persistent immune response shown by circulating measles virus or raised antibody titres in children with ASD who had been vaccinated against measles, mumps and rubella (MMR) compared with controls. DESIGN: Case-control study, community based. METHODS: A community sample of vaccinated children aged 10-12 years in the UK with ASD (n = 98) and two control groups of similar age, one with special educational needs but no ASD (n = 52) and one typically developing group (n = 90), were tested for measles virus and antibody response to measles in the serum. RESULTS: No difference was found between cases and controls for measles antibody response. There was no dose-response relationship between autism symptoms and antibody concentrations. Measles virus nucleic acid was amplified by reverse transcriptase-PCR in peripheral blood mononuclear cells from one patient with autism and two typically developing children. There was no evidence of a differential response to measles virus or the measles component of the MMR in children with ASD, with or without regression, and controls who had either one or two doses of MMR. Only one child from the control group had clinical symptoms of possible enterocolitis. CONCLUSION: No association between measles vaccination and ASD was shown

Stereochemical control of the DNA binding affinity, sequence specificity, and orientation preference of chiral hairpin polyamides in the minor groove

Three-ring polyamides containing pyrrole (Py) and imidazole (Im) amino acids covalently coupled by γ-aminobutyric acid (γ) form six-ring hairpins that recognize five-base-pair sequences in the minor groove of DNA. Selective chiral substitution of the ``gamma-turn'' enhances the properties of polyamide hairpins with regard to DNA affinity and sequence specificity. Polyamides of core sequence composition ImPyPy-γ-PyPyPy which differ by selective stereochemical substitution of the prochiral alpha-position in the γ-turn were prepared. The DNA binding properties of two enantiomeric polyamides were analyzed by footprinting and affinity cleavage on a DNA fragment containing two match sites (5'-TGTTA-3' and 5'-ACATT-3') and one 5'-TGTCA-3' mismatch site. Quantitative footprint titrations demonstrate that replacement of γ-aminobutyric acid by (R)-2,4-diaminobutyric acid enhances DNA binding affinity for the 5'-TGTTA-3' match site 13-fold (K_a = 3.8 x 10^9 M-1). The enhanced affinity is achieved without a compromise in sequence selectivity, which in fact increases and is found to be 100-fold higher relative to binding at a single base pair mismatch sequence, 5'-TGTCA-3'. An (S)-2,4-diaminobutyric acid linked hairpin binds with 170-fold reduced affinity relative to the R-enantiomer and only 5-fold sequence specificity versus a 5'-ACATT-3' reversed orientation site. These effects are modulated by acetylation of the chiral amine substituents. This study identifies structural elements which should facilitate the design of new hairpin polyamides with improved DNA binding affinity, sequence specificity, and orientational selectivity

Concurrent codes:a holographic-type encoding robust against noise and loss

Concurrent coding is an encoding scheme with 'holographic' type properties that are shown here to be robust against a significant amount of noise and signal loss. This single encoding scheme is able to correct for random errors and burst errors simultaneously, but does not rely on cyclic codes. A simple and practical scheme has been tested that displays perfect decoding when the signal to noise ratio is of order -18dB. The same scheme also displays perfect reconstruction when a contiguous block of 40% of the transmission is missing. In addition this scheme is 50% more efficient in terms of transmitted power requirements than equivalent cyclic codes. A simple model is presented that describes the process of decoding and can determine the computational load that would be expected, as well as describing the critical levels of noise and missing data at which false messages begin to be generated

The 2001 Mars In-Situ-Propellant-Production Precursor (MIP) Flight Demonstration

The successful performance of the five individual demonstrations of MARS IN-SITU-PROPELLANT-PRODUCTION PRECURSOR (MIP) will provide both knowledge of and confidence in the reliability of this technology. At the completion of this flight demonstration, the MIP Team will be able to: a) recommend preferred hardware configurations for the intake and adsorption of carbon dioxide from the Martian atmosphere; b) understand the performance characteristics of zirconia cells to generate propellant-grade oxygen; c) understand long-term performance characteristics of advanced solar cells/arrays operated in the actual Mars environment; d) evaluate the functionality of methods to mitigate the deposition of airborne dust onto solar arrays; and e) recommend preferred hardware designs for innovative thermal management including the radiation of heat to the outside environment

Cycle polyamide motif for recognition of the minor groove of DNA

Motifs for covalent linkage of side-by-side complexes of pyrrole−imidazole (Py−Im) polyamides in the DNA minor groove provide for small molecules that specifically recognize predetermined sequences with subnanomolar affinity. Polyamide subunits linked by a turn-specific γ-aminobutyric acid (γ) residue form hairpin polyamide structures. Selective amino-substitution of the prochiral α-position of the γ-turn residue relocates the cationic charge from the hairpin C terminus. Here we report the synthesis of pyrrole resin as well as a solid-phase strategy for the preparation of cycle polyamides. The DNA binding properties of two eight-ring cycle polyamides were analyzed on a DNA restriction fragment containing six base pair match and mismatch binding sites. Quantitative footprint titrations demonstrate that a cycle polyamide of sequence composition cyclo-(γ-ImPyPyPy-(R)^(H2N)γ-ImPyPyPy-) binds a 5‘-AGTACT-3‘ site with an equilibrium association constant K_a = 7.6 × 10^(10) M^(-1), a 3600-fold enhancement relative to the unlinked homodimer (ImPyPyPy-β-Dp)_2·5‘-AGTACT-3‘, and an 8-fold enhancement relative to hairpin analogue ImPyPyPy-(R)^(H2N)γ-ImPyPyPy-C3−OH·5‘-AGTACT-3‘. Replacement of a single nitrogen atom with a C−H (Im→Py) regulates affinity and specificity of the cycle polyamide by 2 orders of magnitude. The results presented here suggest that addition of a chiral γ-turn combined with placement of a second γ-turn within the hairpin structure provides a cycle polyamide motif with favorable DNA binding properties

Analysis of hairpin polyamide complexes having DNA binding sites in close proximity

The binding of two hairpin polyamide ligands at adjacent sites on DNA has been studied using NMR spectroscopy. The ligands ImPyPy-γ-PyPyPy-Gly-Dp and Ac-ImPyPy-γ-PyPyPy-Gly-Dp were studied binding to oligomers containing one or two matched binding sites:  5‘-XGTTA-3‘ and 5‘-TAACX_NGTTA-3‘, where X is G, C, or A and N = 0, 1 or 2. At these sites the C-terminal ring shows an equilibrium between normal and inverted conformations. Better binding was observed with the ligand running 5‘ to 3‘ along the contacted strand than in the opposite direction. Complexes of DNAs with two binding sites indicated that at least one spacing base pair was required, and that the identity of this base pair was not critical. Binding with 5‘ to 3‘ contact is again preferred. Demonstrated binding at adjacent sites indicates that it may be possible to engineer cooperative binding for enhanced specificity or affinity

Analysis of hairpin polyamide complexes having DNA binding sites in close proximity

The binding of two hairpin polyamide ligands at adjacent sites on DNA has been studied using NMR spectroscopy. The ligands ImPyPy-γ-PyPyPy-Gly-Dp and Ac-ImPyPy-γ-PyPyPy-Gly-Dp were studied binding to oligomers containing one or two matched binding sites:  5‘-XGTTA-3‘ and 5‘-TAACX_NGTTA-3‘, where X is G, C, or A and N = 0, 1 or 2. At these sites the C-terminal ring shows an equilibrium between normal and inverted conformations. Better binding was observed with the ligand running 5‘ to 3‘ along the contacted strand than in the opposite direction. Complexes of DNAs with two binding sites indicated that at least one spacing base pair was required, and that the identity of this base pair was not critical. Binding with 5‘ to 3‘ contact is again preferred. Demonstrated binding at adjacent sites indicates that it may be possible to engineer cooperative binding for enhanced specificity or affinity