S. V. Santhana Mariappan

STRUCTURE OF A TUMOR ASSOCIATED ANTIGEN CONTAINING A TANDEMLY REPEATED IMMUNODOMINANT EPITOPE

Human mucins are T or S glycosylated tandem repeat proteins. In breast cancer, mucins become under or unglycosylated. Two-dimensional nuclear magnetic resonance experiments are performed on chemically synthesized mucin tandem repeat polypeptides, (PDTRPAPGST-APPAHGVTSA)n the unglycosylated form for n=1,3 where (APDTR) constitutes the antigenic sites for the antibodies isolated form the tumors in the breast cancer patients. These studies demonstrate how the tandem repeats assemble in space giving rise to the overall tertiary structure, and the local structure and presentation of the antigenic site(APDTR) at the junction of two neighboring repeats. The NMR data reveal repeating knob-like structures connected by extended spacers. The knobs protrude away from the long-axis of Muc-1 and the predominant antigenic site (APDTR) forms the accessible tip of the knob. Multiple tandem repeats enhance the rigidity and presentation of the knob-like structures.

DNA Repeats in the Human Genome

AbstractRepetitive DNA sequences, interspersed throughout the human genome, are capable of forming a wide variety of unusual DNA structures with simple and complex loopfolding patterns. The hairpin formed by the fragile X repeat, (CCG)n, and the bipartite triplex formed by the Friedreichs ataxia repeat, (GAA)n/(TTC)n, show simple loopfolding. On the other hand, the doubly folded hairpin formed by the human centromeric repeat, (AATGG)n, the hairpin G‐quartet formed by (TTAGGG)n at the 3′ telomere overhang, and the hairpin G‐quartet, and hairpin C+•C paired i‐motif formed by the insulin minisatellite,

A [2+2] cross-photodimerisation of photostable olefins via a three-component cocrystal solid solution.

\left( \begin{gathered}{\text{ACAG}}_{\text{4}} {\text{TGTG}}_{\text{4}} \hfill \\{\text{TGTC}}_{\text{4}} {\text{ACAC}}_{\text{4}} \hfill \\ \end{gathered} \right)

A PCR-based method for uniform 13C/15N labeling of long DNA oligomers

show multiple and complex loopfolding. We have performed high resolution nuclear magnetic resonance (NMR) spectroscopy and in vitro replication to show that unique base-pairing and loopfolding render stability to these unusual structures under physiological conditions. The formation of such stable structures offers a mechanism of unwinding which is advantageous during transcription. For example, the formation of the hairpin G-quartet, and hairpin C2+•C paired i-motif upstream of the insulin gene may facilitate transcription. These unusual DNA structures also provide unique ‘protein recognition motifs’ quite different from a Watson—Crick double helix. For example, the hairpin G-quartet formed by (TTAGGG)n at the 3′ telomere overhang is specifically recognized and stabilized by the human repair protein, Ku70/Ku80 hetero-dimer, which may be important in the stability of the telomere. However, the formation of the same unusual DNA structures during replication is likely to cause instability in the lengths of the DNA repeats. If the altered (generally expanded) length enhances the probability of the unusual structure during the next cycle of replication, it further increases the instability of the repeat causing a ‘dynamic mutation’. In fact, NMR and in vitro replication studies show that the longer the repeat length the higher is the probability of hairpin formation by the fragile X repeat, (CCG)n. In addition, the hairpin of the fragile X repeat, upstream of the FMR-1 gene, is more susceptible to CpG methylation than its duplex thereby leading to methyl-directed suppression of transcription. Thus, the selective advantage of the unusual structures formed by the DNA repeats in the regulation of gene expression may be offset by the genomic instability caused by the same structures during replication. The repeat number is a critical parameter that helps maintain a balance between the advantage gained from an unusual structure during gene expression and the disadvantage posed by the same structure during replication.

Intramolecular [2 + 2] Photodimerization Achieved in the Solid State via Coordination-Driven Self-Assembly.

The oxidation and toxicity of dopamine is believed to contribute to the selective neurodegeneration associated with Parkinson disease. The formation of reactive radicals and quinones greatly contributes to dopaminergic toxicity through a variety of mechanisms. The physiological metabolism of dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL) via monoamine oxidase significantly increases its toxicity. To more adequately explain this enhanced toxicity, we hypothesized that DOPAL is capable of forming radical and quinone species upon oxidation. Here, two unique oxidation products of DOPAL are identified. Several different oxidation methods gave rise to a transient DOPAL semiquinone radical, which was characterized by electron paramagnetic resonance spectroscopy. NMR identified the second oxidation product of DOPAL as the ortho-quinone. Also, carbonyl hydration of DOPAL in aqueous media was evident via NMR. Interestingly, the DOPAL quinone exists exclusively in the hydrated form. Furthermore, the enzymatic and chemical oxidation of DOPAL greatly enhance protein cross-linking, whereas auto-oxidation results in the production of superoxide. Also, DOPAL was shown to be susceptible to oxidation by cyclooxygenase-2 (COX-2). The involvement of this physiologically relevant enzyme in both oxidative stress and Parkinson disease underscores the potential importance of DOPAL in the pathogenesis of this condition.

The stereochemistry of N-methyl and aryl substituents determine the biological activities of 3-aryl-8-methyl-8-azabicyclo[3.2.1]oct-2,3-enes

A ditopic hydrogen-bond-donor template in the form of resorcinol facilitates a [2+2] cross-photodimerisation of 4-Cl-stilbazole and 4-Me-stilbazole in a rare cocrystal solid solution. A photoreaction does not proceed with the olefins individually or as a solid solution composed solely of the two olefins.

Supramolecular Construction of an Aldehyde–Cyclobutane via the Solid State: Combining Reversible Imine Formation and Metal–Organic Self-Assembly

Treatment of an achiral molecular ladder of C2h symmetry composed of five edge-sharing cyclobutane rings, or a [5]-ladderane, with acid results in cis- to trans-isomerization of end pyridyl groups. Solution NMR spectroscopy and quantum chemical calculations support the isomerization to generate two diastereomers. The NMR data, however, could not lead to unambiguous configurational assignments of the two isomers. Single-crystal X-ray diffraction was employed to determine each configuration. One isomer readily crystallized as a pure form and X-ray diffraction revealed the molecule as being achiral based on Ci symmetry. The second isomer resisted crystallization under a variety of conditions. Consequently, a strategy based on a cocrystallization was developed to generate single crystals of the second isomer. Cocrystallization of the isomer with a carboxylic acid readily afforded single crystals that confirmed a chiral ladderane based on C2 symmetry. The chiral ladderane and acid self-assembled to generate a five-component hydrogen-bonded complex that packs to form large solvent-filled homochiral channels of nanometer-scale dimensions. Whereas cocrystallizations are frequently applied to structure determinations of proteins, our study represents the first application of a cocrystallization to confirm the relative configuration of a small-molecule diastereomer generated in a solution-phase organic synthesis.

A solid-state [2 + 2] photodimerization involving coordination of Ag(I) ions to 2-pyridyl groups

A polymerase chain reaction (PCR)‐based method is described for uniform 13C/15N labeling of DNA duplexes. In this method, multiple copies of a blunt‐ended duplex are cloned into a plasmid with each copy containing the sequence of interest and the restriction HincII sequences at the 5′ and 3′ ends. PCR with uniformly 13C/15N‐labeled dNTP precursors results in a labeled DNA duplex containing multiple copies of the sequence of interest. Use of bi‐directional primers, instead of self‐priming [Louis et al. (1998) J. Biol. Chem. 273, 2374–2378], produces a DNA fragment of unique length. Twenty‐four cycles of PCR of this purified product followed by restriction and purification gives (with 30% yield) the uniformly 13C/15N‐labeled duplex sequence for multi‐nuclear magnetic resonance spectroscopy.