Catenanes constructed from organic building blocks have been used in the demonstration of molecular devices such as positional switches, unidirectional motors, and other nanoscale functional materials. 1 In the nucleic acid world, catenanes have long been known; they are found in nature and are common in structural DNA nanotechnology. 2 Designed, all-DNA catenanes have been used as topological labels. 3 Crosslinks with organic linkages have been utilized to probe the structures and properties of DNA duplexes, hairpins, and higher order structures, such as t-RNA and ribozymes, 4 as well as to build DNA nanostructures and nanodevices. 5 In this paper, we describe the synthesis of a macrocycle that is prepared by formation of an amide linkage across one full turn of DNA, forming a tailed DNA/organic catenane containing 5'-and 3'-termini that are available for further functionalization.In prior work, we showed that 2'-pendent amines and carboxylates could be linked to form nylon along the phosphodiester backbone contour. 6 Our inquiry here began when we asked whether longer linkers could be used to attach polymeric components parallel to the helix axis. This target requires bridging approximately 35Å across a nucleic acid turn, which had not been reported previously (Fig. 1A), although there is now a report by Gothelf et al. 7 Tetraethylene glycol was used as a spacer into a carboxylate-functionalized uridine and undecaethylene glycol was incorporated into an aminefunctionalized uridine. The linker distances have not been optimized, although studies with tetraethylene glycol as spacer for both amine and caboxylate were unsuccessful.Three 16-mer ODNs 1, 2 and 3 were synthesized, where two modified uridines were separated by 8, 9 and 10 unmodified nucleotides, respectively. Cross-turn coupling using a DNA hairpin template gave > 97% yields of ODN 1 and 2, as estimated by MALDI-TOF spectra and denaturing gel electrophoresis, whereas the coupling yield of 3 was about 62 % (Table 1, Supporting Information). For further characterization, the coupled product was subjected to complete nuclease digestion followed by analysis to detect linked nucleotides. The detection of a polyethylene glycol (PEG)-amide linked uridine dimer by LCMS corroborated the formation of coupled products (Supporting Information).These data established the formula of the reaction product but did not distinguish between a newly formed linkage parallel to the helix axis (i.e., across the turn) versus along the phosphodiester backbone contour. However, templation of the reaction by a circular DNA template (Fig. 1A) would yield an interlocked complex only if the coupling reaction occurred across the turn. ODNs 1 and 2 paired with a 78-mer DNA circular template were subjected to E-mail: E-mail: james.canary@nyu.edu; E-mail: ned.seeman@nyu.edu. Supporting Information. Full experimental details including: Syntheses of phosphoramidites, MALDI-TOF MS of ODNs, LCMS analysis of complete nuclease digestion, denaturing gel analyses of catenane synthesis, exonucle...
Background: An RNAi screen identified UBE3C as a key player in degradation of a model unfolded protein.Results: UBE3C knockdown results in incomplete degradation of relatively stable substrates. Conclusion: UBE3C enhances proteasome processivity to prevent the accumulation of potentially harmful protein fragments. Significance: This advances our understanding of proteasome processivity and the consequences of defects therein.
We report the development of technology that allows inter-strand coupling across various positions within one turn of DNA. Four 2′-modified nucleotides were synthesized as protected phosphoramidites and incorporated into DNA oligonucleotides. The modified nucleotides contain either 5-atom or 16-atom linker components, with either amine or carboxylic acid functional groups at their termini, forming 10 or 32 atom (11 or 33 bond) linkages. Chemical coupling of the amine and carboxylate groups in designed strands resulted in the formation of an amide bond. Coupling efficiency as a function of trajectory distance between the individual linker components was examined. For those nucleotides capable of forming inter-strand cross-links (ICLs), coupling yields were found to depend on temperature, distance, and linker length, enabling several approaches that can control regioselective linkage. In the most favorable cases, the coupling yields are quantitative. Spectroscopic measurements of strands that were chemically cross-linked indicate that the global structure of the DNA duplex does not appear to be distorted from the B form after coupling. Thermal denaturing profiles of those strands were shifted to somewhat higher temperatures than those of their respective control duplexes. Thus, the robust amide ICLs formed by this approach are site-specific, do not destabilize the rest of the duplex, and only minimally perturb the secondary structure.
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