A Switchable Site-Specific Antibody Conjugate.

Genetic incorporation of unnatural amino acids (UAAs) provides a unique approach to the synthesis of site-specific antibody conjugates that are homogeneous and better defined constructs than random conjugates. Yet, the yield varies for every antibody, and the process is costly and time-consuming. We have developed a switchable αGCN4-Fab conjugate that incorporates UAA p-acetylphenylalanine. The GCN4 peptide is used as a switch, and antibodies fused by GCN4 can direct the αGCN4-Fab conjugate to target different cancer cells for diagnosis, imaging, or therapeutic treatment. More importantly, this switchable conjugate demonstrated an impressive potential for pretargeted imaging in vivo. This approach illustrates the utility of an orthogonal switch as a general strategy to endow versatility to a single antibody conjugate, which should facilitate the application of UAA-based site-specific conjugates for a host of biomedical uses in the future.

RNA switches regulate initiation of translation in bacteria.

A large variety of RNA-based mechanisms have been uncovered in all living organisms to regulate gene expression in response to internal and external changes, and to rapidly adapt cell growth in response to these signals. In bacteria, structural elements in the 5' leader regions of mRNAs have direct effects on translation initiation of the downstream coding sequences. The docking and unfolding of these mRNAs on the 30S subunit are critical steps in the initiation process directly modulating and timing translation. Structural elements can also undergo conformational changes in response to environmental cues (i.e., temperature sensors) or upon binding of a variety of trans-acting factors, such as metabolites, non-coding RNAs or regulatory proteins. These RNA switches can temporally regulate translation, leading either to repression or to activation of protein synthesis.

A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation.

V(D)J recombination and transcription within the TCR alpha/delta locus are regulated by three characterized cis-acting elements: the TCR delta enhancer (Edelta), TCR alpha enhancer (Ealpha), and T early alpha (TEA) promoter. Analysis of enhancer and promoter occupancy and function in developing thymocytes in vivo indicates Edelta and Ealpha to be developmental-stage-specific enhancers, with Edelta "on" and Ealpha "off" in double-negative III thymocytes and Edelta "off" and Ealpha "on" in double-positive thymocytes. Edelta downregulation reflects a loss of occupancy. Surprisingly, Ealpha and TEA are extensively occupied even prior to activation. TCR delta downregulation in double-positive thymocytes depends on two events, Edelta inactivation and removal of TCR delta from the influence of Ealpha by chromosomal excision.

Evidence for a human IgG1 class switch program.

In activated murine B lymphocytes, immunoglobulin class switch recombination occurs as a highly regulated process which is targeted to distinct switch regions. Here we present first evidence that in human B lymphocytes, switch recombination is targeted to distinct switch regions as well. In a panel of clonally unrelated IgG1-expressing human B cells, immortalized by Epstein-Barr virus (EBV) transformation, seven out of nine cells show switch recombination between S mu and S gamma 1 on both alleles, the active and inactive one. The remaining cells show no switch recombination on the inactive IgH locus. The very strong correlation of switch recombination on both alleles of IgG1-expressing cells proves that class switch recombination to IgG1 is not random but directed in human B lymphocytes.