Asymmetric Shorter-duplex siRNA Structures Trigger Efficient Gene Silencing With Reduced Nonspecific Effects

Molecular Therapy (2009) 17 4, 725–732

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi) pathway. The current standard synthetic siRNA structure harbors a 19–base-pair duplex region with 3′ overhangs of 2 nucleotides (the so-called 19+2 form). However, the synthetic 19+2 siRNA structure exhibits several sequence-independent, nonspecific effects, which has posed challenges to the development of RNAi therapeutics and specific silencing of genes in research. In this study, we report on the identification of truncated siRNA backbone structures with duplex regions shorter than 19 bp (referred to as asymmetric shorter-duplex siRNAs or asiRNAs) that can efficiently trigger gene silencing in human cell lines. Importantly, this asiRNA structure significantly reduces nonspecific effects triggered by conventional 19+2 siRNA scaffold, such as sense-strand–mediated off-target gene silencing and saturation of the cellular RNAi machinery. Our results suggest that this asiRNA structure is an important alternative to conventional siRNAs for both functional genomics studies and therapeutic applications.

A Role for RNAi in the Selective Correction of DNA Methylation Defects

Science, 2009, 323, 1600

DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, which suggests that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis (as in cancer) or meiosis, leading to epiallelic variation. We demonstrated the existence of an efficient mechanism that protects against transgenerational loss of DNA methylation in Arabidopsis. Remethylation is specific to the subset of heavily methylated repeats that are targeted by the RNA interference (RNAi) machinery. This process does not spread into flanking regions, is usually progressive over several generations, and faithfully restores wild-type methylation over target sequences in an RNAi-dependent manner. Our findings suggest an important role for RNAi in protecting genomes against long-term epigenetic defects.

Variants of the Antibody Herceptin That Interact with HER2 and VEGF at the Antigen Binding Site

Science, 2009, 323, 1610

The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. An affinity-improved version of bH1 inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models. Such “two-in-one” antibodies challenge the monoclonal antibody paradigm of one binding site, one antigen. They could also provide new opportunities for antibody-based therapy.

Detection of Trace Hg2+ via Induced Circular Dichroism of DNA Wrapped Around Single-Walled Carbon Nanotubes

J. Am. Chem. Soc., 2008, 130, 9190

The strongly induced circular dichroism (ICD) signals of DNA wrapped around single-walled carbon nanotubes (SWCNTs) are shown by the Synchrotron Radiation Facility. In solution, trace amounts of Hg ions have a strong affinity to bind the nucleic bases of DNA-SWCNTs via a pseudo-first-order kinetic reaction. The Hg binding to the bases of DNA results in partial DNA disassociation from the SWCNTs. Such disassociation of DNA from the SWCNTs will decrease the coupling effects of the transition dipole moments between DNA and SWCNTs, thus inducing the ICD signal of DNA-SWCNTs to decrease significantly. Herein, the ICD of DNA-SWCNTs is applied to detect the concentration of Hg ions at nM level.

A pH-driven, reconfigurable DNA nanotriangle

Chem. Commun., 2009, 824

A simple and robust DNA nanotriangle that can be conveniently reconfigured by environmental pH changes is demonstrated.