Deconvolution of a Complex Target Using DNA Aptamers

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 40, pp. 34193–34201

In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets.Wepropose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected
populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to
the analysis of other complex biological targets.

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

Gene Therapy (2007) 14, 682–689

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirusmediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.

A DNA nanomachine that maps spatial and temporal pH changes inside living cells

Mingxu presented this paper on 4/30/09

Nature Nanotechnology 2009

DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upon stimulation by
external triggers. Previously, the performance of DNA devices has been limited to in vitro applications. Here we report the
construction of a DNA nanomachine called the I-switch, which is triggered by protons and functions as a pH sensor based
on fluorescence resonance energy transfer (FRET) inside living cells. It is an efficient reporter of pH from pH 5.5 to 6.8,
with a high dynamic range between pH 5.8 and 7. To demonstrate its ability to function inside living cells we use the
I-switch to map spatial and temporal pH changes associated with endosome maturation. The performance of our DNA
nanodevices inside living systems illustrates the potential of DNA scaffolds responsive to more complex triggers in
sensing, diagnostics and targeted therapies in living systems.

Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins

Nature Biotechnology 27, 378 – 386 (2009)

Although the classification of cell types often relies on the identification of cell surface proteins as differentiation markers, flow
cytometry requires suitable antibodies and currently permits detection of only up to a dozen differentiation markers in a single
measurement. We use multiplexed mass-spectrometric identification of several hundred N-linked glycosylation sites specifically
from cell surface–exposed glycoproteins to phenotype cells without antibodies in an unbiased fashion and without a priori
knowledge. We apply our cell surface–capturing (CSC) technology, which covalently labels extracellular glycan moieties on
live cells, to the detection and relative quantitative comparison of the cell surface N-glycoproteomes of T and B cells, as
well as to monitor changes in the abundance of cell surface N-glycoprotein markers during T-cell activation and the controlled
differentiation of embryonic stem cells into the neural lineage. A snapshot view of the cell surface N-glycoproteins will enable
detection of panels of N-glycoproteins as potential differentiation markers that are currently not accessible by other means.

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.