RNA aptamers generated against oligomeric Abeta40 recognize common amyloid aptatopes with low specificity but high sensitivity.

Aptamers are useful molecular recognition tools in research, diagnostics, and therapy. Despite promising results in other fields, aptamer use has remained scarce in amyloid research, including Alzheimer's disease (AD). AD is a progressive neurodegenerative disease believed to be caused by neurotoxic amyloid beta-protein (Abeta) oligomers. Abeta oligomers therefore are an attractive target for development of diagnostic and therapeutic reagents. We used covalently-stabilized oligomers of the 40-residue form of Abeta (Abeta40) for aptamer selection. Despite gradually increasing the stringency of selection conditions, the selected aptamers did not recognize Abeta40 oligomers but reacted with fibrils of Abeta40, Abeta42, and several other amyloidogenic proteins. Aptamer reactivity with amyloid fibrils showed some degree of protein-sequence dependency. Significant fibril binding also was found for the naïve library and could not be eliminated by counter-selection using Abeta40 fibrils, suggesting that aptamer binding to amyloid fibrils was RNA-sequence-independent. Aptamer binding depended on fibrillogenesis and showed a lag phase. Interestingly, aptamers detected fibril formation with > or =15-fold higher sensitivity than thioflavin T (ThT), revealing substantial beta-sheet and fibril formation undetected by ThT. The data suggest that under physiologic conditions, aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils. Nevertheless, the high sensitivity, whereby aptamers detect beta-sheet formation, suggests that they can serve as superior amyloid recognition tools.

Aptamer-based endocytosis of a lysosomal enzyme.

Enzyme replacement therapy for lysosomal storage diseases is currently based on endocytosis of lysosomal enzymes via the mannose or mannose 6-phosphate receptors. We are developing a technology for endocytosis of lysosomal enzymes that depends on generic, chemically conjugated reagents. These reagents are aptamers (single-stranded nucleic acid molecules) selected to bind to the extracellular domain of the mouse transferrin receptor. After selection, an RNA aptamer and a DNA aptamer were modified with biotin and linked to dye-labeled streptavidin for detection by confocal microscopy. Aptamer-streptavidin conjugates showed saturable uptake into mouse fibroblasts (Ltk(-) cells), which could be inhibited by an excess of free aptamer but not by tRNA, calf thymus DNA, or transferrin. The RNA aptamer-streptavidin conjugate was mouse-specific, as human cells (293T) did not take it up unless first transfected with the mouse transferrin receptor. Some streptavidin separated from the recycling pathway of transferrin and colocalized with lysosomes. After characterization in the model system, the DNA aptamer was conjugated to a lysosomal enzyme, alpha-l-iduronidase, from which mannose 6-phosphate had been removed. The aptamer had been modified by attachment of terminal glycerol for oxidation by periodate and reaction of the resulting aldehyde with amino groups on the protein. Dephospho-alpha-L-iduronidase-aptamer conjugate was taken up in saturable manner by alpha-L-iduronidase-deficient mouse fibroblasts, with half-maximal uptake estimated as 1.6 nM. Endocytosed enzyme-aptamer conjugate corrected glycosaminoglycan accumulation, indicating that it reached lysosomes and was functional in those organelles. Both uptake and correction were inhibited by unconjugated aptamer, confirming the role of the aptamer in receptor-mediated endocytosis.

Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3.

Human epidermal growth factor receptor-3 (HER3) is a member of the type I receptor tyrosine kinase family. Several members of this family are overexpressed in various carcinomas. Specifically, HER2 is found to be overexpressed in 20-30% of breast cancers. In contrast to epidermal growth factor receptor or HER2, the kinasedeficient HER3 self-associates readily at low nanomolar concentrations and in the absence of its ligands, various isoforms of heregulin (hrg). Binding of hrg disrupts HER3 oligomerization and leads to the formation of signaling-competent heterodimers, preferentially with HER2. Elevated levels of HER3 contribute to increased drug resistance observed in HER2-overexpressing cells. We have used the SELEX (systematic evolution of ligands by exponential enrichment) methodology to select RNA aptamers against the oligomeric state of the extracellular domains of HER3 (HER3ECD, monomeric molecular mass 82,000 Da). One of the aptamers, A30, binds with high affinity to a limited number of binding sites in the oligomeric state of HER3ECD. Binding of A30 and hrg are not competitive. Instead, the disruption of HER3 oligomers by hrg results in an approximately 10-fold increase in total binding sites, but the newly created binding sites are of lower affinity. High-affinity binding of A30 inhibits hrg-dependent tyrosine phosphorylation of HER2 and the hrg-induced growth response of MCF7 cells. As an example of an aptamer against a large macromolecular protein complex, A30 can serve as a tool for the analysis of receptor interactions and may serve as a lead compound for the development of inhibitors against overexpressed receptor tyrosine kinases in carcinomas.

Transcription inhibition using modified pentanucleotides.

Inhibition of gene expression was recently achieved by targeting the transcriptionally competent open complex using relatively short, pentameric modified oligonucleotides at approximately 60 microM. Corroborative affinity cleavage experiments using the copper complex of a phenanthroline conjugate provided the impetus to synthesize additional analogues containing substituents at the 2'-position of uridine in a derivative of 5'-GUGGA (-4 to +1), with the purpose of inhibiting transcription at lower concentrations. Conjugates of 5'-GUGGA modified at the 2'-position of uridine were convergently synthesized using a recently reported method. Seven analogues based upon the 5'-GUGGA scaffold were tested for their ability to inhibit transcription of the lac UV-5 operon. The conjugate containing a tethered pyrene showed 70% inhibition at 20 microM, and modest inhibition at as low as 5 microM. This is a significant improvement over previously tested pentanucleotides and provides direction for the preparation of a next generation of inhibitors.

Site-specific DNA cleavage of synthetic NarL sites by an engineered Escherichia coli NarL protein-1,10-phenanthroline cleaving agent.

The NarL response regulatory protein of Escherichia coli has been engineered by covalent modification with 1,10-phenanthroline (OP) to create a set of site-specific DNA-cleaving agents. This was accomplished by introducing single cysteine amino acid replacements at selected locations within the carboxy-terminal DNA-binding domain in or nearby the helix 8 to helix 9 region of the NarL protein using site-directed mutagenesis. Of 18 modified NarL-OP proteins made, 13 retained the ability to bind DNA as evidenced by gel mobility assays, whereas 10 of the 1,10-phenanthroline-modified proteins also exhibited specific cleavage activity for a synthetic NarL recognition sequence. These DNA-cleaving agents were divided into two groups based on the location of the cleavage sites. The first class set cleaved the DNA nearby the center of a synthetic 7-2-7 sequence composed of two NarL heptamer sites separated by a 2-bp spacer element. The second class cut the DNA at the periphery of the 7-2-7 sequence. The cleavage data are consistent with the ability of two NarL monomers to recognize and bind to the DNA in a head-to-head orientation. A second set of DNA-cleaving agents was constructed using the carboxy-terminal domain of NarL called NarL(C). Similar cleavage patterns were observed whether full-length NarL or NarL(C) was used. The availability of 1,10-phenanthroline-modified NarL and NarL(C) proteins opens up the possibility to explore the position, orientation, and number of NarL recognition sites at E. coli promoters predicted to contain multiple and complex arrangements of NarL-binding sites.