Aptamer-based Biosensor screens Air Force personnel and equipment

http://www.bio-medicine.org/biology-news/Aptamer-based-Biosensor-screens-Air-Force-personnel-and-equipment-1089-1/

Air Force personnel will soon know within minutes if they or their equipment are contaminated with a biological agent, thanks to a new technology developed by the Air Force and a national laboratory.

 

Personnel will use the biosensor system to collect and isolate samples, detect and identify agents, and assess the seriousness of the threat.

“The system will provide an increased capability for Air Force Special Operations personnel to rapidly determine the presence of biological warfare agents in a combat environment,” said Dr. Richard Stotts, counterproliferation branch chief within the Air Force research Laboratory’s Human Effectiveness Directorate. “The device is compact, quickly identifies agents, can be used repeatedly and requires very little maintenance to keep it running in the field.”

The system consists of a spray, developed at the directorate’s Brooks City-Base, Texas, facilities, and a hand-held “green box,” which determines if agents are present. The green box, or DNA Capture Element instrument, was developed by researchers at the Department of Energy’s Pacific Northwest National Laboratory. The box uses an Air Force-developed biochemical assay based on aptamers, or single chain DNA fragments.

“We’ve used our lab’s expertise to develop an instrument that’s complementary to the Air Force’s technology and that simultaneously satis? es the speed, specificity, sensitivity, portability, durability, health and safety needs,” said Mike Lind, a senior advisor at PNNL. “The rapid detection capability of this instrument will be useful in a variety of applications, even outside of the armed forces.”

With the prototype system, the user sprays the suspected contaminated area, creating a sample that can be picked up by a swab. The sample material on the swab is suspended in liquid by rinsing it in a container. Once in a liquid form, the sample is injected into a special flow cell, the place where the assay occurs.

 

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Selection of aptamers against live bacterial cells.

 Anal Chem. 2008 Oct 15;80(20):7812-9. Epub 2008 Sep 20

Single-stranded DNA or RNA aptamer molecules have usually been selected against purified target molecules. To eliminate the need of purifying target molecules on the cell surface, we have developed a selection technique using live bacterial cells in suspension as targets, to select for ssDNA aptamers specific to cell surface molecules. Lactobacillus acidophilus cells were chosen to demonstrate proof of principle based on their high abundance of surface molecules (potential targets). Aptamer pools obtained after 6-8 rounds of selection demonstrated high affinity for and selective binding with L. acidophilus cells when tested via flow cytometry, microscopy, and fluorescence measurements. Out of 27 aptamers that were cloned and sequenced, one sequence, hemag1P, was found to bind to L. acidophilus much more strongly and specifically than other cells tested. This aptamer was predicted to have a tight hairpin secondary structure. On average, an estimated 164 +/- 47 aptamer molecules were bound to a target cell with an apparent K d of 13 +/- 3 nM. A likely putative molecular target of hemag1P is the S-layer protein on the cell surface.

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Aptamer-based endocytosis of a lysosomal enzyme

PNAS October 14, 2008 vol. 105no. 41 15908-15913

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, α-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-α-l-iduronidase-aptamer conjugate was taken up in saturable manner by α-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.

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Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins

Journal of Virological Methods Volume 151, Issue 1, July 2008, Pages 107-115 Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrPC, and the pathological isoform, PrPSc. In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were ‘orphan’ clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (Kd) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrPC. Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrPSc following guanidinium denaturation.   D

Single microbead SELEX for efficient ssDNA aptamer generation against botulinum neurotoxin

Chem. Commun., 2008, 1883 – 1885, DOI: 10.1039/b717936g

 

We are interested in the generation of ssDNA aptamers

toward the fabrication of aptasensors for biosecurity applications.

6,7 Herein we describe a novel and efficient approach to

generate ssDNA aptamers through the use of only a single

target-conjugated microbead (Fig. 1a). As opposed to traditional

means exposing the ssDNA library to a column packed

with thousands of target-derivatized microbeads, we exposed

the library to only a single microbead. We hypothesized that

by drastically reducing the amount of target molecules, only

the tightest ssDNA binders are expected to remain bound to

the exposed target after the incubation period (the weak

binders will be displaced by the higher affinity binders). For

this study, we have chosen two Clostridium botulinum neurotoxin

(BoNT)-related targets, namely the BoNT-toxoid (aldehyde-

inactivated toxin) and a short 19 amino acid (aa

1177–1195 of the heavy chain (Hc), Fig. 1b) BoNT Hc-peptide

domain, as targets for ssDNA aptamer generation.

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In vivo efficacy of a phosphodiester TLR-9 aptamer and its beneficial effect in a pulmonary anthrax infection model

Cellular Immunology Volume 251, Issue 2, 2008, Pages 78-85 Immunostimulatory oligonucleotide (ISS-ODN) used as adjuvants are commonly modified with phosphorothioate (PS). The PS backbone prevents nuclease degradation, but confers undesired side effects, including systemic cytokine release. Previously, R10–60, a phosphodiester (PO) ISS-ODN, was structurally optimized as an intracellular Toll-like receptor-9 agonist. Here intravenous, intradermal and intranasal administration of PO R10–60 elicit local or adaptive immune responses with minimal systemic effects compared to a prototypic PS ISS-ODN in mice. Furthermore, prophylactic intranasal administration of PO R10–60 significantly delayed death in mice exposed to respiratory anthrax comparable to the PS ISS-ODN. The pattern of cytokine release suggested that early IL-1β production might contribute to this protective effect, which was replicated with recombinant IL-1β injections during infection. Hence, the transient effects from a PO TLR-9 agonist may be beneficial for protection in a bacterial bioterrorism attack, by delaying the onset of systemic infection without the induction of a cytokine syndrome.   D

Systematic Investigation of Optimal Aptamer Immobilization for Protein−Microarray Applications

Anal. Chem., 80 (19), 7372–7378, 2008. 10.1021/ac801081v 

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to a wide range of target molecules with high affinity and specificity. As nucleic acids, aptamers can undergo denaturation, but the process is reversible. As a result of this stability and the possibility of automated selection of aptamers, these oligonucleotides are highly promising capture molecules in microarray formats. In this study, his-tagged proteins and an aptamer directed against the his-tag were chosen as a model system. Different factors affect the activity of aptamers immobilized on a solid support like a microarray surface. The orientation of the immobilized aptamer plays an important role in correct aptamer folding and, thus, in effective binding of the corresponding target. Other important parameters identified in this work are the microarrays’ surface charge as well as the length of the spacer between aptamer and solid support. These parameters were investigated systematically, resulting in the development of an aptamer-based microarray for detection of his-tagged proteins. The general applicability of the developed immobilization strategy was demonstrated by utilization of three different aptamers.

 

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Aptamers in Immunotherapy Review

HUMAN GENE THERAPY 19:443–450 (May 2008)

Review summarizing aptamers that have been selected for membrane receptors on the immune cells, as well as cytokines and chemokines. 

 

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Novel Dual Inhibitory Function Aptamer–siRNA Delivery System for HIV-1 Therapy

Molecular Therapy (2008) 16 8, 1481–1489 doi:10.1038/mt.2008.92

 

The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. In this study, we demonstrate cell type–specific delivery of anti-human immunodeficiency virus (anti-HIV) siRNAs through fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1-infected cells, allowing binding and internalization of the aptamer–siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer–siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and that the appended siRNA is processed by Dicer; this releases an anti-tat/revsiRNA which, in turn, inhibits HIV replication. We show for the first time a dual functioning aptamer–siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also show that gp120 expressed on the surface of HIV-infected cells can be used for aptamer-mediated delivery of anti-HIV siRNAs.

 

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Preliminary development of DNA aptamer-Fc conjugate opsonins

J Biomed Mater Res A. 2008 Jul 31

 

 

Encapsulated bacteria such as virulent strains of Bacillus anthracis impair phagocytosis with their capsules unless opsonized by antibodies. Poly-gamma-D-glutamic acid (gamma-PDGA) is the major component of the B. anthracis capsule. In this work, poly-alpha-D-glutamic acid (alpha-PDGA)-coated magnetic beads (MBs) were used as surrogates to simulate vegetative B. anthracis cells and avoid the hazards of working with virulent bacteria. DNA aptamers were developed against the alpha-linked PDGA-MBs and sequenced. Four of the most frequent candidate aptamer sequences in the pool were coupled at their 5′ ends to Fc fragments of murine IgG to act as artificial antibodies. The effects of candidate aptamer-Fc conjugate addition on macrophage attachment and internalization of alpha-PDGA-MBs were tested on P388D1 and RAW 264.7 murine macrophage lines by spectrofluorometric and image analysis techniques. P388D1 cells were not able to internalize the alpha-PDGA-MBs, but attachment to alpha-PDGA-MBs was enhanced by the conjugates to varying degrees. Ingestion of alpha-PDGA-MBs by RAW 264.7 cells in the presence of several different candidate aptamer-Fc conjugates demonstrated a statistically significant (p < 0.01) increase in phagocytic index (P.I.) up to threefold in the first 30 min of exposure to alpha-PDGA-MBs. This preliminary study using alpha-linked instead of gamma-linked PDGA provides proof-of-concept for future work in the new area of hybrid DNA aptamer-protein constructs as potential opsonins. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

 

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