Aptamers and nanoparticles
Larry H. Bernstein, MD, FCAP, Curator
LPBI
What’s new at Base Pair?
Proprietary, new software algorithms power our ability to identify novel aptamer sequences and structural motifs.New methods developed and optimized over the past year in our laboratory allow us to not only develop aptamers directed against as many as thirty different targets at the same time, but also to design screens that identify aptamers recognizing overlapping sets of targets or that selectively recognize a single target while ignoring others. Finally, our new methods allow us to select for aptamers directed against your target, in the very same type of sample that you would like to use for your eventual assay, avoiding matrix background and non-specific binding problems.
Sensitive detection of MRSA
Early detection, enabling early intervention, is a critical goal for infectious disease agents and sensitive assays are crucial for early detection. One of Base Pair’s aptamers (catalog number ATW0032) targeted against penicillin binding protein 2a ,the protein responsible for the antibiotic resistance of methicillin resistant Staphyloccus aureus (MRSA), , is featured in a recently accepted publication by Adhikari et al. [1] in this month’s issue of Analytical Chemistry. In the paper entitled, “Aptamer-phage reporters for ultrasensitive lateral flow assays” the authors utilize an innovative combination of extensively labeled phage particle reporters and aptamers for molecular recognition and improve the sensitivity of 2 lateral flow assays for MRSA by ~100-fold.
We introduce the modification of bacteriophage particles with aptamers for the use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ~100 times lower than those of previously reported IgE assays.
Multivalent aptamers
While it is generally thought that because of their small size, DNA aptamers are univalent and thus have lower affinities and faster off rates than antibodies, this may not be the case. In an article published in RSC Advances, Wang et al. [2] utilized molecular modeling to understand observed experimental performance of a Base Pair aptamer directed against tetracycline, as well as a previously published aptamer directed against the same target. Interestingly, through modeling the authors predict that both aptamers are multivalent for tetracycline, and that each likely have 3 binding sites for the small molecule. This may be a generalized phenomenon for tight binders and Base Pair would be interested in hearing from researchers who have observed similar results.
Conformational structure-dependent molecular recognition of two aptamers for tetracycline
Different aptamers towards one target molecule can be selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), however, not all aptamers have real world practicability. In this study, conformational structure-dependent molecular recognition of two aptamers towards tetracycline (TC), 76 mer and 40 mer, was studied both quantitatively and computationally. Two formats of competitive enzyme-linked aptamer assay (ELAA), a molecular docking module and Isothermal Titration Calorimeter (ITC) analysis were used to further investigate the two selected aptamers. With longer strand length, more G, C bases, and more recognition sites for TC, the 76 mer aptamer showed better performance than the 40 mer aptamer. Deciphering the relevance of aptamers with different molecular characteristics towards one target molecule can furnish as a referral guidance for aptamer selection and further practical application.
In June of 2015 Somin Lee et al. of Lawrence Berkeley National Laboratory published a paper in ACS Nano Letters entitled, “Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules” [3]. Plasmon rulers are pairs of gold nanoparticles that are coupled via a linker. They exhibit a shorter wavelength light scattering peak when further apart and longer wavelength light scattering peak when closer together. The distance between the particles can be measured as a function of scattering wavelength, thus creating a ruler. By attaching two particles together through a DNA aptamer, Lee and colleagues were able to create a plasmon resonance based sensor for aptamer binding. The Base Pair aptamer ATW0062, which recognizes matrix metalloproteinase-3 (MMP-3) was used to link two gold nanoparticles together. In order to facilitate synthesis, Base Pair provided the aptamer with a thiol on one end and a biotin on the other end. Using the resulting nanoparticle-aptamer-nanoparticle constructs, the authors demonstrated detection of MMP-3 in cell culture with single molecule sensitivity and full reversibility of the sensor. Unlike fluorescence-resonance energy transfer (FRET), such a sensor can be used to continuously monitor dynamic changes in biological systems without concern for photobleaching.
Anal Chem. 2015 Oct 12. [Epub ahead of print]
References:
2. Wang S, Liu J, Dong Y, Su H, Tan T: Conformational structure-dependent molecular recognition of two aptamers for tetracycline. RSC Adv 2015, 5:53796–53801.
3. Lee SE, Chen Q, Bhat R, Petkiewicz S, Smith JM, Ferry VE, Correia AL, Alivisatos AP, Bissell MJ: Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules. Nano Lett 2015, 15:4564–4570.
Pairs of affinity reagents (typically antibodies) are frequently used in a variety of research-use-only and clinical diagnostic assays. These are commonly known as “sandwich pairs” – i.e. they bind non-overlapping epitopes on their target protein. Unfortunately, screening for complementary pairs of sandwich antibodies is tedious and resource intensive, requiring expression of multiple monoclonal antibodies and empirical testing of pairwise compatibility.
In contrast, Base Pair has developed a proprietary workflow leveraging high-content arrays to rapidly discover complementary sandwich pairs of DNA aptamers. These pairs are then validated for their binding in a quantitative manner by Microscale Thermophoresis (MST) or backscattering interferometry(BSI). As an example, ATW0077 and ATW0083 is a pair of aptamers demonstrated to bind to both human and murine interleukin-6 (IL6). IL6 is an important cytokine involved in many phase of inflammation and immune response. Therefore, these aptamers represent a useful reagent pair for any number of prototype assay or biosensor development projects. We will gladly synthesize these aptamers or others with immobilization and detection modifications to meet your specific requirements. More information can be found in our Aptamers that Work ™ online catalog, or contact us for custom sandwich pair requirements.
We can tailor solutions for your unique platform or for use with standard instrumentation. Our custom aptamer development services can also be deployed to design and develop new affinity agent pairs to address your objectives.
Mass spectroscopy is one of the most important available tools for studying and quantifying post-translational modifications. Quantitative workflows such as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies), or “multi-reaction monitoring” (MRM-MS) have become the workhorse of advanced proteomic studies. Unfortunately, any affinity-based mass spectroscopy approach is limited by the quality of the affinity reagent itself. In many cases, the availability of an adequately performing antibody can be a limiting factor.
As an example, development of antibodies specific to modified peptides (a frequently desired analyte) can be challenging by conventional immunization. Under funding from the National Cancer Institute (NIH, U.S.) Base Pair has recently demonstrated its patented multiplex approach to confer aptamer specificity to phosphorylated peptides over their non-phosphorylated counterparts. The Aptamers that Work ™ online catalog has multiple peptide-specific aptamers to ERK1 and ERK2, which are extracellular-signal-regulated kinases that play important roles in regulating meiosis, mitosis and postmiotic functions in differentiated cells and which are part of one of the most important known signal transduction cascades. These aptamers were selected to recognize both phosphorylated and non-phosphorylated peptides with validation data to both for added confidence. More information on our modification specific aptamers can be found in our Technical Note Phosphorylation Site Specific Aptamers for Cancer Biomarker Peptide Enrichment and Detection in Mass Spectrometry Based Proteomics.
The synthesis and silane functionalization of magnetic iron nanoparticles is greatly facilitated by the use of the automated Globe system, as it offers improved sensitivity and simple operation.
The magnetic particle-linked antibody for immunoassay of progesterone has great potential to supersede the traditional enzyme-linked immunosorbent assay for progesterone determination.
Chemisens Calorimeter Helps Max Planck Researchers
Researchers at Germany’s Max Planck Institute of Colloids and Interfaces have provided a testimonial about the Chemisens Calorimeter, praising it for generating reliable, reproducible data and for being really easy to use.
They use the calorimeter to quantitatively evaluate polymerization reaction profiles and accurately determine end points. Klaus Tauer, Leader of the Department of Colloid Chemistry’s Heterophase Polymerization – Polymer Dispersions Group, explains: “We rely on a Chemisens Calorimeter for this work, using it almost daily for three main purposes; heterophase polymerizations, to follow reaction kinetics, and as a tool for polymer synthesis.”
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