Towards a competitive, aptamer-mediated DNA amplification assay for ultrasensitive protein quantitation. Ilavarasi Gandhi, Mithil Soni, Christopher Quan, and ...
Towards a competitive, aptamer-mediated DNA amplification assay for ultrasensitive protein quantitation Ilavarasi Gandhi, Mithil Soni, Christopher Quan, and George W. Jackson BioTex, Inc. (and subsidiary, Ice Nine Biotechnologies), Houston, TX, USA www.iceninebio.com
Abstract Herein a novel protein detection method based on aptamer-mediated, competitive amplification of a synthetic DNA template is demonstrated. A nonextendable aptamer which we have termed a 'NExA' blocks amplification of a DNA template in the absence of the protein analyte. When analyte is present, the NExA binds the protein of interest releasing the template for amplification. Using glycated bovine serum albumin as a model analyte in conjunction with a glyBSA-specific aptamer, both rolling circle (RCA) and conventional DNA amplification were investigated. The sensitivity of the assay was studied by gel electrophoresis to separate products and reactants, and the expected product yield was quantified by NIH-IMAGEJ software. While not yet fully optimized, the assay has the potential to rival so-called “immuno-PCR” in terms of sensitivity, and should be suitable to any of the large number of analytes to which aptamers can be evolved. Figure 3 : 1.5 % Agarose Gel [A] and Corresponding IMAGEJ Plot [B] of Amplification Product under Decreasing Concentrations of NExA (Blocker) (No Involvement of Proteins). As the blocker concentration decreases, the amount of amplification product increases.
Introduction There is a continued need for clinical diagnostics having greater sensitivity, specificity, and cost-effective reagents. In general, antibody-based methods such the enzyme linked immuno-sorbent assay (ELISA) are sensitive, however antibodies may be difficult to produce and are heterogeneously expressed often resulting in a “batch-to-batch” variance in performance. Gene expression levels as measured by real-time/quantitative polymerase chain reaction (qPCR) can not measure post-translational modifications to proteins, and protein amounts may not always correlate well with mRNA levels. An assay leveraging the ready amplification of signal afforded by DNA amplification combined with antibody-like molecular recognition of proteins would be of great utility. The dual nature of aptamers as DNA and protein-binding reagents can address these challenges. Aptamers are single stranded DNA or RNA oligonucleotides selected to have unique three dimensional folding structure for binding to a variety of targets such as proteins, peptides, and even small molecules with affinity and specificity rivaling that of antibodies. They are generated by a method called Systematic Evolution of Ligands by EXponential enrichment or „SELEX‟ [1,2] (Figure 1). They have been used as molecular probes [3], and in a variety of biosensors [4] as well as therapeutics [5].
Figure 2: Novel Assay Principle Leveraging the Dual Nature of an Aptamer as Protein Recognition Element and Base-pairing Agent. [Panel A] A non extendible DNA aptamer (NExA- blue with red X) is hybridized to its DNA complement. The Stoffel fragment of Taq DNA polymerase (Hatched Pie Chart) cannot remove the aptamer by normal exonuclease activity. [Panel B] Upon exposure to the aptamer’s protein target, the template is free for amplification. Not all PCR components are shown.
Results Potential Advantages: • “All DNA assay for proteins” – more stable, deployable reagents • Once optimized, may rival the sensitivity of so-called „immuno-PCR‟ [6]. • No complex bioconjugations • Thermal cycling or isothermal implementations feasible In order to test the feasibility of this approach, we first demonstrated the dependence of final product on NExA blocker concentration (Figure 3) as a surrogate for varied protein concentration. We then tested the full assay cocktail with a variety of protein:blockingaptamer ratios (Figure 4). In addition to the data presented here, we have tested the general reaction scheme above under a variety of specific implementations including:
Figure 1: Overview of SELEX for Production of Aptamers: Initially, a library containing sequences which have a randomized middle region (typically 30-40 bases) flanked by two constant regions for PCR priming. The library is allowed to bind with the target. Following binding, bound and unbound populations are partitioned, and bound nucleic acids are amplified for the next round. Following repeated (typically 6-12) rounds of selection and enrichment, high affinity ligands emerge which are cloned and sequenced.
Linear PCR with both dsDNA template and ssDNA template: We obtained DNA amplification using both single-stranded (ssDNA) and double-stranded (dsDNA) templates with product inversely proportional to blocking “NExA”. Rolling Circle Amplification (RCA): In this DNA amplification format, complementary ssDNA is first circularized and this circular ssDNA is used for isothermal amplification. RCA cascade: In this augmented version of RCA, additional primers complementary to the initial amplified product are included, so that a cascade of amplification takes place. Figure 3 was obtained using an RCA cascade reaction. RCA cascade with T4 ligase activity: RCA is extremely sensitive to an initial priming event. To prevent “leaky” amplification, we first ligate the initiating primer and NExA using T4 ligase. Figure 4 was obtained using this approach.
Figure 4 : 1.5 % Agarose Gel [A] and Corresponding IMAGEJ Plot [B] Involving Protein and NExA. Constant blocker concentration (denoted as ‘B’) was employed in all reactions. With increasing protein, ‘P’, the amplification product also increased, demonstrating that the NExA has shifted from binding complementary nucleic acid to the protein analyte.
Discussion • While the assay is far from optimized, these results demonstrate the feasibility of a completely new approach to protein detection utilizing inexpensive reagents and allowing for large amounts of signal amplification. • Given the wide range of arbitrary targets that aptamers may be „evolved‟ against, we have developed a platform detection scheme for virtually any analyte. • We are presently further optimizing the reaction and characterizing aptamer binding (equilibrium dissociation constant, Kd) to better match that with the DNA templates (which can be designed with mismatches) employed in the reactions.
References 1. Ellington, A.D. & Szostak, J.W. (1990). Nature, 346 (6287), 818-22. 2. Tuerk, C. & Gold, L. (1990). Science, 249 (4968), 505-10. 3. Shangguan, D., et al. (2006). Proc Natl Acad Sci U S A, 103 (32), 11838-43. 4. Strehlitz , B., et al. (2008). Sensors, 8, 4296-4307. 5. Ng, E.W.M., & Adamis, A. P. (2006). Nature Reviews Drug Discovery, 5, 123-132. 6. Sano, T., et al. (1992). Science , 258 (5079),120-122.
Acknowledgements Special thanks to Drs. Katerina Kourentzi and Ulrich Strych of the University of Houston for helpful discussions.
