14th April 2020

Although the world is focused on the COVID-19 virus, it is the envelope proteins that are helping companies to develop all important tests.

Benefits of Aptamers for Protein Applications

Proteins play significant roles in the physiological and pathological mechanisms of living organisms including human beings. There is an increased demand for convenient methodologies for detecting, isolating and measuring the levels of specific proteins in biological and environmental samples because their detection, identification and quantification can be very complex, expensive and time consuming. Until now, antibodies have been the used for the above applications in most cases; however, they come with limitations such as:

  • Lengthy development process
  • Sensitive to temperature
  • Denaturation over the time
  • Background binding, therefore lack of specificity
  • Batch-to-batch variability

Aptamers, since their introduction in 1990 and undergoing numerous modifications in SELEX technology, have shown to address the above limitations due to their properties such as:

  • Facile and reproducible synthesis in vitro
  • Chemical stability
  • Comparable affinity and selectivity to antibodies.
  • Facilitated conjugation or modification with different functional moieties
  • Easily produced in bulk

In addition, the key advantages of advanced SELEX technology that has aided for successful end applications of protein detection are that it:

  • Does not require a purification process from a crude bio-sample
  • Allows for screening the functional states of target proteins
  • Facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples
  • Enables the efficient screening of aptamers with a low SELEX cycle by subtractive selection or competitive selection

Taking advantage of highly specific aptamers, following researchers have selected aptamers against proteins using advanced SELEX that can be used for various applications such as biomarker identification and protein purification.

Aptamers as biosensors for detecting protein biomarkers in serum

Zou et al., 2018 developed a simple enzyme free and sensitive sensing platform for the detection of a cancer biomarker PDGF-BB in human serum. The strategy was based on the combination of target binding – induced conformational change of aptamers and toehold-mediated strand displacement reaction (TSDR, a powerful method to control DNA hybridizations). The binding of target PDGF-BB with the fluorescent aptamer resulted in conformational switching of the aptamer hairpin probes. This further triggered the TSDR to recycle the target proteins to generate significantly amplified fluorescent signals (Figure 1). The detection limit (dl) was as low as 3.2 pM for the target PDGF-BB.

Similarly, Qin et al., 2019 and Zhang et al., 2020 independently developed ultrasensitive aptamer based label free fluorescent detection of mucin1 (dl 35 fM) and thrombin (dl 3.6 fM) respectively with the capability of discriminating target molecules against other non-specific protein in the serum samples.

Figure 1. A. Schematic principle of the autonomous aptamer machine biosensor for enzyme-free and amplified detection of PDGF-BB. B. Selectivity of the sensing method for the target PDGF-BB against other control proteins. C. Detection of PDGF-BB in real serum samples.

Having the advantage of being enzyme and nanomaterial free for signal amplification, the developed approach holds a great potential for the construction of various aptasensors for detecting various proteins or small molecules at low levels.

Aptamer facilitates protein purification

Aptamers have been used for affinity chromatography with effective results and advantages over most advanced protein separation technologies. Inomata et al., 2017 demonstrated the advantages of using alkaline tolerant RNA aptamer against the Fc region of human IgG for purification of acid sensitive antibodies from the human sera. The IgG aptamer required divalent cations for binding to IgG and bound IgG dissociated easily upon treatment with chelating reagent such as EDTA under neutral conditions. Overall, the advantage of using aptamer columns were:

  • Milder elution conditions – This was advantageous for maintaining active conformations of therapeutic antibodies operated at neutral pH, versus acidic conditions that are prone to form antibody aggregates
  • Usable for all IgG subclasses
  • Stable under caustic conditions usually adopted for column cleaning

Using this approach, Seifert et al., 2019 demonstrated successful purification of plasma proteins such as fibrinogen and immunoglobulin G using aptamer based affinity chromatography (Figure 2).

Figure 2. SDS-PAGE showing purification of human plasma proteins Protein G, Fibrinogen and IgG (down arrows) using aptamer mediated affinity chromatography (Adapted from Perret and Boschetti, 2019).

In addition to the above applications, aptamers have been demonstrated as promising agents in imaging, activation/inhibition of signaling pathways in cells, targeted delivery for diagnostics, and therapeutic applications as molecular targeted drugs.

At Aptamer Group (AG), we have developed our selection process tailored to customer requirements such as introducing specific buffers, matrices, and most importantly counter-selection steps to ensure that the aptamers are specific to the target of interest. Because of such robust process, we have successfully selected aptamers that have shown to discriminate closely related proteins, such as Post Translational Modifications. Moreover, our automated parallel processing platform allows multiple targets and variables thus increasing the throughput.

For more information regarding the applications of aptamers in protein detection and how it can be best utilized in your research, please contact us using the form below.

References:

Inomata, E., Tashiro, E., Miyakawa, S., Nakamura, Y., & Akita, K. (2017). Alkaline-tolerant RNA aptamers useful to purify acid-sensitive antibodies in neutral conditions. Biochimie, 145, 113-124.

Goto S., Tsukakoshi K & Ikebukuro K (2017). Development of aptamers against unpurified proteins. Biotechnology Bioeng. 2017 Dec;114(12):2706-2716. doi: 10.1002/bit.26389

Perret G, Boschetti E (2019). Aptamer-Based Affinity Chromatography for Protein Extraction and Purification. Advances in Biochemical Engineering/biotechnology. DOI: 10.1007/10_2019_106.

Qin Y. et al., 2020. Cascaded multiple recycling amplifications for aptamer-based ultrasensitive fluorescence detection of protein biomarkers. Analyst. 4;144 (22):6635-6640. doi: 10.1039/c9an01674k.

Seifert A (2019) Anti-fibrinogen aptamers and uses thereof WO 2018/007530

Seifert A (2019) Anti-immunoglobulin G aptamers and uses thereof. WO 2018/019538

Zhang T et al., 2019. Polymerization nicking-triggered LAMP cascades enable exceptional signal amplification for aptamer-based label-free detection of trace proteins in human serum. Anal Chim Acta. 2020 Feb 15;1098:164-169. doi: 10.1016/j.aca.2019.11.044.

Zou M, Li D, Yuan R and Xiang Y (2018). A target-responsive autonomous aptamer machine biosensor for enzyme-free and sensitive detection of protein biomarkers Mater. Chem. B, 6, 4146–4150.

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