Shuo Ren

A cross-contamination-free SELEX platform for a multi-target selection strategy

Multi-target aptamer selection, known as multiplex systematic evolution of ligands by exponential enrichment (SELEX), is rapidly drawing interest because of its potential to enable high-speed, high-throughput aptamer selection. The parallelization of chemical processes by integrating microfluidic unit operations is a key strategy for developing a multiplex SELEX process. One of the potential problems with on-chip multiplexing chemical processes is cross-contamination. In order to avoid this, we propose a microfluidic network platform that uses pneumatic valves to allow the serial loading and incubation of aptamers with sol-gel entrapped target proteins. After target binding inside the sol-gels, the cross-contamination-free parallel elution of specifically bound aptamers is performed. The platform allows selective binding with five different targets immobilized in sol-gel spots. When eluting bound species, cross-contamination is avoided by sealing the adjacent elution chambers from each other using the pneumatic microvalves. Consequently, we demonstrate specific aptamer binding to the respective protein target and subsequent aptamer elution without any cross-contamination. This proof of concept opens the way to increased automation and microscale parallel processing of the SELEX methodology.

Acousto-microfluidics for screening of ssDNA aptamer

We demonstrate a new screening method for obtaining a prostate-specific antigen (PSA) binding aptamer based on an acoustofluidic separation (acoustophoreis) technique. Since acoustophoresis provides simultaneous washing and separation in a continuous flow mode, we efficiently obtained a PSA binding aptamer that shows high affinity without any additional washing step, which is necessary in other screening methods. In addition, next-generation sequencing (NGS) was applied to accelerate the identification of the screened ssDNA pool, improving the selecting process of the aptamer candidate based on the frequency ranking of the sequences. After the 8th round of the acoustophoretic systematic evolution of ligands by exponential enrichment (SELEX) and following sequence analysis with NGS, 7 PSA binding ssDNA aptamer-candidates were obtained and characterized with surface plasmon resonance (SPR) for affinity and specificity. As a result of the new SELEX method with PSA as the model target protein, the best PSA binding aptamer showed specific binding to PSA with a dissociation constant (Kd) of 0.7 nM.

Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2′F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of 1.3 × 106 CFU/ml. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate (5 × 104 CFU/ml). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of 2 × 104 CFU/mL of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5.

Linkage-specific polyubiquitination regulates many cellular processes. The N-terminal fragment of Rabex-5 (Rabex-5(9-73)) contains tandem ubiquitin binding domains: A20_ZF and MIU. The A20_ZF-MIU of Rabex-5 is known to bind monoubiquitin but molecular details of polyubiquitin binding affinity and linkage selectivity by Rabex-5(9-73) remain elusive. Here we report that Rabex-5(9-73) binds linear, K63- and K48-linked tetraubiquitin (Ub(4)) chains with K(d) of 0.1-1 μM, determined by biolayer interferometry. Mutational analysis of qualitative and quantitative binding data reveals that MIU is more important than A20_ZF in linkage-specific polyubiquitin recognition. MIU prefers binding to linear and K63-linked Ub(4) with sub μM affinities. However, A20_ZF recognizes the three linkage-specific Ub(4) with similar affinities with K(d) of 3-4 μM, unlike ZnF4 of A20. Taken together, our data suggest differential physiological roles of the two ubiquitin binding domains in Rabex-5.

Low efficient generation of ssDNA aptamer using chemical modified spacer primer

Aptamers are oligonucleotides (ssDNA or RNA) with an appropriate size of 100 bps that bind with high affinity and specificity to a wide range of target molecules, including virtually any class of protein, drugs or small organic/inorganic molecules. The in vitro selection process referred to as SELEX provides a powerful tool to identify specific aptamers with high affinity and even discriminate between closely related targets. Aptamers have various applications such as analytical tools, disease diagnosis and prediction, pharmaceutical research, drug development, therapy and even for environmental monitoring. Nowadays, with the development of SELEX methods, generation of aptamer becomes more efficient, less time consuming and even automatically. The whole SELEX process includes binding, separation, and nucleic acid amplification. As amplification of nucleotides is an important process in successive SELEX, we will compare several methods for generation of aptamer in this report.

Synthesis of UV active 2-methylisoborneol for water pollutant detection

Abstract2-Methylisoborneol and geosmin are metabolites of blue-green algae actinomycete bacteria or fungi. They are the main reason for unpleasant smell in public water supplies and certain fish. As the odor threshold is as low as 10 ng/L (ppt), it is necessary to detect them from public drinking water supplies. Since traditional detection technology needs sample extraction or critical condition for assay, they are not able to give specific and fast detection. In this report we synthesized 2-MIB from camphor with chiral structures and attached the UV active group which enables UV detection. Using these modified chemical structures, a biosensor system will be applied and developed for high throughput detection of water pollutants directly from tap water with specificity. Moreover, this novel detection system could be applied to the universal detection of water pollutant with high sensitivity.