Aptamer-based Cell Recognition and Detection

: Cells, regarded as the structural and functional units of organisms, have become one of the most important objects in many research areas. Specific recognition and detection of malignant cells are critical for disease diagnosis, therapy and prognosis. Aptamers are short; single-stranded oligonucleotides screened from a random library by an in vitro technology termed “Systematic Evolution of Ligands by Exponential Enrichment” (SELEX) on the basis of their specific binding to target cargos. With the advantages of small size, easy synthesis, convenient modification, high chemical stability and low immunogenicity, aptamers have attracted broad attention in bioanalysis. Using intact living cells as the selection target, the cell-SELEX technology enables the generation of many aptamers that can specifically recognize molecular signatures of target cells. These aptamers have been extensively utilized in various cell-based research. In this mini-review, we focus on recent advances in aptamer-based recognition and detection of cells, particularly circulating tumor cells (CTCs).

Chaperones drive in vitro evolution of uracil glycosylase towards misfolded states

Evolution is driven by random mutations, whose fitness outcome is tested over time. In vitro evolution of a library of a randomly mutated protein mimics this process, however, on a short time scale, driven by a specific outcome (such as binding to a bait). Here, we used directed in vitro evolution to investigate the role of molecular chaperones in curbing promiscuity in favor of specificity of protein-protein interactions. Using yeast surface display, we generated a random library of the E. coli protein Uracil glycosylase (UNG), and selected it against various baits. Those included the purified chaperones GroEL, DnaK+DnaJ+ATP, or total protein extracts from WT or delta DnaK+DnaJ cells. We show that in-vitro evolution differs from natural evolution in cells, both physically and thermodynamically. We found that chaperones, whether purified or as part of the protein extract, select for and thus enrich uracil glycosylase (UNG) misfolded species during this in vitro evolution process. In a more general context, our results show that chaperones purge promiscuous misfolded clones from the system, and thereby avoiding their detrimental effects, such as forming wrong interactions with other macromolecules, including proteins, which can harm proteostasis.

A Phage Display-Identified Short Peptide Capable of Hydrolyzing Calcium Pyrophosphate Crystals—The Etiological Factor of Chondrocalcinosis

Chondrocalcinosis is a metabolic disease caused by the presence of calcium pyrophosphate dihydrate crystals in the synovial fluid. The goal of our endeavor was to find out whether short peptides could be used as a dissolving factor for such crystals. In order to identify peptides able to dissolve crystals of calcium pyrophosphate, we screened through a random library of peptides using a phage display. The first screening was designed to select phages able to bind the acidic part of alendronic acid (pyrophosphate analog). The second was a catalytic assay in the presence of crystals. The best-performing peptides were subsequently chemically synthesized and rechecked for catalytic properties. One peptide, named R25, turned out to possess some hydrolytic activity toward crystals. Its catalysis is Mg2+-dependent and also works against soluble species of pyrophosphate.

The role of libraries in distance learning during COVID-19

As a form of education, distance learning currently shows great promise and the role of online libraries in delivering off-campus information services increases. This paper presents a comparative analysis of measures undertaken by traditional academic libraries in China and Italy in response to pandemic restrictions on educational operations during the lockdown period. The study utilized a specially developed questionnaire, carried out through an online survey, as a means of collecting data from library users (students and teachers). The study population includes 102 random library patrons, including 64 students and 38 teachers who were using services of academic libraries in China at the time. Through comparison with other studies, the quarantine responses of Chinese libraries were identified. The results of the study show that COVID-19 responses of libraries in China went in a direction similar to those in Italy, but the final decisions varied because of different levels of technological development. This knowledge may be used by libraries to identify areas for improvement. The results show that Chinese libraries were more effective in overcoming quarantine restrictions than those in Italy. The overwhelming majority of respondents reported that they had positive experience learning remotely and would not mind if distance learning programs continue to function after the pandemic is over.

Aptamer-targeting of Aleutian mink disease virus (AMDV) can be an effective strategy to inhibit virus replication

Aleutian mink disease (AMD), which is caused by Aleutian mink disease virus (AMDV), is an important contagious disease for which no effective vaccine is yet available. AMD causes major economic losses for mink farmers globally and threatens some carnivores such as skunks, genets, foxes and raccoons. Aptamers have exciting potential for the diagnosis and/or treatment of infectious viral diseases, including AMD. Using a magnetic beads-based systemic evolution of ligands by exponential enrichment (SELEX) approach, we have developed aptamers with activity against AMDV after 10 rounds of selection. After incubation with the ADVa012 aptamer (4 μM) for 48 h, the concentration of AMDV in the supernatant of infected cells was 47% lower than in the supernatant of untreated cells, whereas a random library of aptamers has no effect. The half-life of ADVa012 was ~ 32 h, which is significantly longer than that of other aptamers. Sequences and three dimensions structural modeling of selected aptamers indicated that they fold into similar stem-loop structures, which may be a preferred structure for binding to the target protein. The ADVa012 aptamer was shown to have an effective and long-lasting inhibitory effect on viral production in vitro.

Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of NaV1.7 Inhibitors

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.

Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

<div> <div> <div> <p>Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains a sluggish process, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib (funclib-weizmann.ac.il) is a novel automated computational procedure which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of a stability metric. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this a particularly challenging system to optimize. Yet, experimental screening of a very small number of active-site, multi-point variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (~2·104 M-1 s-1 and ~102 s-1) that compare well with modern natural enzymes, and that approach the catalysis levels for the best Kemp eliminases derived from extensive screening. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ~2 kcal·mol-1 and indicate that the improvements in activity are linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by EVB-based computational predictions of catalytic activity, as a generalized approach for computational enzyme design. </p> </div> </div> </div>

Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

<div> <div> <div> <p>Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains a sluggish process, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib (funclib-weizmann.ac.il) is a novel automated computational procedure which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of a stability metric. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this a particularly challenging system to optimize. Yet, experimental screening of a very small number of active-site, multi-point variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (~2·104 M-1 s-1 and ~102 s-1) that compare well with modern natural enzymes, and that approach the catalysis levels for the best Kemp eliminases derived from extensive screening. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ~2 kcal·mol-1 and indicate that the improvements in activity are linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by EVB-based computational predictions of catalytic activity, as a generalized approach for computational enzyme design. </p> </div> </div> </div>