Enzyme screening with synthetic multifunctional pores: Focus on biopolymers

Abstract

This report demonstrates that a single set of identical synthetic multifunctional pores can detect the activity of many different enzymes. Enzymes catalyzing either synthesis or degradation of DNA (exonuclease III or polymerase I), RNA (RNase A), polysaccharides (heparinase I, hyaluronidase, and galactosyltransferase), and proteins (papain, ficin, elastase, subtilisin, and pronase) are selected to exemplify this key characteristic of synthetic multifunctional pore sensors. Because anionic, cationic, and neutral substrates can gain access to the interior of complementarily functionalized pores, such pores can be the basis for very userfriendly screening of a broad range of enzymes. There are compelling reasons to believe that the ''universal enzyme sensor,'' a user-friendly, noninvasive device that can detect all existing enzyme activities, belongs to the world of fiction and, despite functional proteomics, will never become reality (1, 2, ‡). However, it would be erroneous to conclude that efforts to maximize adaptability of noninvasive enzyme sensors to as many enzymes as possible are a simple waste of time. In this study, selected examples from biopolymer enzymology are used to demonstrate that a set of identical synthetic multifunctional pores (SMPs) can be used for noninvasive detection of the activity of many different enzymes (Fig. 1).In brief, if substrates bind and block the pore better than products, enzyme activity gradually removes the blocking agent and the pore can open. On the other hand, if products bind and block the pores better than the substrates, enzyme activity gradually produces blocking agents that can block the pores (Fig. 2). Thus, if there is substantial molecular recognition of either substrate or product by the same pore, the only prerequisite for detecting enzyme activities with SMP sensors is a simple method to distinguish between blocked and unblocked pores.As SMPs we introduced rigid-rod ␤-barrels 1 and 2 ( Fig. 2C) (2). Whereas various other rigid-rod ␤-barrel SMPs are available to cope with particular sensing requirements, SMPs other than rigid-rod ␤-barrels remain underexplored (3-9), particularly when compared with the remarkable progress made with bioengineered multifunctional pores as stochastic sensors of single analytes (10-13).The syntheses of barrel-stave supramolecules 1 (14) and 2 (15) from commercial biphenyl and amino acid derivatives in 19 steps overall each have been described. The characteristics of pores formed by rigid-rod ␤-barrels 1 and 2 in spherical and planar lipid bilayer membranes have also been reported (2, 14-16). Even without optimization, the extraordinary permeabilizing activity of these p-octiphenyl ␤-barrels makes it possible to perform Ͼ300,000 enzyme assays per mg of polypeptide (1).L-histidine (H) and L-arginine (R) residues at the inner barrel surface account for the multifunctionality of pore 1. Lining the ion-conducting pathway of SMP 1, these cationic residues recognize anionic substrates and products Ͼ10,000 times better than biological po...

Nanocellulose-Based Biosensors: Design, Preparation, and Activity of Peptide-Linked Cotton Cellulose Nanocrystals Having Fluorimetric and Colorimetric Elastase Detection Sensitivity

“…The molecular basis of some of these sensor motifs include peptide cross-linked dextran hydrogels with a tunable quartz crystal microbalance detection [51], chromogenic peptide substrates tethered to collagen, polyamide polyesters, silica gel [52], ethoxyacrylate resin [53], and cellulose [54], and DNA-based aptasensors [55]. A variety of sensor design motifs have also been employed, and these include: 1) a microchip integrated with reagent-release capillaries is reported as a "drop-and-sip" technique, which utilizes a single microliter droplet of HNE-containing solution with fluorescence image analysis of the hydrolyzed substrate product [56]; 2) fluorometric detection of HNE activity with synthetic supramolecular pore sensors [57,58] the covalent immobilization of HNE on biosensor chips having surface plasma resonance capability has been employed for analysis of HNE inhibitors [59]; 3) a microdialysis sampling assay of HNE activity where the substrate is delivered through the microdialysis probe to external solutions containing HNE, and the product, pNA, is recovered back into the probe [60].…”

On selectivity and sensitivity of synthetic multifunctional pores as enzyme sensors: Discrimination between ATP and ADP and comparison with biological pores

“…Internal crowding at the p-octiphenyl "turns" guides the terminal amino acid residues to the outer barrel surface ( Figure 1, B for ␤ 1 -barrel 1, 7 B and D for ␤ 3 -barrels 2 8 -12 19,12,[22][23][24][25] ). The ␤-sheet conformation positions the adjacent amino acid residues at the inner barrel surface (C for 2-3, C and E for 4-7), and if present, the following ones again at the outer barrel surface (D with 4-7).…”

“…29 The possibility of internal pore design beyond the structural constraints of bioengineering is attractive because it provides unprecedented access to adaptable synthetic multifunctional pores (SMPs). With the objective to couple molecular translocation across with molecular recognition and transformation within synthetic multifunctional pores, rigid-rod ␤-barrels 2-7 with internal L-lysine (K, Lys), 8 -12 L-histidine (H, His), [12][13][14][15][16][17][18][19][20][21][22][23] L-arginine (R, Arg), 18 -23 and L-aspartate (D, Asp) 12,19,22-25 residues were prepared after an initial proof of principle with "minibarrel" 1 ( Figure 1). 7 Molecular recognition of guests by SMP hosts has been studied extensively using guests reaching from inorganic cations to polymers like DNA, RNA, polysaccharides, polypetides, polyacetylenes, and polarized lipid bilayer membranes.…”

Artificial tongues and leaves

“…For this purpose, the applicability of synthetic multifunctional pores as optical transducers of reactions is ideal [26]. With sensitivity toward changes in charge or bulk, optical transduction with synthetic pores is compatible with a broad range of reac-tions [27]. In practice, their general use is similar to chromatographic methods such as high-performance liquid chromatography (HPLC) or thin layer chromatography (TLC) with regard to both scope and simplicity.…”

“…3 and 4) [26]. The broad compatibility of synthetic pores with many reactions assures detectability of the activity of many different enzymes [27]. This adaptability assures applicability of pore transducers in various fields, including drug discovery (enzyme inhibitor screening) and diagnostics.…”