Selection of aptamers using ß-1,3-glucan recognition protein-tagged proteins and curdlan beads.

RNA aptamersare nucleic acids that are obtained using the systematic evolution of ligands by exponential enrichment (SELEX) method. When using conventional selection methods to immobilize target proteins on matrix beads using protein tags, sequences are obtained that bind not only to the target proteins but also to the protein tags and matrix beads. In this study, we performed SELEX using ß-1,3-glucan recognition protein (GRP)-tags and curdlan beads to immobilize the acute myeloid leukaemia 1 (AML1) Runt domain (RD) and analysed the enrichment of aptamers using high-throughput sequencing. Comparison of aptamer enrichment using the GRP-tag and His-tag suggested that aptamers were enriched using the GRP-tag as well as using the His-tag. Furthermore, surface plasmon resonance analysis revealed that the aptamer did not bind to the GRP-tag and that the conjugation of the GRP-tag to RD weakened the interaction between the aptamer and RD. The GRP-tag could have acted as a competitor to reduce weakly bound RNAs. Therefore, the affinity system of the GRP-tagged proteins and curdlan beads is suitable for obtaining specific aptamers using SELEX.

Composition of Minerals and Volatile Organic Components of Non-Centrifugal Cane Sugars from Japan and ASEAN Countries.

Non-centrifugal cane sugar (NCS) is an unrefined dehydrated form of sugar syrup produced worldwide. To date, there is a lack of differentiation in the key nutrients and flavor qualities of NCS products among countries, which makes it difficult for interested parties to select NCSs suitable for their needs. This study aimed to evaluate the minerals and volatile organic components (VOCs) in NCS products from Japan and ASEAN countries. Mineral components were determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES). VOCs and their aroma profiles were examined using gas chromatography-mass spectrophotometry (GC-MS) and MS-e-nose analyses, respectively. The total minerals content in Japanese NCSs ranged from 228.58 to 1347.53 mg/100 g, comprising K, Ca, Mg, P, and Na (69.1, 16.6, 7.9, 4.5, and 3.2%, respectively); their average total amounts were as high as those of Malaysia and Indonesia origins (962.87, 984.67, and 928.47 mg/100 g, respectively). Forty-four VOCs were identified, of which concentrations of pyrazines, furans, and pyranones varied significantly among the NCSs. Additionally, the MS-e-nose analysis provided a multivariate differentiation profile of the NCS products based on differences in the intensities of the VOC ion masses. Nine statistical clusters were presented, wherein certain NCS products of ASEAN origin had volatile profiles comparable to those of the Japanese products. These outcomes suggest that the origin of production greatly influences the mineral and VOC compositions of NCS, affecting their quality traits.

Nucleic acid ligands act as a PAM and agonist depending on the intrinsic ligand binding state of P2RY2.

G protein-coupled receptors (GPCRs) play diverse roles in physiological processes, and hence the ligands to modulate GPCRs have served as important molecules in biological and pharmacological approaches. However, the exploration of novel ligands for GPCR still remains an arduous challenge. In this study, we report a method for the discovery of nucleic acid ligands against GPCRs by an advanced RNA aptamer screening technology that employs a virus-like particle (VLP), exposing the GPCR of interest. An array of biochemical analyses coupled with a cell-based assay revealed that one of the aptamers raised against purinergic receptor P2Y2 (P2RY2), a GPCR, exhibits an activation potency to unliganded receptor and prohibits a further receptor activation by endogenous ligand, behaving like a partial agonist. However, the aptamer enhances the activity of intrinsic ligand-binding P2RY2, thereby acting as a positive allosteric modulator (PAM) to liganded receptor. Our findings demonstrate that the nucleic acid aptamer conditionally exerts PAM and agonist effects on GPCRs, depending on their intrinsic ligand binding state. These results indicate the validity of our VLP-based aptamer screening targeting GPCR and reemphasize the great potential of nucleic acid ligands for exploring the GPCR activation mechanism and therapeutic applications.

Specific inhibition of FGF5-induced cell proliferation by RNA aptamers.

Fibroblast growth factor 5 (FGF5) is a crucial regulator of hair growth and an oncogenic factor in several human cancers. To generate FGF5 inhibitors, we performed Systematic Evolution of Ligands by EXponential enrichment and obtained novel RNA aptamers that have high affinity to human FGF5. These aptamers inhibited FGF5-induced cell proliferation, but did not inhibit FGF2-induced cell proliferation. Surface plasmon resonance demonstrated that one of the aptamers, F5f1, binds to FGF5 tightly (Kd = 0.7 ± 0.2 nM), but did not fully to FGF1, FGF2, FGF4, FGF6, or FGFR1. Based on sequence and secondary structure similarities of the aptamers, we generated the truncated aptamer, F5f1_56, which has higher affinity (Kd = 0.118 ± 0.003 nM) than the original F5f1. Since the aptamers have high affinity and specificity to FGF5 and inhibit FGF5-induced cell proliferation, they may be candidates for therapeutic use with FGF5-related diseases or hair disorders.

Altered-specificity mutants of the HIV Rev arginine-rich motif-RRE IIB interaction.

Arginine-rich motifs (ARMs) bind RNA structures with high affinity and specificity, and the human immunodeficiency virus (HIV) exploits ARM-RNA interactions to regulate its lifecycle. The expression of HIV structural genes relies on recognition between the ARM of its Rev protein and its primary binding site, an internal loop in the viral RNA, the Rev-response element region IIB (IIB). Many functional variants of the Rev ARM-IIB interaction have been discovered, yet how easily it can evolve new specificities is poorly explored. A double mutant of Rev ARM, R35G-N40 V, uses an unknown strategy to recognize IIB. Here, isothermal titration calorimetry and gel shift assays show that the R35G-N40V-IIB interaction has high affinity and specificity in vitro and a larger unfavorable entropy change upon binding than that of wild-type Rev ARM-IIB. In stark contrast with the critical dependence of wild-type Rev on Arg35, Arg39, Asn40, and Arg44, mutational profiling shows R35G-N40V is highly mutable at positions 40 and 44 and dependent on Gly35, Arg38, Arg39, Arg42, and Arg43. Affinity measurements in vitro and reporter assay measurements in vivo are consistent with the wild-type Rev ARM and R35G-N40V maintaining their recognition strategies when binding IIB mutants specific to wild-type Rev ARM and R35G-N40V, respectively. Some single amino acid mutants of wild-type Rev ARM and R35G-N40V have enhanced specificity, recognizing mutant IIBs yet not wild-type IIB. These results provide another example of viral ARM-RNA interactions evolving new specificities with few mutations, consistent with neutral theories of evolution.

Kinetic and Thermodynamic Analyses of RNA-Protein Interactions.

Recently created biophysical methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), have been widely used to quantitatively study biomolecule interactions. The dissociation constant of the interaction with kinetic parameters, such as association rate constant and dissociation rate constant, can be obtained using SPR analysis. With thermodynamic parameters, such as enthalpy change and entropy change, the dissociation constant can be obtained by ITC analysis. Both methods differ not only in the type of information obtained but also in throughput and sample concentration. Analyzing the biophysical parameters of RNA-protein interactions will help us understand their functions in biological processes. In this chapter, we describe step-by-step SPR and ITC protocols suitable to study the kinetics and thermodynamics of RNA-protein interactions.

ITC Measurement for High-Affinity Aptamers Binding to Their Target Proteins.

Aptamers are nucleic acid molecules that bind to a target molecule with high affinity and specificity, which are generated by a process known as systematic evolution of ligands by exponential enrichment (SELEX). Because of their high affinity and specificity, aptamers were developed as therapeutic agents. Although aptamers are investigated as promising therapeutic agents, the mechanism of their high affinity and specificity is not clear. Therefore, structural and biophysical studies are important to know that. To date, ITC is increasingly being used to study the thermodynamic basis of aptamer-target protein interactions. Understanding the mechanism of aptamer binding would contribute to their development for therapeutic applications. In this chapter, we describe the protocol to study the thermodynamics of aptamer-protein interactions.

Characterisation of an aptamer against the Runt domain of AML1 (RUNX1) by NMR and mutational analyses.

Since the invention of systematic evolution of ligands by exponential enrichment, many short oligonucleotides (or aptamers) have been reported that can bind to a wide range of target molecules with high affinity and specificity. Previously, we reported an RNA aptamer that shows high affinity to the Runt domain (RD) of the AML1 protein, a transcription factor with roles in haematopoiesis and immune function. From kinetic and thermodynamic studies, it was suggested that the aptamer recognises a large surface area of the RD, using numerous weak interactions. In this study, we identified the secondary structure by nuclear magnetic resonance spectroscopy and performed a mutational study to reveal the residue critical for binding to the RD. It was suggested that the large contact area was formed by a DNA-mimicking motif and a multibranched loop, which confers the high affinity and specificity of binding.

Single-round isolation of diverse RNA aptamers from a random sequence pool.

Aptamers are oligonucleotide ligands with specific binding affinity to target molecules. Generally, RNA aptamers are selected from an RNA pool with random sequences, using the technique termed SELEX, in which the target-binding RNA molecules are repeatedly isolated and exponentially amplified. Despite several advantages, SELEX often produces uncertain results during the iterative amplifications of the rare target-binding RNA molecules. Here, we develop a non-repeated, primer-less and target immobilization-free isolation method for generating RNA aptamers, which is robust to experimental noise. Uniquely, this method focuses on finding and removal of non-aptamer sequences from the RNA pool by RNase digestion leaving target-bound aptamer molecules, and thus is independent of aptamer types. The undigested RNA sequences remaining are so few in number that they must be mixed with a large excess of a known sequence for further manipulations and this sequence is then removed by restriction digestion followed by high-throughput sequencing analysis to identify aptamers. Using this method, we generated multiple RNA aptamers targeting α-thrombin and TGFß1 proteins, independently. This method potentially generates thousands of sequences as aptamer candidates, which may enable us to predict a common average sequence or structural property of these aptamers that is different from input RNA.

Conjugation of two RNA aptamers improves binding affinity to AML1 Runt domain.

To develop a high-affinity aptamer against AML1 Runt domain, two aptamers were conjugated based on their structural information. The newly designed aptamer Apt14 was generated by the conjugation of two RNA aptamers (Apt1 and Apt4) obtained by SELEX against AML1 Runt domain, resulting in improvement in its binding performance. The residues of AML1 Runt domain in contact with Apt14 were predicted in silico and confirmed by mutation and NMR analyses. It was suggested that the conjugated internal loop renders additional contacts and is responsible for the enhancement in the binding affinity. Conjugation of two aptamers that bind to different sites of the target protein is a facile and robust strategy to develop an aptamer with higher performance.

NMR monitoring of the SELEX process to confirm enrichment of structured RNA.

RNA aptamers are RNA molecules that bind to a target molecule with high affinity and specificity using uniquely-folded tertiary structures. RNA aptamers are selected from an RNA pool typically comprising up to 1015 different sequences generated by iterative steps of selection and amplification known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Over several rounds of SELEX, the diversity of the RNA pool decreases and the aptamers are enriched. Hence, monitoring of the enrichment of these RNA pools is critical for the successful selection of aptamers, and several methods for monitoring them have been developed. In this study, we measured one-dimensional imino proton NMR spectra of RNA pools during SELEX. The spectrum of the initial RNA pool indicates that the RNAs adopt tertiary structures. The structural diversity of the RNA pools was shown to depend highly on the design of the primer-binding sequence. Furthermore, we demonstrate that enrichment of RNA aptamers can be monitored using NMR. The RNA pools can be recovered from the NMR tube after measurement of NMR spectra. We also can monitor target binding in the NMR tubes. Thus, we propose using NMR to monitor the enrichment of structured aptamers during the SELEX process.

Two stems with different characteristics and an internal loop in an RNA aptamer contribute to spermine-binding.

Though polyamines (putrescine, spermidine, and spermine) bind to the specific position in RNA molecules, interaction mechanisms are poorly understood. SELEX procedure has been used to isolate high-affinity oligoribonucleotides (aptamers) from randomized RNA libraries. Selected aptamers are useful in exploring sequences and/or structures in RNAs for binding molecules. In this study, to analyze the interaction mechanism of polyamine to RNA, we selected RNA aptamers targeted for spermine. Two spermine-binding aptamers (#5 and #24) were obtained and both of them had two stem-loop structures. The 3' stem-loop of #5 (SL_2) bound to spermine more effectively than the 5' stem-loop of #5 did. A thermodynamic analysis by an isothermal titration calorimetry revealed that the dissociation constant of SL_2 for spermine was 27.2 µM and binding ratio was nearly 1:1. Binding assay with base-pair replaced variants showed that two stem regions and an internal loop in SL_2 were important for their spermine-binding activities. NMR analyses proposed that a terminal-side and a loop-side stem in SL_2 take a loose and a stable structure, respectively and a conformational change of SL_2 is induced by spermine. It is conclusive that two stems with different characteristics and an internal loop in SL_2 contribute to the specific spermine-binding.

Kinetic and Thermodynamic Analyses of Interaction between a High-Affinity RNA Aptamer and Its Target Protein.

AML1 (RUNX1) protein is an essential transcription factor involved in the development of hematopoietic cells. Several genetic aberrations that disrupt the function of AML1 have been frequently observed in human leukemia. AML1 contains a DNA-binding domain known as the Runt domain (RD), which recognizes the RD-binding double-stranded DNA element of target genes. In this study, we identified high-affinity RNA aptamers that bind to RD by systematic evolution of ligands by exponential enrichment. The binding assay using surface plasmon resonance indicated that a shortened aptamer retained the ability to bind to RD when 1 M potassium acetate was used. A thermodynamic study using isothermal titration calorimetry (ITC) showed that the aptamer-RD interaction is driven by a large enthalpy change, and its unfavorable entropy change is compensated by a favorable enthalpy change. Furthermore, the binding heat capacity change was identified from the ITC data at various temperatures. The aptamer binding showed a large negative heat capacity change, which suggests that a large apolar surface is buried upon such binding. Thus, we proposed that the aptamer binds to RD with long-range electrostatic force in the early stage of the association and then changes its conformation and recognizes a large surface area of RD. These findings about the biophysics of aptamer binding should be useful for understanding the mechanism of RNA-protein interaction and optimizing and modifying RNA aptamers.

The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element.

AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5'-NNCCAC-3' and 5'-GCGMGN'N'-3' (M:A or C; N and N' form Watson-Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences.

Mineralization of alkylbenzenesulfonates in water by means of contact glow discharge electrolysis.

Mineralization of aqueous alkylbenzenesulfonates (ABS) was investigated by means of contact glow discharge electrolysis (CGDE). Toluenesulfonic and dodecylbenzenesulfonic acids in a neutral phosphate buffer solution were smoothly degraded and eventually converted to inorganic carbon and sulfate ion when CGDE was conducted under the applied DC voltage of 500 V and current of ca. 90 mA. As the intermediate products, some phenolic compounds were detected as well as carboxylic acids such as oxalate, formate and so on. It was demonstrated that the decay of ABS followed the first-order rate law. Based on the detailed analysis of the hydroxylation products and kinetic consideration, it was assumed that hydroxyl radicals would play a crucial role in the oxidative degradation of aqueous ABS. The features of CGDE as a tool for the removal of organic pollutants in water were discussed in comparison with other physicochemical methods.