Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small molecule

In this contribution, an electrochemical aptameric sensing scheme for the sensitive detection of small molecules is proposed using adenosine as a target model. A ferrocene (Fc)-functionalized thiolated aptamer probe is adapted and immobilized onto an electrode surface. Introducing a recognition site for EcoRI into the aptamer sequence not only suppresses the peak current corresponding to blank sample but also provides a signal-on response mechanism. In the absence of adenosine, the aptamer can fold into a hairpin structure and form a cleavable double-stranded region. Fc is capable of being removed from electrode surface by treatment with endonuclease, and almost no peak current is observed. The adenosine/aptamer binding induces the conformational transition of designed aptamer, dissociating the cleavable double-stranded segment. Therefore, the integrated aptamer sequence is maintained when exposing to endonuclease, generating a peak current of Fc. Utilizing the present sensing scheme, adenosine even at a low concentration can give a detectable current signal. Thus, a detection limit of 10⁻¹⁰ M and a linear response range from 3.74 × 10⁻⁹ to 3.74 × 10⁻⁵ M are achieved. The proposed proof-of-principle of a novel electrochemical sensing is expected to extend to establish various aptameric platforms for the analysis of a broad range of target molecules of interest.     

Abundance of correctly folded RNA motifs in sequence space, calculated on computational grids

Although functional RNA molecules are known to be biased in overall composition, the effects of background composition on the probability of finding a particular active site by chance has received little attention. The probability of finding a particular motif has important implications both for understanding the distribution of functional RNAs in ancient and modern organisms with varying genome compositions and for tuning SELEX pools to optimize the chance of finding specific functions. Here we develop a new method for calculating the probability of finding a modular motif containing base-paired regions, and use a computational grid to fold several hundred million random RNA sequences containing the core elements of the isoleucine aptamer and the hammerhead ribozyme to estimate the probability that a sequence containing these structural elements will fold correctly when isolated from background sequences of different compositions. We find that the two motifs are most likely to be found in distinct regions of compositional space, and that the regions of greatest abundance are influenced by the probability of finding the conserved bases, finding the flanking helices, and folding, in that order of importance. Additionally, we can refine our estimates of the number of random sequences required for a 50% probability of finding an example of each site in unbiased random pools of length 100 to 4.1 × 10⁹ for the isoleucine aptamer and 1.6 × 10¹⁰ for the hammerhead ribozyme. These figures are consistent with the facile recovery of these motifs from SELEX experiments.     

Folding of the cocaine aptamer studied by EPR and fluorescence spectroscopies using the bifunctional spectroscopic probe Ç

The cocaine aptamer is a DNA molecule that binds cocaine at the junction of three helices. The bifunctional spectroscopic probe Ç was incorporated independently into three different positions of the aptamer and changes in structure and dynamics upon addition of the cocaine ligand were studied. Nucleoside Ç contains a rigid nitroxide spin label and can be studied directly by electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy after reduction of the nitroxide to yield the fluoroside Ç^f. Both the EPR and the fluorescence data for aptamer 2 indicate that helix III is formed before cocaine binding. Upon addition of cocaine, increased fluorescence of a fully base-paired Ç^f, placed at the three-way junction in helix III, was observed and is consistent with a helical tilt from a coaxial stack of helices II and III. EPR and fluorescence data clearly show that helix I is formed upon addition of cocaine, concomitant with the formation of the Y-shaped three-way helical junction. The EPR data indicate that nucleotides in helix I are more mobile than nucleotides in regular duplex regions and may reflect increased dynamics due to the short length of helix I.     

Aptamer Selection Based on G4-Forming Promoter Region

We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as “G4 promoter-derived aptamer selection (G4PAS).” Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K _d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10⁻⁷ M, 6.3 × 10⁻⁹ M, and 4.4 × 10⁻⁷ M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.     

Probing l-Pyruvate Kinase Regulatory Phosphorylation Site by Mutagenesis

The activity of L-type pyruvate kinase (L-PK, ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is regulated by phosphorylation of serine residue 12 of the N-terminal regulatory domain MEGPAGYLRR¹⁰AS ¹² VAQLTQEL²⁰GTAFF of the protein. In this report we studied the effect of the point mutations around this phosphorylation site on the catalytic properties of this enzyme, by introducing amino acids A, L, K, Q and E into positions 9, 10 and 13 of this peptide sequence. It was found that some of these mutations in positions 9 and 10, although occurring at great distances from the enzyme??s active site, affected the enzyme??s activity by decreasing the effectiveness of phosphoenolpyruvate binding (PEP) with the enzyme, but had practically no influence on the binding effectiveness of the second substrate ADP. A similar asymmetric effect on the binding of these substrates was previously observed after phosphorylation of the enzyme regulatory N-domain peptide, and also after proteolytic truncation of the same N-terminal part of L-PK. All these results could be explained by the internal complex formation between the N-domain peptide and the enzyme??s main body. The present study delineated the specificity of the internal binding site and revealed the possibility that the regulatory effect could be modulated by selecting mutation sites and amino acids introduced into the N-terminal domain structure.     

Low-fidelity DNA synthesis by human DNA polymerase theta

Human DNA polymerase theta (pol θ or POLQ) is a proofreading-deficient family A enzyme implicated in translesion synthesis (TLS) and perhaps in somatic hypermutation (SHM) of immunoglobulin genes. These proposed functions and kinetic studies imply that pol θ may synthesize DNA with low fidelity. Here, we show that when copying undamaged DNA, pol θ generates single base errors at rates 10- to more than 100-fold higher than for other family A members. Pol θ adds single nucleotides to homopolymeric runs at particularly high rates, exceeding 1% in certain sequence contexts, and generates single base substitutions at an average rate of 2.4 × 10⁻³, comparable to inaccurate family Y human pol κ (5.8 × 10⁻³) also implicated in TLS. Like pol κ, pol θ is processive, implying that it may be tightly regulated to avoid deleterious mutagenesis. Pol θ also generates certain base substitutions at high rates within sequence contexts similar to those inferred to be copied by pol θ during SHM of immunoglobulin genes in mice. Thus, pol θ is an exception among family A polymerases, and its low fidelity is consistent with its proposed roles in TLS and SHM.     

Rapid binding and release of Hfq from ternary complexes during RNA annealing

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼10⁸ M⁻¹s⁻¹) observed with Cy3-Hfq was followed by a slow transition (0.5 s⁻¹) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions.     

Fabrication and characterization of RNA aptamer microarrays for the study of protein-aptamer interactions with SPR imaging

RNA microarrays were created on chemically modified gold surfaces using a novel surface ligation methodology and employed in a series of surface plasmon resonance imaging (SPRI) measurements of DNA–RNA hybridization and RNA aptamer–protein binding. Various unmodified single-stranded RNA (ssRNA) oligonucleotides were ligated onto identical 5′-phosphate-terminated ssDNA microarray elements with a T4 RNA ligase surface reaction. A combination of ex situ polarization modulation FTIR measurements of the RNA monolayer and in situ SPRI measurements of DNA hybridization adsorption onto the surface were used to determine an ssRNA surface density of 4.0 × 10¹² molecules/cm² and a surface ligation efficiency of 85 ± 10%. The surface ligation methodology was then used to create a five-component RNA microarray of potential aptamers for the protein factor IXa (fIXa). The relative surface coverages of the different aptamers were determined through a novel enzymatic method that employed SPRI measurements of a surface RNase H hydrolysis reaction. SPRI measurements were then used to correctly identify the best aptamer to fIXa, which was previously determined from SELEX measurements. A Langmuir adsorption coefficient of 1.6 × 10⁷ M⁻¹ was determined for fIXa adsorption to this aptamer. Single-base variations from this sequence were shown to completely destroy the aptamer–fIXa binding interaction.     

Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon

An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K_m = 47.1 μM and a k_cat = 0.151 s⁻¹, which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA^Cys with the GCA anticodon (26.9 μM and 0.111 s⁻¹). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.     

A High-Resolution View of Genome-Wide Pneumococcal Transformation

Transformation is an important mechanism of microbial evolution through which bacteria have been observed to rapidly adapt in response to clinical interventions; examples include facilitating vaccine evasion and the development of penicillin resistance in the major respiratory pathogen Streptococcus pneumoniae. To characterise the process in detail, the genomes of 124 S. pneumoniae isolates produced through in vitro transformation were sequenced and recombination events detected. Those recombinations importing the selected marker were independent of unselected events elsewhere in the genome, the positions of which were not significantly affected by local sequence similarity between donor and recipient or mismatch repair processes. However, both types of recombinations were sometimes mosaic, with multiple non-contiguous segments originating from the same molecule of donor DNA. The lengths of the unselected events were exponentially distributed with a mean of 2.3 kb, implying that recombinations are stochastically resolved with a fixed per base probability of 4.4×10⁻⁴ bp⁻¹. This distribution of recombination sizes, coupled with an observed under representation of large insertions within transferred sequence, suggests transformation has the potential to reduce the size of bacterial genomes, and is unlikely to act as an efficient mechanism for the uptake of accessory genomic loci.     

Detection of phosphodiester adducts formed by the reaction of benzo[a]pyrene diol epoxide with 2'-deoxynucleotides using collision-induced dissociation electrospray ionization tandem mass spectrometry

In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE) with 2′-deoxynucleoside 3′-monophosphates. The B[a]PDE plus 2′-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M−H]⁻ consistent with B[a]PDE adducted (either on the base or phosphate group) 2′-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)−H]⁻, resulting from cleavage of the glycosidic bond between the 2′-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 2′-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 2′-deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)−H]⁻ and [(2′-deoxyribose+phosphate+B[a]Ptriol)−H]⁻, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 2′-deoxynucleotides.     

Heparan Sulfate Proteoglycan Binding by Herpes Simplex Virus Type 1 Glycoproteins B and C, Which Differ in Their Contributions to Virus Attachment, Penetration, and Cell-to-Cell Spread

Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB’s role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK⁻ also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK⁻ gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK⁻. Compared with wild-type virus, KgBpK⁻ showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the lacZ gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK⁻gC⁻) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK⁻ mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.     

DNA repair and sequence context affect 1O2-induced mutagenesis in bacteria

Electronic excited molecular oxygen (singlet oxygen, ¹O₂) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by ¹O₂ in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a ¹O₂-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G→T and G→C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with ¹O₂-induced DNA damage are linked to the context of the damaged sequence.     

Synthesis of long-chain fatty acid derivatives as a novel anti-Alzheimer’s agent

In order to develop new drugs for Alzheimer’s disease, we prepared 17 fatty acid derivatives with different chain lengths and different numbers and positions of double bonds by using Wittig reaction and stereospecific hydrogenation of triple bonds as key reactions. Among them, (4Z,15Z)-octadecadienoic acid (10) and (23Z,34Z)-heptatriacontadienoic acid (16) showed the most potent neurite outgrowth activities on Aβ(25–35)-treated rat cortical neurons, which activities were comparable to that of a positive control, NGF. Both fatty acids 10 and 16 possess two (Z)-double bonds at the n-3 and n-14 positions, which might be important for the neurite outgrowth activity.     

Sequence-function relationships provide new insight into the cleavage site selectivity of the 8-17 RNA-cleaving deoxyribozyme

Many sequence variations of the 8–17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8–17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5′ rN₁₈-N_1.1 3′) cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3′ position (N_1.1) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5′ position (rN₁₈) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8–17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000-fold over background. Three optimal 8–17 variants, identified from ~75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of ≥0.1 min⁻¹, and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.     

Nucleotide sequence of an attenuated mutant of coxsackievirus B3 compared with the cardiovirulent wildtype: assessment of candidate mutations by analysis of a revertant to cardiovirulence

Background: Coxsackievirus B3 (CVB3) causes myocarditis in the SWR (H^2q) mouse model and persistence of CVB3 in myocardium disposes to the development of dilated cardiomyopathy. An attenuated strain of CVB3 has been isolated, sequenced and several candidate mutations for attenuation identified. Derivation of a revertant to cardiovirulence allows the significance of these mutations to be assessed.Objectives: To ascertain which candidate mutation(s) determine(s) the attenuated phenotype.Study design: A revertant to cardiovirulence was isolated following passage through severe combined immunodeficient disease (SCID) mouse heart. The 5′-non-translated region (NTR) and region coding for capsid proteins were sequenced and compared to the wildtype and attenuant.Results: There are five candidates for attenuation: (1) A–G at base 580 in the 5′-NTR; (2) A–T at base 690 in the 5′-NTR; (3) CG–GC at bases 1401/2 (Thr to Ser at amino acid 151 in VP2); (4) AA–GT at bases 2691/2 (Lys to Ser at amino acid 80 in VP1); (5) A–G at base 2916 (Asp to Gly at amino acid 155 in VP1). It was shown previously that mutations at 580, 690 and 2691/2 are not important in attenuation. Additionally, there are three novel mutations in the coding region of the revertant and one in the 5′-NTR which are unlikely to be relevant for attenuation as they are not present in the attenuant. Of nucleotide changes seen at 1401/2 and 2916 in the attenuant, only 2916 reverts to the wildtype sequence and so is a strong candidate for a determinant of attenuation.Conclusions: The A–G mutation at 2916 (Asp to Gly at amino acid 155 in VP1) is a strong candidate for attenuation. It is located at the top of the receptor binding cleft and mutation of the Asp to a Gly may destabilise the receptor binding site.     

Evolutionary Landscapes for the Acquisition of New Ligand Recognition by RNA Aptamers

The evolution of ligand specificity underlies many important problems in biology, from the appearance of drug resistant pathogens to the re-engineering of substrate specificity in enzymes. In studying biomolecules, however, the contributions of macromolecular sequence to binding specificity can be obscured by other selection pressures critical to bioactivity. Evolution of ligand specificity in vitro—unconstrained by confounding biological factors—is addressed here using variants of three flavin-binding RNA aptamers. Mutagenized pools based on the three aptamers were combined and allowed to compete during in vitro selection for GMP-binding activity. The sequences of the resulting selection isolates were diverse, even though most were derived from the same flavin-binding parent. Individual GMP aptamers differed from the parental flavin aptamers by 7 to 26 mutations (20 to 57% overall change). Acquisition of GMP recognition coincided with the loss of FAD (flavin-adenine dinucleotide) recognition in all isolates, despite the absence of a counter-selection to remove FAD-binding RNAs. To examine more precisely the proximity of these two activities within a defined sequence space, the complete set of all intermediate sequences between an FAD-binding aptamer and a GMP-binding aptamer were synthesized and assayed for activity. For this set of sequences, we observe a portion of a neutral network for FAD-binding function separated from GMP-binding function by a distance of three mutations. Furthermore, enzymatic probing of these aptamers revealed gross structural remodeling of the RNA coincident with the switch in ligand recognition. The capacity for neutral drift along an FAD-binding network in such close approach to RNAs with GMP-binding activity illustrates the degree of phenotypic buffering available to a set of closely related RNA sequences—defined as the sets functional tolerance for point mutations—and supports neutral evolutionary theory by demonstrating the facility with which a new phenotype becomes accessible as that buffering threshold is crossed.     

Synthesis, structure, DNA binding and cleavage properties of ternary amino acid Schiff base-phen/bipy Cu(II) complexes

Ternary Cu(II) complexes [Cu(II)(saltrp)(B)] (1,2), (saltrp = salicylidene tryptophan, B = 1,10 phenathroline (1) or 2,2′ bipyridine (2)) were synthesized and characterized. Complex 2 was structurally characterized by single crystal X-ray crystallography. The molecular structure shows a distorted square pyramidal coordination geometry (CuN₃O₂) in which the ONO donor Schiff base is bonded to the Cu(II) in the basal plane. The N,N donor heterocyclic base displays an axial-equatorial binding mode. CT-DNA binding studies revealed that the complexes show good binding propensity (Intrinsic binding constant, K_b = 3.32 × 10⁵ M⁻¹ for 1 and K_b = 3.10 × 10⁵ M⁻¹ for 2). The catalytic role of these complexes in the oxidative and hydrolytic cleavage of DNA was studied in detail. Complex 1 binds and cleaves DNA more efficiently as compared to 2. From the kinetic experiments, rate constants for the hydrolysis of phosphodiester bond of DNA backbone were determined as 1.94 h⁻¹ and 1.05 h⁻¹ for 1 and 2 respectively. It amounts to (2.93-5.41) × 10⁷ fold rate enhancement compared to uncatalyzed double stranded DNA, which is impressive as compared to related Cu(II) Schiff base complexes.