Phosphate End Research Articles

Mechanism of RNA 2′,3′-cyclic phosphate end healing by T4 polynucleotide kinase–phosphatase

T4 polynucleotide kinase–phosphatase (Pnkp) exemplifies a family of enzymes with 5′-kinase and 3′-phosphatase activities that function in nucleic acid repair. The polynucleotide 3′-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3′-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid–base catalyst Asp167. We report that Pnkp also has RNA 2′-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2′,3′-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA > p to RNAOH, mutant D167N converts RNA > p to RNA 3′-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNAOH. Our results suggest that healing of 2′,3′-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3′-phosphoaspartyl)-Pnkp → RNA3′p + Pnkp → RNAOH + phosphoaspartyl-Pnkp → Pi + Pnkp.

Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation

Chlorella virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3'-OH and 5'-PO(4) termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PP(i) and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5'-phosphate to form DNA-adenylate; 3) the 3'-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg(2+). Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (k(step3) = 25 s(-1)) exceeds that for DNA adenylylation (k(step2) = 2.4 s(-1)) and that Mg(2+) binds with similar affinity during step 2 (K(d) = 0.77 mM) and step 3 (K(d) = 0.87 mM). The rates of DNA adenylylation and phosphodiester synthesis respond differently to pH, such that step 3 becomes rate-limiting at pH ≤ 6.5. The pH profiles suggest involvement of one and two protonation-sensitive functional groups in catalysis of steps 2 and 3, respectively. We suggest that the 5'-phosphate of the nick is the relevant protonation-sensitive moiety and that a dianionic 5'-phosphate is necessary for productive step 2 catalysis. Motif VI, located at the C terminus of the OB-fold domain of ChVLig, is a conserved feature of ATP-dependent DNA ligases and GTP-dependent mRNA capping enzymes. Presteady state and burst kinetic analysis of the effects of deletion and missense mutations highlight the catalytic contributions of ChVLig motif VI, especially the Asp-297 carboxylate, exclusively during the ligase adenylylation step.

Identification of the catalytic residues of sequence-specific and histidine-free ribonuclease colicin E5

Colicin E5 cleaves tRNAs for Tyr, His, Asn and Asp in their anticodons to abolish protein synthesis in Escherichia coli. We previously showed how its C-terminal RNase domain, E5-CRD, recognizes the anticodon bases but the catalytic mechanism remained to be elucidated. Although the reaction products with 5'-OH and 2',3'-cyclic phosphate ends suggested a similar mechanism to those of RNases A and T1, E5-CRD does not have the His residues necessary as a catalyst in usual RNases. To identify residues important for the catalytic reaction, mutants as to all residues within 5 Å from the central phosphorus of the scissile phosphodiester bond were prepared. Evaluation of the killing activities of the mutant colicins and the RNase activities of the mutant E5-CRDs suggested direct involvement of Arg33, Lys25, Gln29 and Lys60 in the reaction. Particularly, Arg33 plays a critical role and Ile94 provides a structural support of Arg33. Crystal structure of the complex of E5-CRD(R33Q)/dGpdUp showed structural and binding functional integrity of this mutant protein, suggesting involvement of Arg33 in the catalytic reaction. The structure of the free E5-CRD, we also determined, showed great flexibility of a flap region, which facilitates the access of Lys60 to the substrate in an induced-fit manner.

Detection of Ligation Products of DNA Linkers with 5′-OH Ends by Denaturing PAGE Silver Stain

To explore if DNA linkers with 5′-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5–1% of linkers A–B and E–F, and 0.13–0.5% of linkers C–D could be joined by T4 DNA ligases. About 0.25–0.77% of linkers A–B and E–F, and 0.06–0.39% of linkers C–D could be joined by E. coli DNA ligases. A 1-base deletion (-G) and a 5-base deletion (-GGAGC) could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025–0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5′-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i) about 0.025–0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3′-OH ends of other linkers; and (ii) the linkers could delete one or more nucleotide(s) at their 5′-ends and thereby generated some 5′-phosphate ends, and then these 5′-phosphate ends could be joined to the 3′-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5′-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK.

The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent

The RNA ligase RtcB splices broken RNAs with 5′-OH and either 2′,3′-cyclic phosphate or 3′-phosphate ends. The 3′-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5′-OH end attacks the 3′-phosphorus of the N(3′)pp(5′)G end to form a 3′,5′-phosphodiester and release GMP. Scheme 2 posits that the N(3′)pp(5′)G end is converted to a 2′,3′-cyclic phosphodiester, which is then attacked directly by the 5′-OH RNA end to form a 3′,5′-phosphodiester. Here we show that the sealing of a 2′,3′-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB–GMP adduct, and involves a kinetically valid RNA(3′)pp(5′)G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2′,3′-cyclic phosphate to a 3′-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB–GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3′-phosphate ligase steps of the RNA splicing pathway.

Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

AbstractIn this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through AuS bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10−13 to 1.0×10−6 mol·L−1 with a detection limit of 3.5×10−14 mol·L−1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.

Ferrocene-functionalized SWCNT for electrochemical detection of T4 polynucleotide kinase activity

A novel electrochemical strategy for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK) is developed by use of titanium ion (Ti 4+) mediated signal transition coupled with signal amplification of single wall carbon nanotubes (SWCNTs). In this method, a DNA containing 5′-hydroxyl group is self-assembled onto the gold electrode and used as substrate for PNK. The biofunctionalized SWCNTs with anchor DNA and ferrocene are chosen as the signal indicator by virtue of the intrinsic 5′-phosphate end of anchor DNA and the high loading of ferrocene for electrochemical signal generation and amplification. The 5′-hydroxyl group of the substrate DNA on the electrode is phosphorylated by T4 PNK in the presence of ATP, and the resulting 5′-phosphoryl end product can be linked with the signal indicator by Ti 4+. The redox ferrocene group on the SWCNTs is grafted to the electrode and generates the electrochemical signal, the intensity of which is proportional to the activity of T4 PNK. This assay can measure activity of T4 PNK down to 0.01 U mL −1. The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.

Structures of RNA 3′-phosphate cyclase bound to ATP reveal the mechanism of nucleotidyl transfer and metal-assisted catalysis

RNA 3'-phosphate cyclase (RtcA) synthesizes RNA 2',3' cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-(histidinyl-Nε)-AMP intermediate; transfer of adenylate to an RNA 3'-phosphate to form RNA(3')pp(5')A; and attack of the vicinal O2' on the 3'-phosphorus to form a 2',3' cyclic phosphate and release AMP. Here we report the crystal structures of RtcA•ATP, RtcA•ATP•Mn(2+), and RtcA•ATP•Co(2+) substrate complexes and an RtcA•AMP product complex. Together with the structures of RtcA apoenzyme and the covalent RtcA-AMP intermediate, they illuminate the mechanism of nucleotidyl transfer, especially the stereochemical transitions at the AMP phosphate, the critical role of the metal in orienting the PP(i) leaving group of ATP during step 1, and the protein conformational switches that accompany substrate binding and product release. The octahedral metal complex of RtcA•ATP•Mn(2+) includes nonbridging oxygens from each of the ATP phosphates, two waters, and Glu14 as the sole RtcA component. Whereas the RtcA adenylylation step is metal-catalyzed, the subsequent steps in the cyclization pathway are metal-independent.

Novel Mechanism of RNA Repair by RtcB via Sequential 2′,3′-Cyclic Phosphodiesterase and 3′-Phosphate/5′-Hydroxyl Ligation Reactions

RtcB enzymes are a newly discovered family of RNA ligases, implicated in tRNA splicing and other RNA repair reactions, that seal broken RNAs with 2',3'-cyclic phosphate and 5'-OH ends. Parsimony and energetics would suggest a one-step mechanism for RtcB sealing via attack by the O5' nucleophile on the cyclic phosphate, with expulsion of the ribose O2' and generation of a 3',5'-phosphodiester at the splice junction. Yet we find that RtcB violates Occam's razor, insofar as (i) it is adept at ligating 3'-monophosphate and 5'-OH ends; (ii) it has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. Thus, RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP, which reacts with RtcB in the presence of manganese to form a covalent RtcB-guanylate adduct. This adduct is sensitive to acid and hydroxylamine but resistant to alkali, consistent with a phosphoramidate bond.

The Structural Basis for Substrate Recognition by Mammalian Polynucleotide Kinase 3′ Phosphatase

Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.

RtcB, a Novel RNA Ligase, Can Catalyze tRNA Splicing and HAC1 mRNA Splicing in Vivo

RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.

RtcB Is the RNA Ligase Component of an Escherichia coli RNA Repair Operon

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.

RNA 3′-Phosphate Cyclase (RtcA) Catalyzes Ligase-like Adenylylation of DNA and RNA 5′-Monophosphate Ends

RNA 3'-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2',3'-cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP(i); transfer of AMP from Rtc to an RNA 3'-phosphate to form an RNA(3')pp(5')A intermediate; and attack by the terminal nucleoside O2' on the 3'-phosphate to form an RNA 2',3'-cyclic phosphate product and release AMP. The chemical transformations of the cyclase pathway resemble those of RNA and DNA ligases, with the key distinction being that ligases covalently adenylylate 5'-phosphate ends en route to phosphodiester synthesis. Here we show that the catalytic repertoire of RNA cyclase overlaps that of ligases. We report that Escherichia coli RtcA catalyzes adenylylation of 5'-phosphate ends of DNA or RNA strands to form AppDNA and AppRNA products. The polynucleotide 5' modification reaction requires the His(309) nucleophile, signifying that it proceeds through a covalent RtcA-AMP intermediate. We established this point directly by demonstrating transfer of [(32)P]AMP from RtcA to a pDNA strand. RtcA readily adenylylated the 5'-phosphate at a 5'-PO(4)/3'-OH nick in duplex DNA but was unable to covert the nicked DNA-adenylate to a sealed phosphodiester. Our findings raise the prospect that cyclization of RNA 3'-ends might not be the only biochemical pathway in which Rtc enzymes participate; we discuss scenarios in which the 5'-adenylyltransferase of RtcA might play a role.

A sensitive fluorimetric biosensor for detection of DNA hybridization based on Fe/Au core/shell nanoparticles

In this study, we reported a sensitive fluorescent biosensor for detection of DNA hybridization based on Fe/Au core/shell (Fe@Au) nanoparticles (NPs). First, Fe@Au NPs were synthesized using a reverse micelle method, with gold as the shell and iron as the core. The nanoparticle size was confirmed by transmission electron microscopy (TEM). Scanning electron microscopy (SEM) was performed in order to elucidate the morphology of the Fe@Au NPs. Then probe DNA with -SH at the 5'-phosphate end was covalently immobilized onto the surface of the Fe@Au NPs. The DNA hybridization event can be detected by a fluorescent method and methylene blue (MB) as the fluorescent probe. The decline of the fluorescence intensity of MB (ΔF) was linear with the concentration of the complementary DNA from 3.0 × 10(-13) to 1.0 × 10(-9) M with a detection limit of 1.0 × 10(-13) M (S/N = 3). In addition, this approach of DNA detection exhibited excellent selectivity, even for single-mismatched DNA detection.

Oxidation of a guanine derivative coordinated to a Pt(iv) complex initiated by intermolecular nucleophilic attacks

In this study we report that fac-[Pt(IV)(dach)(9-EtG)Cl(3)](+) (dach = d,l-1,2-diaminocyclohexane, 9-EtG = 9-ethylguanine) in high pH (pH 12) or phosphate solution (pH 7.4) produces 8-oxo-9-EtG and Pt(II) species. The reaction in H(2)(18)O revealed that the oxygen atom in hydroxide or phosphate ends up at the C8 position of 8-oxo-G. The kinetics of the redox reaction was first order with respect to both Pt(IV)-G and free nucleophiles (OH(-) and phosphate). The oxidation of G initiated by hydroxide was approximately 30∼50 times faster than by phosphate in 100 mM NaCl solutions. The large entropy of activation of OH(-1) (ΔS(‡) = 26.6 ± 4.3 J mol(-1) K(-1)) due to the smaller size of OH(-) is interpreted to be responsible for the faster kinetics compared to phosphate (ΔS(‡) = -195.5 ± 11.1 J mol(-1) K(-1)). The enthalpy of activation for phosphate reaction is more favorable relative to the OH(-) reaction (ΔH(‡) = 35.4 ± 3.5 kJ mol(-1) for phosphate vs. 96.6 ± 11.4 kJ mol(-1) for OH(-1)). The kinetic isotope effect of H8 was determined to be 7.2 ± 0.2. The rate law, kinetic isotope effect, and isotopic labeling are consistent with a mechanism involving proton ionization at the C8 position as the rate determining step followed by two-electron transfer from G to Pt(IV).

The number and not the molecular structure of DNA double-strand breaks is more important for the formation of chromosomal aberrations: A hypothesis

DNA double-strand breaks (DSB) are known to cause chromosomal aberrations (CA). The ends of DSB have different molecular structures depending on the inducing agent. Restriction endonucleases, DNase I and benzon nuclease produce DSB with 3′-OH and 5′-phosphate ends which should be easily rejoinable (“clean” DSB). Other DSB-inducing agents, such as neocarzinostatin, bleomycin and ionizing radiation induce DSB with chemically modified ends (“dirty” DSB) which are not religatable before enzymatic pruning to make them “clean”. Both “clean” and “dirty” DSB lead to CA whose quantities and distributions are quite similar. We conclude that the number and not the molecular structures of DSB are essential for the production of CA. All DSB-inducing agents discussed in this review induce exchange events in the G1 phase of the cell cycle which look like sister chromatid exchanges (SCE) and are therefore called “false” SCE. “False” SCE seem to be mainly intrachanges and would therefore outweigh the frequencies of interchanges. Consequently, the factor F of inter- to intrachanges would then be <1, indicating that the majority of DSB would lead to CA inside the chromosome domains in which they were induced and not between different chromosomes.

Structure of the RNA 3′-Phosphate Cyclase-Adenylate Intermediate Illuminates Nucleotide Specificity and Covalent Nucleotidyl Transfer

RNA 3'-phosphate cyclase (RtcA) synthesizes RNA 2',3' cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-AMP intermediate; transfer of adenylate to an RNA 3'-phosphate to form RNA(3')pp(5')A; and attack of the vicinal O2' on the 3'-phosphorus to form a 2',3' cyclic phosphate. Here we report the 1.7 A crystal structure of the RtcA-AMP intermediate, which reveals the mechanism of nucleotidyl transfer. Adenylate is linked via a phosphoamide bond to the His309 Nepsilon atom. A network of hydrogen bonds to the ribose O2' and O3' accounts for the stringent ribonucleotide preference. Adenine is sandwiched in a hydrophobic pocket between Tyr284 and Pro131 and the preference for adenine is enforced by Phe135, which packs against the purine C2 edge. Two sulfates bound near the adenylate plausibly mimic the 3'-terminal and penultimate phosphates of RNA. The structure illuminates how the four alpha2/beta4 domains contribute to substrate binding and catalysis.

Structure–activity relationships in human RNA 3′-phosphate cyclase

RNA 3'-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2',3' cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP(i); transfer of AMP from Rtc1 to an RNA 3'-phosphate to form an RNA(3')pp(5')A intermediate; and attack by the terminal nucleoside O2' on the 3'-phosphate to form an RNA 2',3' cyclic phosphate product and release AMP. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human RNA cyclase Rtc1. An alanine scan defined seven conserved residues as essential for the Rtc1 RNA cyclization and autoadenylylation reactions. Structure-activity relationships were clarified by conservative substitutions. Our results are consistent with a mechanism of adenylate transfer in which attack of the Rtc1 His320 nucleophile on the ATP alpha phosphorus is facilitated by proper orientation of the PP(i) leaving group via contacts to Arg21, Arg40, and Arg43. We invoke roles for Tyr294 in binding the adenine base and Glu14 in binding the divalent cation cofactor. We find that Rtc1 forms a stable binary complex with a 3'-phosphate terminated RNA, but not with an otherwise identical 3'-OH terminated RNA. Mutation of His320 had little impact on RNA 3'-phosphate binding, signifying that covalent adenylylation of Rtc1 is not a prerequisite for end recognition.

Rational design and performance testing of aptamer-based electrochemical biosensors for adenosine

This work emphasizes on the design criteria of aptamer-based biosensors for adenosine and the comparison of their performances by using both electrochemical and biochemical methods. The adenosine biosensors have been typically prepared by hybridizing a short bridging DNA (modified with a thiol for the attachment to gold electrode) to the aptamer strand that releases upon adenosine binding. We have carefully compared the two “isomeric” designs, one with the thiol-linker tethered to the 3′-hydroxyl and the other to the 5′-phosphate end of the bridging DNA. It has been demonstrated through electrochemical and gel electrophoresis experiments that the 3′-thiol bridged sensor shows much better performance than its 5′-counterpart on surface, whereas in solution phase the two sensor systems are equally sensitive. We believe that the “transient” secondary structure of the aptamer sequence restricts the freedom of the strand displacement upon analyte binding, leading to a sluggish release of the aptamer strand from the 5′-thiol-bridging DNA.

Properties of plant nuclease 1 purified from Tradescantia leaves by binding to Concanavalin A-Sepharose

A nuclease with 3′-nucleotidase activity was purified at least 2,500-fold from Tradescantia paludosa leaves by a process that included chromatography on Concanavalin A-Sepharose. The preparation contains several active isozymes without impurities as judged by gel electrophoresis, but immunological tests revealed one or two major and a few minor inactive components. Phosphatase, phosphodiesterase and 5′-nucleotidase were absent or minimal, as were the known glycoproteins, peroxidase and esterase. The nuclease was highly active on RNA and denatured DNA, and less active on native DNA; it hydrolyzed the substrates in endonuclease manner. The mononucleotides had 5′-phosphate ends. Nuclease activity was optimal at pH 5.5–6.5 in tris-acetate buffers, 0.05–0.1 M. DNase activity was insensitive to EDTA and not stimulated by Mg2+ or Ca2+. Poly A and poly U were good and poly C and poly G poor substrates for the nuclease, but (d)GMP was the predominant mononucleotide from digests of nucleic acids. 3′-AMP, GMP and UMP were about equally good substrates for the nucleotidase at pH 6 but 3′-CMP was degraded at 1/5th the rate with 3-AMP, and any activity on deoxynucleotides was very slight.