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1.
Gani OA Adekoya OA Giurato L Spyrakis F Cozzini P Guccione S Winberg JO Sylte I 《Biophysical journal》2008,94(4):1412-1427
Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD+ complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors. 相似文献
2.
Olga V. Belyaeva Seung-Ah LeeOleg V. Kolupaev Natalia Y. Kedishvili 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster.Methods
Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.Results
A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.Conclusions
Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.General significance
The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs. 相似文献3.
Annotations of protein or gene sequences from large scale sequencing projects are based on protein size, characteristic binding motifs, and conserved catalytic amino acids, but biochemical functions are often uncertain. In the large family of short-chain dehydrogenases/reductases (SDRs), functional predictions often fail. Putative tropinone reductases, named tropinone reductase-like (TRL), are SDRs annotated in many genomes of organisms that do not contain tropane alkaloids. SDRs in vitro often accept several substrates complicating functional assignments. Cochlearia officinalis, a Brassicaceae, contains tropane alkaloids, in contrast to the closely related Arabidopsis thaliana. TRLs from Arabidopsis and the tropinone reductase isolated from Cochlearia (CoTR) were investigated for their catalytic capacity. In contrast to CoTR, none of the Arabidopsis TRLs reduced tropinone in vitro. NAD(H) and NADP(H) preferences were relaxed in two TRLs, and protein homology models revealed flexibility of amino acid residues in the active site allowing binding of both cofactors. TRLs reduced various carbonyl compounds, among them terpene ketones. The reduction was stereospecific for most of TRLs investigated, and the corresponding terpene alcohol oxidation was stereoselective. Carbonyl compounds that were identified to serve as substrates were applied for modeling pharmacophores of each TRL. A database of commercially available compounds was screened using the pharmacophores. Compounds identified as potential substrates were confirmed by turnover in vitro. Thus pharmacophores may contribute to better predictability of biochemical functions of SDR enzymes. 相似文献
4.
DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a γ-phosphate attack mechanism. ADP-Pi and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-Pi to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by Pi release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-Pi-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases. 相似文献
5.
Tim Smilda Anne H. Kamminga Peter Reinders Wia Baron Johan E.T. van Hylckama Vlieg Jaap J. Beintema 《Journal of molecular evolution》2001,52(5):457-466
Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases
from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several
secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short
ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity
for R(−) alcohols. At a high concentration of R(−) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those
at low substrate concentrations.
Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved
residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived
from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved
in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic
domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar.
Received: 23 May 2000 / Accepted: 4 January 2001 相似文献
6.
7.
Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae
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Jung-Kul Lee Bong-Seong Koo Sang-Yong Kim Hyung-Hwan Hyun 《Applied microbiology》2003,69(8):4438-4447
Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (kcat = 823 s−1, Km = 28.0 mM, and kcat/Km = 29.4 mM−1 s−1) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants. 相似文献
8.
The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ∼5%) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE; ∼1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, probably after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese lipoxygenase (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-MnLOX with larger and smaller hydrophobic residues interconverted the regiospecific oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion. 相似文献
9.
An improved procedure is described for the recovery and purification of the coenzyme A-synthesizing protein complex (CoA-SPC) of Saccharomyces cerevisiae (bakers' yeast). The molecular mass of the CoA-SPC, determined prior to and following its purification, is estimated by Sephacryl S-300 size exclusion chromatography to be between 375 000–400 000. Two previously unreported catalytic activities attributed to CoA-SPC have been identified. One of these is CoA-hydrolase activity which catalyzes the hydrolysis of CoA to form 3′,5′-ADP and 4′-phosphopantetheine, and the other is dephospho-CoA-pyrophosphorylase activity which catalyzesa reaction between 4′-phosphopantetheine and ATP to form dephospho-CoA. The dephospho-CoA then reacts with ATP, catalyzed by the dephospho-CoA-kinase. to reform CoA. This sequence of reactions, referred to as the CoA/4′-phosphopantethiene cycle, provides a mechanism by which the 4′-phosphopantetheine can be recycled to form CoA. Each turn of the cycle utilizes two mol of ATP and produces one mol of ADP, one mol of PPi, and one mol of 3′,5′-ADP. Other than the hydrolysis of CoA by CoA-SPC, the 4′-phosphopantetheine for the cycle apparently could be supplied by alternate sources. One alternate source may be the conventional pathway of CoA biosynthesis. Intact CoA-SPC has been separated into two segments. One segment is designated apo-CoA-SPC and the other segment is referred to as the 10 000–15 000 Mr subunit. The 5′-ADP-4′-pantothenic acid-synthetase, 5′-ADP-4′-pantothenylcysteine-synthetase, 5′-ADP-4′-pantothenylcysteine-decarboxylase, and CoA-hydrolase activities reside in the apo-CoA-SPC segment of CoA-SPC. Whereas the dephospho-CoA-kinase and the dephospho-CoA-pyrophosphorylase activities reside in the 10 000–15 000 Mr subunit. Thus, the 10 000–15 000 Mr subunit mimics the bifunctional enzyme complex that catalyzes the final two steps in the conventional pathway of CoA biosynthesis. 相似文献
10.
11.
Alexey A. Zeifman Fedor N. Novikov Victor S. Stroylov Oleg V. Stroganov Ghermes G. Chilov Alexander Y. Skoblov Anatoly I. Miroshnikov Yuri S. Skoblov 《FEBS letters》2014
2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with Ki = 250 μM (95% CI: 106–405 μM) and dose-dependently increased the rate of the ATP hydrolysis with KM = 112 μM (95% CI: 28–195 μM). The kinetic scheme consistent with this experimental data is proposed. 相似文献
12.
Michaël L. Cartron John D. Olsen Melih Sener Philip J. Jackson Amanda A. Brindley Pu Qian Mark J. Dickman Graham J. Leggett Klaus Schulten C. Neil Hunter 《BBA》2014
Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc1–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities. 相似文献
13.
Deoxyribozymes (DNAzymes) are important catalysts for potential therapeutic RNA destruction and no DNAzyme has received as much notoriety in terms of therapeutic use as the Mg2+-dependent RNA-cleaving DNAzyme 10–23 (Dz10–23). As such, we have investigated the synthetic modification of Dz10–23 with a guanidinium group, a functionality that reduces the anionic nature and can potentially enhance the membrane permeability of oligonucleotides. To accomplish this, we synthesized a heretofore unknown phosphoramidite, 5-(N,N′-biscyanoethoxycarbonyl)-guanidinoallyl-2′-deoxyuridine and then incorporated it into oligonucleotides via solid phase synthesis to study duplex stability and its effect on Dz10–23. This particular modification was chosen as it had been used in the selection of Mg2+-free self-cleaving DNAzymes; as such this will enable the eventual comparison of modified DNAzymes that do or do not depend on Mg2+ for catalysis. Consistent with antecedent studies that have incorporated guanidinium groups into DNA oligonucleotides, this guanidinium-modified deoxyuridine enhanced the thermal stability of resulting duplexes. Surprisingly however, Dz10–23, when synthesized with modified residues in the substrate binding regions, was found to be somewhat less active than its non-modified counterpart. This work suggests that this particular system exhibits uniform binding with respect to ground state and transition state and provides insight into the challenge of re-engineering a Mg2+-dependent DNAzyme with enhanced catalytic activity. 相似文献
14.
Yashavanthi Niranjan Daniela Ungureanu Henrik Hammarén Arturo Sanz-Sanz Adrie H. Westphal Jan Willem Borst Olli Silvennoinen Riet Hilhorst 《Analytical biochemistry》2013
Förster resonance energy transfer (FRET) between the fluorescent ATP analogue 2′/3′-(N-methyl-anthraniloyl)-adenosine-5′-triphosphate (MANT–ATP) and enzymes is widely used to determine affinities for ATP–protein binding. However, in analysis of FRET fluorescence data, several important parameters are often ignored, resulting in poor accuracy of the calculated dissociation constant (Kd). In this study, we systematically analyze factors that interfere with Kd determination and describe methods for correction of primary and secondary inner filter effects that extend the use of the FRET method to higher MANT nucleotide concentrations. The interactions of the fluorescent nucleotide analogues MANT–ATP, MANT–ADP [2′/3′-O-(N-methylanthraniloyl) adenosine diphosphate], and MANT–AMP [2′/3′-O-(N-methylanthraniloyl) adenosine monophosphate] with the JAK2 tyrosine kinase domain are characterized. Taking all interfering factors into consideration, we found that JAK2 binds MANT–ATP tightly with a Kd of 15 to 25 nM and excluded the presence of a second binding site. The affinity for MANT–ADP is also tight with a Kd of 50 to 80 nM, whereas MANT–AMP does not bind. Titrations of JAK2 JH1 with nonhydrolyzable ATP analogue MANT–ATP-γ-S [2′/3′-O-(N-methylanthraniloyl) adenosine-5′-(thio)- triphosphate] yielded a Kd of 30 to 50 nM. The methods demonstrated here are applicable to other enzyme–fluorophore combinations and are expected to help improve the analysis of steady-state FRET data in MANT nucleotide binding studies and to obtain more accurate results for the affinities of nucleotide binding proteins. 相似文献
15.
One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar 总被引:1,自引:0,他引:1
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Sakaki N Shimo-Kon R Adachi K Itoh H Furuike S Muneyuki E Yoshida M Kinosita K 《Biophysical journal》2005,88(3):2047-2056
F1-ATPase is a rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α3β3-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the β-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F1 rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F1 molecules that carried a bead duplex on the γ-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 × 107 M−1s−1. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F1 produced the same torque of ~40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme. 相似文献
16.
Seth PP Yu J Allerson CR Berdeja A Swayze EE 《Bioorganic & medicinal chemistry letters》2011,21(4):1122-1125
The synthesis and biophysical properties of R-6′-Me-α-l-LNA, which has a methyl group in the (R) configuration on the 2′,4′-bridging substituent of α-l-LNA, is reported. The synthesis of the uracil nucleobase phosphoramidite was efficiently accomplished in 14 steps and 8 chromatographic purifications starting from a known sugar intermediate. Biophysical evaluation revealed that substitution along the edge of the major groove does not impair the high affinity duplex forming ability of α-l-LNA modified oligonucleotides. 相似文献
17.
Yoshiyuki Hari Takashi Osawa Yutaro Kotobuki Aiko Yahara Ajaya R. Shrestha Satoshi Obika 《Bioorganic & medicinal chemistry》2013,21(14):4405-4412
Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized, and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides preferred single-stranded RNA (ssRNA) to single-stranded DNA (ssDNA) in duplex formation. Both amide- and N-methylamide-modified oligonucleotides led to a significant increase in the binding affinity to ssRNA by up to +4.7 and +3.7 °C of the Tm value per modification, respectively, compared with natural oligonucleotide. In addition, their oligonucleotides showed high stability against 3′-exonuclease. 相似文献
18.
Mizuki Sekiya Ryosuke Hisasaka Atsuko Iwamoto-Kihara Masamitsu Futai Mayumi Nakanishi-Matsui 《Biochemical and biophysical research communications》2014
ATP synthase (F-ATPase) function depends upon catalytic and rotation cycles of the F1 sector. Previously, we found that F1 ATPase activity is inhibited by the dietary polyphenols, curcumin, quercetin, and piceatannol, but that the inhibitory kinetics of curcumin differs from that of the other two polyphenols (Sekiya et al., 2012, 2014). In the present study, we analyzed Escherichia coli F1 ATPase rotational catalysis to identify differences in the inhibitory mechanism of curcumin versus quercetin and piceatannol. These compounds did not affect the 120° rotation step for ATP binding and ADP release, though they significantly increased the catalytic dwell duration for ATP hydrolysis. Analysis of wild-type F1 and a mutant lacking part of the piceatannol binding site (γΔ277–286) indicates that curcumin binds to F1 differently from piceatannol and quercetin. The unique inhibitory mechanism of curcumin is also suggested from its effect on F1 mutants with defective β–γ subunit interactions (γMet23 to Lys) or β conformational changes (βSer174 to Phe). These results confirm that smooth interaction between each β subunit and entire γ subunit in F1 is pertinent for rotational catalysis. 相似文献
19.
F1-ATPase is a rotary molecular motor in which the central γ subunit rotates inside a cylinder made of α3β3 subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting γ angle defined as 0°, phosphate is released at ∼200°, and ADP is released during quick rotation between 240° and 320° that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly. 相似文献
20.
Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues
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The base-pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5′-appended acylamido caps that enhance the stability and base-pairing fidelity of oligonucleotides with a 5′-terminal 2′-deoxyadenosine residue using combinatorial synthesis and MALDI-monitored nuclease selections. This provided the residue of 4-(pyren-1-yl)butyric acid as a lead. Lead optimization gave (S)-N-(pyren-1-ylmethyl)pyrrolidine-3-phosphate as a cap that increases duplex stability and base-pairing fidelity. For the duplex of 5′-AGGTTGAC-3′ with its fully complementary target, this cap gives an increase in the UV melting point Tm of +10.9°C. The Tm is 6.3–8.3°C lower when a mismatched nucleobase faces the 5′-terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3′-terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double-terminal mismatches that is 5.7–5.9°C greater than that for the unmodified control duplex. 相似文献