Pentopyranosyl Oligonucleotide Systems,Communication No. 10 , The α‐L‐Lyxopyranosyl‐(4′→2′)‐oligonucleotide System

To determine whether the remarkable chemical properties of the pyranosyl isomer of RNA as an informational Watson‐Crick base‐pairing system are unique to the pentopyranosyl‐(4′→2′)‐oligonucleotide isomer derived from the RNA‐building block D ‐ribose, studies on the entire family of diastereoisomeric pyranosyl‐(4′→2′)‐oligonucleotide systems deriving from D ‐ribose, L ‐lyxose, D ‐xylose, and L ‐arabinose were carried out. The result of these extended studies is unambiguous: not only pyranosyl‐RNA, but all members of the pentopyranosyl‐(4′→2′)‐oligonucleotide family are highly efficient Watson‐Crick base‐pairing systems. Their synthesis and pairing properties will be described in a series of publications in this journal. The present paper describes the α‐L ‐lyxopyranosyl‐(4′→2′)‐system.     

Pentopyranosyl Oligonucleotide Systems. Communication No. 13

Among the members of a family of diastereoisomeric pentopyranosyl‐(4′→2′)‐oligonucleotide systems derived from D ‐ribose, D ‐xylose, L ‐lyxose, and L ‐arabinose, the α‐arabinopyranosyl system shows by far the strongest Watson? Crick base pairing. The system is, in fact, one of the strongest oligonucleotide‐type base‐pairing systems known. It undergoes efficient cross‐pairing with all the other members of the pentopyranosyl family, but not with RNA and DNA. The paper describes the synthesis and pairing of the properties of α‐L ‐arabinopyranosyl‐(4′→2′)‐oligonucleotides.     

寡聚核苷酸/单链阳离子表面活性剂囊泡与沉淀共存(英文)

DNA(包括寡聚核苷酸)和阳离子表面活性剂可形成难溶复合物.本文通过浊度测试和透射电子显微镜观察,发现单链阳离子表面活性剂可以诱使寡聚核苷酸/单链阳离子表面活性剂沉淀转变成为寡聚核苷酸/单链阳离子表面活性剂囊泡,且寡聚核苷酸/单链阳离子表面活性剂囊泡可以与寡聚核苷酸/单链阳离子表面活性剂沉淀共存.在寡聚核苷酸/单链阳离子表面活性剂沉淀向囊泡的转变过程中,表面活性剂和沉淀之间的疏水作用力发挥了重要作用.此外,当体系温度达到寡聚核苷酸开始融解的温度后,寡聚核苷酸/单链阳离子表面活性剂体系更容易形成囊泡.因此,寡聚核苷酸的链越伸展,越易于寡聚核苷酸/单链阳离子表面活性剂囊泡的生成.据我们所知,有关寡聚核苷酸/阳离子表面活性剂囊泡的报道尚不多见.因此,考虑到DNA(包括寡聚核苷酸)/两亲分子体系在医学、生物学、药学和化学中的重要性,该研究应该有助于我们进一步了解该体系并对其进行更合理有效的应用.     

Bridging the Two Worlds: A Universal Interface between Enzymatic and DNA Computing Systems

Molecular computing based on enzymes or nucleic acids has attracted a great deal of attention due to the perspectives of controlling living systems in the way we control electronic computers. Enzyme‐based computational systems can respond to a great variety of small molecule inputs. They have the advantage of signal amplification and highly specific recognition. DNA computing systems are most often controlled by oligonucleotide inputs/outputs and are capable of sophisticated computing as well as controlling gene expressions. Here, we developed an interface that enables communication of otherwise incompatible nucleic‐acid and enzyme‐computational systems. The enzymatic system processes small molecules as inputs and produces NADH as an output. The NADH output triggers electrochemical release of an oligonucleotide, which is accepted by a DNA computational system as an input. This interface is universal because the enzymatic and DNA computing systems are independent of each other in composition and complexity.     

Synthesis of new OBAN's and further studies on positioning of the catalytic group

Two new zinc ion dependent oligonucleotide based artificial nucleases (OBAN's) have been synthesized. These consist of 2'-O-methyl modified RNA oligomers conjugated to 5-amino-2,9-dimethylphenanthroline (neocuproine)via a urea linker. OBAN 4 carries the catalytic group on a linker extending from the C-4 of an internal cytosine moiety. OBAN 5 has two neocuproine units attached, each to linkers extending from the C-5 position of uridine moieties, one placed internally and the other at the at the 5'-end of the oligonucleotide. The key step in the synthesis of the OBAN systems is conjugation of the catalytic group to the respective amino linkers of the modified oligonucleotides. This is achieved by first converting the 5-amino-2,9-dimethylphenanthroline to the phenylcarbamate. The reaction of this neocuproine phenylcarbamate with the oligonucleotide carrying one or two primary aliphatic amines in aqueous buffer (at pH 8.5) leads to nearly quantitative formation of the urea-linked conjugates. Both OBAN systems were found to cleave RNA in the bulged out regions formed from the non-complementary part of the target sequences, in the presence of Zn(II) ions. Differences in efficiency between these and previously reported systems are discussed.     

Optical detection and discrimination of cystic fibrosis-related genetic mutations using oligonucleotide–nanoparticle conjugates

Novel methods for application of oligonucleotide–gold nanoparticle conjugates to selective colorimetric detection and discrimination of cystic fibrosis (CF) related genetic mutations in model oligonucleotide systems are presented. Three-strand oligonucleotide complexes are employed, wherein two probe oligonucleotide–gold nanoparticle conjugates are linked together by a third target oligonucleotide strand bearing the CF-related mutation(s). By monitoring the temperature dependence of the optical properties of the complexes, either in solution or on silica gel plates, melting behaviors may be accurately and reproducibly compared. Using this approach, fully complementary sequences are successfully distinguished from mismatched sequences, with single base mismatch resolution, for F 508, M470V, R74W and R75Q mutations.     

Cationic oligonucleotide-peptide conjugates with aggregating properties enter efficiently into cells while maintaining hybridization properties and enzymatic recognition

Oligonucleotide delivery is a crucial issue for therapeutical purposes and is often addressed by conjugation to short cationic peptides although with controversial results. To further examine this mechanism, a 15-mer anionic oligonucleotide was conjugated to a cationic peptide in order to obtain a diblock compound with an overall positive charge with aggregation properties. These microaggregates were efficiently internalized in cells via the expeditious pathway used by commercial gene delivery systems. Moreover, stability of the duplex formed with the complementary sequence increased without inhibiting oligonucleotide enzyme recognition as shown by the properties of the conjugate to prime chain elongation by Taq DNA polymerase in a linear amplification/sequencing process.     

Diffusion of Oligonucleotides from within Iron‐Cross‐Linked,Polyelectrolyte‐Modified Alginate Beads: A Model System for Drug Release

An analytical model to describe diffusion of oligonucleotides from stable hydrogel beads is developed and experimentally verified. The synthesized alginate beads are Fe³⁺‐cross‐linked and polyelectrolyte‐doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross‐linking, that is, the absence of a surface‐layer barrier in this case. Furthermore, the results suggest a new simple approach to measuring the diffusion coefficient of mobile oligonucleotide molecules inside hydrogels. The considered alginate beads provide a model for a well‐defined component in drug‐release systems and for the oligonucleotide‐release transduction steps in drug‐delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate, which induces full oligonucleotide release with nondiffusional kinetics.     

Synthesis of fluorescent oligonucleotide--EYFP conjugate: towards supramolecular construction of semisynthetic biomolecular antennae

A novel species of DNA--protein conjugate was synthesized by chemically linking DNA oligonucleotides to Aequorea victoria green fluorescent protein mutant EYFP. An additional cysteine was added to the C-terminus of the EYFP by genetic engineering and used to covalently attach amino-modified oligonucleotide with the aid of the heterobifunctional crosslinker sSMCC. EYFP maintained its fluorescence upon conjugation. The oligonucleotide provides an additional binding site to the fluorescent protein, and hence, the EYFP conjugate could be specifically hybridized with both complementary DNA-protein conjugates in-solution as well as immobilized at capture oligonucleotides attached to a solid substrate. These studies are paving the way for future applications in the self-assembly of photoactive supramolecular complexes, such as artificial light-harvesting systems.     

Fluorescence detection of potassium ion using the G-quadruplex structure

Oligonucleotides with sequences of human telomere DNA or thrombin binding aptamer (TBA) are known to form tetraplex structures upon binding the K(+) ion. Structural changes associated with the formation of tetraplex assemblies led to the development of potassium-sensing oligonucleotide (PSO) probes, in which two fluorescent dyes were attached to both termini of particular oligonucleotide. The combination of dyes included fluorescence resonance energy transfer (FRET) and excimer emission approaches, and the structural changes upon binding K(+) ion could be monitored by a fluorescence technique. These systems showed a very high preference for K(+) over Na(+) ion, which was suitable for fluorescence imaging of the potassium concentration gradient in a living cell. In the case of human telomere DNA, it was also possible to follow the polymorphism of its tetraplex structures.     

Oligonucleotide‐Based Luminescent Detection of Metal Ions

Metal ions are prevalent in biological systems and are critically involved in essential life processes. However, excess concentrations of metals can pose a serious danger to living organisms. Oligonucleotides represent a versatile sensing platform for the detection of various molecular entities including metal ions. This review summarizes the recent advances in the development of oligonucleotide‐based luminescent detection methods for metal ions.     

Rigid polymerics: the future of oligonucleotide analysis and purification

A family of rigid macroporous HPLC materials, reversed phase and anion exchange, has been evaluated for the analysis and purification of a range of de-protected, dimethoxytrityl-off, oligonucleotides. A 25-base pair (bp) double-stranded DNA ladder was used to determine the resolving range for the four pore sizes of reversed-phase media. The 100 A pore size resolves up to 50-75 bp, the 300 A up to 250-300 bp, the 1000 A up to 400-450 bp and the 4000 A pore size is capable of resolving in excess of 500 bp. The dynamic capacity of these four pore sizes was also determined using a synthetic oligonucleotide with two ion-pairing agents at ambient and 60 degrees C. The dynamic capacity was shown to decrease with increasing pore size and that with the triethylammonium acetate ion-pairing agent there was negligible temperature dependency. The dynamic capacity was higher when tetrabutylammonium bromide was used at elevated temperature. A strong anion-exchange functionality on a pH-stable polymeric particle was used to investigate the selectivity and resolution of the technique. Using a poly-T-oligonucleotide size standard, resolution of full length oligonucleotide (n) from the truncated species due to coupling failure (n-1, n-2, etc.) was demonstrated up to at least the 30mer. Resolution of a phospho diester contaminant from a phospho thioate oligonucleotide and a truncated sequence was demonstrated using anion-exchange HPLC at high pH.     

Amphipathic DNA Origami Nanoparticles to Scaffold and Deform Lipid Membrane Vesicles

We report a synthetic biology‐inspired approach for the engineering of amphipathic DNA origami structures as membrane‐scaffolding tools. The structures have a flat membrane‐binding interface decorated with cholesterol‐derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions leading to the formation of ordered arrays on the membrane. Such a tight and regular arrangement makes our DNA origami capable of deforming free‐standing lipid membranes, mimicking the biological activity of coat‐forming proteins, for example, from the I‐/F‐BAR family.     

Switching of the enzymatic activity synchronized with signal recognition by an artificial DNA receptor on a liposomal membrane

We constructed a supramolecular system on a liposomal membrane that is capable of activating an enzyme via DNA hybridization. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, providing a platform for the system, was prepared by the self-assembly of an oligonucleotide lipid, a phospholipid and a cationic synthetic lipid. The enzyme was immobilized on the liposomal surface through electrostatic interactions. Selective recognition of DNA signals was achieved by hybridizing the DNA signals with the oligonucleotide lipid embedded in the liposome. The hybridized DNA signal was sent to the enzyme by a copper ion acting as a mediator species. The enzyme then amplified the event by the catalytic reaction to generate the output signal. In addition, our system demonstrated potential for the discrimination of single nucleotide polymorphisms.     

A Family of “Click” Nucleosides for Metal‐Mediated Base Pairing: Unravelling the Principles of Highly Stabilizing Metal‐Mediated Base Pairs

A family of artificial nucleosides has been developed by applying the Cu^I‐catalyzed Huisgen 1,3‐dipolar cycloaddition. Starting from 2‐deoxy‐β‐D ‐glycosyl azide as a common precursor, three bidentate nucleosides have been synthesized. The 1,2,3‐triazole involved in all three nucleobases is complemented by 1,2,4‐triazole ( TriTri ), pyrazole ( TriPyr ), or pyridine ( TriPy ). Molecular structures of two metal complexes indicate that metal‐mediated base pairs of TriPyr may not be fully planar. An investigation of DNA oligonucleotide duplexes comprising the new “click” nucleosides showed that they can bind Ag^I to form metal‐mediated base pairs. In particular the mispair formed from TriPy and the previously established imidazole nucleoside is significantly stabilized in the presence of Ag^I. A comparison of different oligonucleotide sequences allowed the determination of general factors involved in the stabilization of nucleic acids duplexes with metal‐mediated base pairs.     

Efficient deanilidation of phosphoranilidates by the use of nitrites and acetic anhydride

Reagent systems of sodium– and tetrabutylammonium nitrite–acetic anhydride were proved to be extremely efficient for the deanilidation of nucleoside 3′-phosphoranilidates, whose reactions were rapid with the former and instant with the latter. It was further found that the reagent system is applicable to oligonucleotide synthesis provided that the exocyclic amino groups of 2′-deoxyadenosine, 2′-deoxyguanosine, and 2′-deoxycytidine were protected by succinylation.     

Optimizing processing parameters for signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy

Signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy was achieved by optimizing chip processing parameters. The parameters investigated were fabrication, blocking and guide dot concentration, probe concentration and modification, print buffer, humidity during arraying, slide agitation, spot volume and spotter compatibility. The optimum oligonucleotide concentration was 20 microM, while the optimum protein concentration was 0.05 mg/ml. Amino-modified oligonucleotides were best able to be bound to the resin's epoxy groups at pH 8, whereas thiol-modified oligonucleotides displayed an optimum coupling value of pH 7. So as to avoid background (BG) contamination of probes around bright guide dots, the concentration of fluorescent guide dots was set to 1 muM. The most suitable print buffers for oligonucleotide arrays using both piezo- and contact-printing systems proved to be 3 x SSC/1.5 M betaine and commercial ArrayLink. When 0.01% monochlortriazinyl-beta-cyclodextrin sodium salt (MCT) was added, the hybridization signal doubled in strength as compared to plain buffer. The optimum print buffer for proteins was 0.1 N phosphate buffer, pH 8/10% glycerine. The optimum humidity for arraying oligonucleotides was 60% and for proteins 40%. Initially agitating slides for 15 min was found just as effective as agitating slides over the total hybridization period (2.5 h), and this resulted in a three times stronger signal.     

Structural and kinetic characterization of an acyl transferase ribozyme

We have previously isolated, by in vitro selection, an acyl-transferase ribozyme that is capable of transferring a biotinylated methionyl group from the 3' end of a hexanucleotide substrate to its own 5'-hydroxyl. Comparison of the sequences of a family of evolved derivatives of this ribozyme allowed us to generate a model of the secondary structure of the ribozyme. The predicted secondary structure was extensively tested and confirmed by single-mutant and compensatory double-mutant analyses. The role of the template domain in aligning the acyl-donor oligonucleotide and acyl-acceptor region of the ribozyme was confirmed in a similar manner. The significance of different domains of the ribozyme structure and the importance of two tandem G:U wobble base pairs in the template domain were studied by kinetic characterization of mutant ribozymes. The wobble base pairs contribute to the catalytic rate enhancement, but only in the context of the complete ribozyme; the ribozyme in turn alters the metal binding properties of this site. Competitive inhibition experiments with unacylated substrate oligonucleotide are consistent with the ribozyme acting to stabilize substrate binding to the template, while negative interactions with the aminoacyl portion of the substrate destabilize binding.     

A micropreparative chromatographic strategy for the purification and sequence analysis of murine granulocyte-colony stimulating factor (mG-CSF)

Summary A micropreparative chromatographic strategy using short (<5 cm) 1 and 2 mm ID HPLC columns for preparing pure mG-CSF and its peptide fragments is described. The proteins and peptides are used to obtain both N-terminal and internal amino acid sequence data for the purpose of constructing sequence specific oligonucleotide probes for use in the isolation of the gene coding for the protein of interest, thereby ultimately enabling amplified amounts of protein to be generated using prokaryotic expression systems.     

Effects of oligonucleotide adsorption on the physicochemical characteristics of a nanoparticle-based model delivery system for antisense drugs

Cationic polystyrene nanoparticles, as a model drug carrier system for nucleic acids, are capable of binding negatively charged oligonucleotides by multiple electrostatic interactions. The effect of the adsorption of phosphorothioate oligonucleotides on the physicochemical properties of the carrier system was investigated for uncoated and sterically stabilized latex particles. Turbidity measurements and photon-correlation spectroscopy indicate that the colloidal stability of the nanoparticle-oligonucleotide conjugates is influenced by the number of oligonucleotides adsorbed on the carrier. Especially in the case of the uncoated material, a destabilizing effect has been observed up to oligonucleotide concentrations of 2.7 μmol/g polymer. Strikingly, at higher concentrations the latexes exhibit colloidal stability similar to the oligonucleotide-free samples. These results were correlated to zeta-potential measurements demonstrating a reversal from positive to negative values of the zeta potential with increasing oligonucleotide concentration. The points of zero charge of the particles are in the region of maximum coagulation. These findings were compared to adsorption studies and calculations based on the random sequential adsorption model. It appears that at first the colloidal stability of the carrier systems is diminished with increasing oligonucleotide adsorption, while higher surface coverages lead to a significant reduction in coagulation. At the saturation level the surface coverage can be considered as a monolayer of “side-on” adsorbed molecules and the conjugates exhibit colloidal stability similar to the bare particles without adsorbed molecules. Received: 20 April 1998 Accepted: 16 July 1998