Aldehydic Oligonucleotide: A Key Intermediate for the Preparation of Oligonucleotide Conjugates Through Oxime Bond Formation

Oligonucleotides functionalized with an aldehyde group are the key intermediates used for the preparation of peptide-oligonucleotide conjugates through the formation of an oxime linkage. Herein, we describe a brief overview of various synthetic protocols developed in our laboratory for the preparation of aldehyde containing oligonucleotides and their subsequent conjugation with peptides.     

Picomole Syntheses of High Quality Oligonucleotide Primers in Combination with the Preparation of Oligonucleotide Arrays

Abstract

The establishment of a new synthesis procedure for the preparation of oligonucleotide arrays is described. A modified phosphoramidite chemistry allowed the in situ synthesis of oligomer arrays on specially derivatized polypropylene membranes which can be used both for hybridisation experiments and for the isolation of the individual oligonucleotides.     

Mast Cells Contain Large Quantities of Secretagogue-Sensitive N-Acetylaspartate

Abstract: Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve-mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide-containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N -acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease.     

Evaluation of Sulfur-Transfer Reagents for Large Scale Synthesis of Oligonucleotide Phosphorothioate Analogues

Abstract

Several sulfur-transfer reagents have been evaluated for large scale synthesis of oligonucleotide phosphorothioate analogues, in which 3-ethoxy-1, 2-dithiazoline-5-one (EDITH, 5) shows potential as an alternative to Beaucage reagent.     

Improved Synthesis of Trinucleotide Phosphoramidites and Generation of Randomized Oligonucleotide Libraries

A new method to produce a set of 20 high quality trinucleotide phosphoramidites on a 5–10 g scale each was developed. The procedure starts with condensation reactions of P-components with N-acyl nucleosides, bearing the 3 ′-hydroxyl function protected with 2-azidomethylbenzoyl, to give fully protected dinucleoside phosphates 13. Upon cleavage of dimethoxytrityl group from 13, dinucleoside phosphates 16 are initially transformed into trinucleoside diphosphates 19 and then the 2-azidomethylbenzoyl is selectively removed under neutral conditions to generate trinucleoside diphosphates 5 in excellent yield. Subsequent 3 ′-phosphitylation affords target trinucleotide phosphoramidites 7. When mutagenic oligonucleotides are synthesized employing mixtures of building blocks 7 as well as following the new synthetic protocol, representative oligonucleotide libraries are generated in good yields.     

Procedure for the Preparation of Milligram Quantities of Adenovirus Messenger Ribonucleic Acid

Late after adenovirus 2 infection (18 hr), nearly all newly synthesized polysomal messenger ribonucleic acid (mRNA) is viral specified. Large amounts of adenovirus mRNA have been purified by utilizing membrane filtration at high ionic strength. With this procedure, molecules that contain polyadenylic acid [poly (A)] tracts are bound selectively, and ribosomal RNA can be separated from the viral mRNA which contains poly(A). Polysomal RNA synthesized 18 hr after infection was labeled in the presence of 0.02 mug of actinomycin D per ml and extracted at pH 9.0. This RNA annealed 40% to 3 mug of adenovirus 2 deoxyribonucleic acid; the RNA selected by membrane filtration bound 80% under the same conditions. The RNA eluted from membrane filters was 80 to 90% greater than 18S and contained species migrating as 31, 27, and 24S. Binding of polysomal RNA to individual membrane filters was linear, using as much as 300 mug of RNA per membrane. A 1.1-mg amount of viral RNA was prepared from 17.7 mg of polysomal RNA that had been purified by extraction at pH 9.0.     

鱼类LZF-IDNA指纹探针的制备

利用Ｏｌｉｇｏ１０００ＤＮＡ合成仪（Ｂｅｃｋｍａｎ）合成了长度为４５ｂｐ的寡核苷酸单链,经纯化后用同位素γ－３２Ｐ－ＡＴＰ作５′末端标记后,制备成鱼类ＬＺＦ－ＩＤＮＡ指纹探针。通过对鱼类的群体实验、亲子鉴定实验、组织细胞的稳定性实验和鱼类种类的适用范围实验后,测得：（１）ＬＺＦ－ＩＤＮＡ指纹探针属多位点寡核苷酸探针;（２）ＬＺＦ－ＩＤＮＡ指纹探针在鱼类种群中的鉴别机率为９．２３×１０－１６;（３）ＬＺＦ－ＩＤＮＡ指纹探针在鱼类亲子鉴定实验中的父系概率为０．９９９９６２;（４）ＬＺＦ－ＩＤＮＡ指纹探针,是一种稳定的,既具有个体识别能力,又具有一定种属特异性的、适用于鱼类ＤＮＡ指纹图研究的基因指纹探针。     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.     

A Rapid Method for the Preparation of a One Dimensional Sequence-Overlapping Oligonucleotide Library

Abstract

A new synthesis modul was constructed to prepare one-dimensional libraries of sequentally overlapping oligonucleotides on surface modified polypropylene tapes. The use of such a library for the diagnostic screening of a point mutation in the regulatory protein gene p53 is described.     

Comparison of Complex DNA Mixtures with Generic Oligonucleotide Microchips

Abstract

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to VD-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.     

用于寡核苷酸芯片制备载体的醛基载玻片的制备方法优化及性质研究

优化了醛基载玻片的制备方法 ,探讨了醛基修饰载玻片固定寡核苷酸探针的性质。研究发现氨基硅烷试剂的浓度是影响载玻片荧光背景的主要因素 ;2 %氨基化试剂处理 16min、戊二醛处理 30min可以得到荧光背景较低、固定效果较好的醛基载玻片。寡核苷酸固定过程中 ,末端氨基修饰没有明显的特异性 ,但是可以提高被固定探针的杂交容量。在较低的浓度 (小于 10 μmol L)时 ,探针的浓度与杂交信号趋近线性关系 ,浓度为 2 0 μmol L时杂交信号达到饱和     

A Model for the Complex Between the Helix Destabilizing Protein GP32 of Bacteriophage T4 and Single-Stranded DNA

Abstract

A model for the structure of the complex between the helix-destabilizing protein of bacteriophage T4, GP32, and single-stranded DNA is proposed. In this model the bases are arranged in a helix, that is characterized by a relatively large distance between successive bases, a substantial base tilt, in combination with a small rotation per base. This helix is further organized into a tertiary structure, possibly a superhelix, of which the corresponding protein shell corresponds to the relatively rigid and rod-like structure that is observed in hydrodynamic experiments. It is proposed that similar structural features apply to other single-stranded DNA binding proteins in complex with polynucleotides.     

A New Approach to the Synthesis of Trinucleotide Phosphoramidites - Synthons for the Generation of Oligonucleotide/Peptide Libraries

Abstract

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites - trinucleotide phosphotriesters - have been prepared using the phosphotriester approach without protection of the 3′-hydroxyl function. More than 10 oligonucleotides up to 90 bases long have been synthesized by a phosphite-triester approach using new synthons. The 67-mer (12 random codons) has been used to generate a display library of 2 × 10⁸ complexity.     

Online Community Detection for Large Complex Networks

Complex networks describe a wide range of systems in nature and society. To understand complex networks, it is crucial to investigate their community structure. In this paper, we develop an online community detection algorithm with linear time complexity for large complex networks. Our algorithm processes a network edge by edge in the order that the network is fed to the algorithm. If a new edge is added, it just updates the existing community structure in constant time, and does not need to re-compute the whole network. Therefore, it can efficiently process large networks in real time. Our algorithm optimizes expected modularity instead of modularity at each step to avoid poor performance. The experiments are carried out using 11 public data sets, and are measured by two criteria, modularity and NMI (Normalized Mutual Information). The results show that our algorithm''s running time is less than the commonly used Louvain algorithm while it gives competitive performance.     

Mass Culturing of Ditylenchus dipsaci to Yield Large Quantities of Inoculum

Methods are described for rearing large quantities of Ditylenchus dipsaci on alfalfa tissues. Nematodes and alfalfa seed were disinfected and nematodes were reared in quantities sufficient to provide a continuous supply of inoculum for our alfalfa-breeding program. Nematodes reproduced best in darkness at 20-25 C. Cultures reached maximum numbers in 3-6 wk.     

小鼠细胞因子相关基因表达检测寡核苷酸芯片的制备及分析

生物芯片技术用于基因表达谱研究是近年来发展起来的一项新技术 ,该方法本质上是基于对一玻璃片或膜表面上固定的ｃＤＮＡ或寡核苷酸的分子杂交 ,这一新技术可同时测定成千上万个基因的作用方式 ,几周获得的信息用其它方法可能要几年才能得到 ,是以定量方式同时监测大量基因相对表达的强有力的新方法[1 ,2 ] 。国内外目前主要采用ｃＤＮＡ芯片进行基因表达的检测 ,芯片制备所用的ＤＮＡ探针一般为已知基因ｃＤＮＡ克隆的ＰＣＲ扩增产物或ＥＳＴ的扩增产物[3～ 8] 。对基因的表达检测来说 ,ｃＤＮＡ芯片技术是一条非常适用的检测方法 ,但在有…     

A Novel Technique for Efficient Construction of Large scFv Libraries

The development of patient-tailored anti-tumor therapies requires large-scale production of antibodies for the purpose of screening specific tumor antibodies. Thus, the generation of a single chain fragment of variable region (scFv) library with a large repertoire is required for the selection of specific antibodies with high affinity. Presently, the generation of large scFv libraries is impeded by the low efficiency of cloning PCR fragments into phage display vectors, which is due to the low efficiency of the restriction digestion step required in the process. The aim of this study was to increase the efficiency of this critical step. We found methods that are formally believed to facilitate the digestion efficiency, such as adding longer tails to the primers or prolonging the incubation time, were inefficient. We then investigated the feasibility of using T/A cloning to improve the efficiency of cloning and found that when PCR fragments were first cloned into T-vector, and then subsequently subcloned into a phagemid, the cloning efficiency was dramatically increased. Our findings show that by utilizing this method the construction of a large scFv library can be easily accomplished. Aizhi Zhao is the co-first author.