Extracellular Circulating Nucleic Acids in Human Plasma in Health and Disease

The concentration of extracellular DNA and RNA in blood plasma of healthy donors, trauma patients, patients with breast and lung cancer, nonmalignant breast tumors and nonmalignant lung diseases were estimated. Significant amounts of extracellular RNA were found in plasma of trauma patients. The concentration of DNA and RNA in plasma of trauma patients correlates with the extent of posttraumatic organ failure. Extracellular RNA was not found in the plasma of breast cancer patients and patients with nonmalignant breast tumors, whereas a very high concentration of extracellular RNA was found in patients with malignant and nonmalignant diseases of lung.     

Plasma Processing Conditions Substantially Influence Circulating microRNA Biomarker Levels

Circulating, cell-free microRNAs (miRNAs) are promising candidate biomarkers, but optimal conditions for processing blood specimens for miRNA measurement remain to be established. Our previous work showed that the majority of plasma miRNAs are likely blood cell-derived. In the course of profiling lung cancer cases versus healthy controls, we observed a broad increase in circulating miRNA levels in cases compared to controls and that higher miRNA expression correlated with higher platelet and particle counts. We therefore hypothesized that the quantity of residual platelets and microparticles remaining after plasma processing might impact miRNA measurements. To systematically investigate this, we subjected matched plasma from healthy individuals to stepwise processing with differential centrifugation and 0.22 µm filtration and performed miRNA profiling. We found a major effect on circulating miRNAs, with the majority (72%) of detectable miRNAs substantially affected by processing alone. Specifically, 10% of miRNAs showed 4–30x variation, 46% showed 30-1,000x variation, and 15% showed >1,000x variation in expression solely from processing. This was predominantly due to platelet contamination, which persisted despite using standard laboratory protocols. Importantly, we show that platelet contamination in archived samples could largely be eliminated by additional centrifugation, even in frozen samples stored for six years. To minimize confounding effects in microRNA biomarker studies, additional steps to limit platelet contamination for circulating miRNA biomarker studies are necessary. We provide specific practical recommendations to help minimize confounding variation attributable to plasma processing and platelet contamination.     

利用胎儿核酸进行产前诊断的研究进展

本文分别介绍了孕妇血浆中胎儿DNA和RNA的特性以及近年采利用胎儿DNA和胎儿RNA进行产前基因诊断技术的研究进展,并且着重介绍了其中三种产前基因诊断技术:多重连结探针扩增法(MLPA)、单碱基延伸-质谱分析(SABER-MS)和SNP-RNA等位基因比率法。综述了利用胎儿核酸进行产前诊断技术在临床上的应用。     

14种蜂花粉的DNA和RNA分析

本文使用紫外光吸收法,分析了芝麻、葵花、泡桐等１４种蜂花粉中核酸（ＤＮＡ和ＲＮＡ）的含量,以进一步探讨花粉的抗衰老作用。结果说明不论那一种花粉,均含有丰富的核酸,但种类不同的花粉其中核酸的含量是不完全相同的。     

血浆中游离核酸:分子诊断的新靶点

1948年Mandel和Metais首次报道在病人的血浆中有游离形式的核酸存在,当时并未引起人们的广泛关注,很长一段时间对于这些核酸的研究处于搁浅状态;如今,人们已经能够在血浆中检测得到肿瘤源﹑胎儿源以及移植物源的核酸,不仅可以将其应用于胎儿性别﹑RHD血型、Y染色体连锁的遗传疾病的诊断与预防,还可以应用这些游离形式的核酸检测肿瘤﹑癌症的发生以及骨髓移植后嵌合状态的研究。     

Synthesis and Nucleic Acids Binding Properties of Diastereomeric Aminoethylprolyl Peptide Nucleic Acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the “cis-” homothymine aepPNA decamers with (2′R,4′R) and (2′S,4′S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2′R,4′S) and (2′S,4′R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2′R,4′R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

Nucleic Acids of Entamoeba histolytica

This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 178 (0.8 kDa) and 58 rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 258 rRNA: guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 28 RNA relative to 178 RNA. The 258 RNA is “nicked” (apparently during nuclear processing) and dissociates readily into 1 78 (0.7 kDa) and 168 (0.6 kDa) species, and a more rigidly bound 5.88 species. A small amount of “unnicked” 258 RNA was recovered with guanidine. Two DNA-dcpendent RNA polymerases (I and II) with a pronounced preference for denatured DNA as template were eluted from DEAE-Sephadex in reverse order of what occurs in other eukaryotes, except Physarum polycephalum. This conclusion was based on salt optima and alpha-amanitin sensitivity studies. Initial characterization of DNA isolated with a procedure capable of isolating > 100-kbp Leishmania DNA showed that undigested DNA migrates as a broad band between markers 6 and 24 kbp. The persistent recovery of such a “band” by us and Perez-Mutul et al. no larger than ca. 24 kbp (with the exception of >48 kbp DNA isolated by Hernandez et al. using an in situ lysis technique which did not include a proteinase) may be due to nicks introduced during isolation; or, perhaps much of the amebal DNA exists in vivo as gene sized fragments. However, preliminary data generated using orthogonal pulse-field agarose gel electrophoresis do suggest that amebal DNA may be in small chromosomes.     

Isolation and Analysis of the Nucleic Acids and Polysaccharides from Clostridium welchii

A method previously described for the use of bentonite in the isolation of the nucleic acids from two gram-positive organisms was applied to the isolation of the nucleic acids from two strains of Clostridium welchii. The nucleic acids were separated from polysaccharides by the fractional precipitation of their cetyltrimethyl-ammonium salts from sodium chloride solution, and the base composition of the nucleic acids was determined. One strain of C. welchii investigated (NCTC 10578) was shown to produce considerable quantities of an acidic and also a weakly acidic or neutral polysaccharide; the other strain (ATCC 10543) gave very small quantities of the latter but none of the former polysaccharide. The monosaccharide composition of these polysaccharides was determined and the acidic polysaccharide was shown to resemble dermatan sulfate.     

Experimental and Theoretical Volumes of Nucleic Acids

Abstract

Further evidence of the need to incorporate both experimental and theoretical results in estimates of the volumes of molecules is presented. The volumes of a series of alkyllated purines and pyrimidines, with experimentally known volumes, were estimated by van der Waals and Semi-empirical quantum mechanical methods. In all instances, the van der Waals methods failed to reproduce the experimental volumes. The Semi-empircal methods were able to reproduce the experimental volumes consistentally.     

Circuit Topology of Proteins and Nucleic Acids

1. Download : Download high-res image (231KB)
2. Download : Download full-size image

     

Intercalating Nucleic Acids: The Influence of Linker Length and Intercalator Type on Their Duplex Stabilities

Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.     

The Enzymatic Phosphorylation of Nucleic Acids and Its Application to End-Group Analysis

Polynucleotide kinase catalyzes the transfer of a phosphate group from ATP to the 5'-hydroxyl termini of polynucleotides. Selective labeling of the 5'-hydroxyl termini of DNA with polynucleotide kinase has been used to study the number and the identity of the 5'-terminal residues of bacteriophage DNA's, and to examine the nature of the phosphodiester bond cleavages produced by endonucleases and by sonic irradiation. The intact strands of T7 DNA bear 5'-phosphoryl end-groups; only deoxyadenylate and deoxythymidylate are present as 5'-terminal residues. The intact strands of native λ-DNA bear 5'-hydroxyl end-groups. M13 DNA, a circular molecule, cannot be phosphorylated. End-group labeling of DNA provides a method for determination of molecular weight; calibration against other DNA preparations is not required. The molecular weight of a single strand of T7 DNA, determined by end-group labeling, is 13.1 x 10⁶; the molecular weight of a single strand of λ-DNA is 16.0 x 10⁶. These values are in agreement with molecular weight estimates by sedimentation analysis and electron microscopy. Sonic irradiation of DNA has been shown to favor the production of polynucleotides terminated by 5'-phosphomonoester groups. All four deoxyribonucleotides are present as 5'-terminal residues of sonicated DNA.     

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer.Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample.     

Structural Pre-organization of Peptide Nucleic Acids

Abstract

Introduction of constraint via chemical bridging in the aegPNA leads to the five or six membered cyclic structures that may contribute towards maintaining the balance between rigidity and flexibility of the PNA backbone. The significant promise of our approach to use the naturally occurring trans-4-hydroxy-L-proline to arrive at different chirally pure cyclic PNA analogs and their DNA binding properties will be presented.     

Study on the Interaction Between Nucleic Acids and Acetamiprid

Abstract

The interaction between deoxyribonucleic acid (DNA) and acetamiprid was studied. It was found that the fluorescence of acetamiprid could be enhanced in the presence of DNA in sulfuric acid solution. The excitation and emission wavelength of acetamiprid was 291 nm and 587 nm, respectively. Under optimal conditions, the calibration graph is over the range of 0.1–10 µg mL^?1. The calibration limit is 0.06 µg mL^?1 (S/N = 3). The determination results of DNA in yeast cell and golden staphylococcus samples by this method were satisfactory. The mechanism of the reaction is discussed.     

The Resurgence of Acyclic Nucleic Acids

This review examines acyclic nucleoside analogs as therapeutic agents, potential progenitor candidates to RNA, and novel building blocks for nucleic‐acid nanotechnology. Together, these areas of research provide new insights into the structural and functional properties of nucleic acids and suggest new paradigms for nucleic acid self‐assembly.