Structural Studies of Heterochiral DNA/DNA,RNA/RNA,AND DNA/RNA Duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

菜用大豆种子活力与DNA,RNA及蛋白质合成的关系

菜用大豆种子随着其活力的下降,对DNA,RNA和蛋白质前体的吸收,以及合成这些大分子的能力都明显下降,已丧失合成DNA和蛋白质能力的失活种子,仍能进行微弱的RNA合成。高活力种子在吸胀初期DNA合成速率较低,然后增加,至16h达高峰;RNA的合成速率在吸胀一开始就很高,在整个吸胀过程中均保持较高水平;蛋白质的合成速率则在开始较高,并随着吸胀过程呈增强趋势。     

The evolutionary transition from RNA to DNA in early cells

Summary The evolution of genetic material can be divided into at least three major phases: first, genomes of nucleic acid-like molecules; secondly, genomes of RNA; and finally, double-stranded DNA genomes such as those present in all contemporary cells. Using properties of nucleic acid molecules, we attempt to explain the evolutionary transition from RNA alone as a cellular informational macromolecule prior to the evolution of cell systems based on double-stranded DNA. The idea that ribonucleic acid-based cellular genomes preceded DNA is based on the following: (1) protein synthesis can occur in the absence of DNA but not of RNA; (2) RNA molecules have some catalytic properties; (3) the ubiquity of purine and pyridine nucleotide coenzymes as well as other similar ribonucleotide cofactors in metabolic pathways; and (4) the fact that the biosynthesis of deoxyribonucleotides always proceeds via the enzymatic reduction of ribonucleotides.The RNA prior to DNA hypothesis can be further developed by understanding the selective pressures that led to the biosynthesis of deoxyribose, thymine, and proofreading DNA polymerases. Taken together these observations suggest to us that DNA was selected as an informational molecule in cells to stabilize earlier RNA-protein replicating systems. These arguments include the facts that (1) the 2-deoxy-containing phosphodiester backbone is more stable in aqueous conditions and in the presence of transition metal ions (such as Zn²⁺) than its ribo-equivalents; (2) the absence of proofreading activity in RNA polymerases leads to a higher rate of mutation in RNA genomes relative to DNA; (3) information in RNA degrades because of the tendency of cytosine to deaminate to uracil and the lack of a correcting enzyme; and (4) UV irradiation produces a larger number of photochemical changes in RNA molecules relative to double-stranded DNA. The absence of atmospheric UV attenuation during the early Earth environment (Hadean and early Archean) would have imposed an intense selection pressure favoring duplex DNA over other genetic information storage systems.If RNA preceded DNA as a reservior of cellular genetic information, then an RNA-replicating oligopeptide must have been one of the earliest protoenzymes from which RNA polymerase presumably evolved. We conclude that RNA polymerases are among the oldest classes of enzymes.     

通过PCR进行长片段DNA的合成

利用PCR合成DNA长片段(Synthesis Large Frament DNA using PCR,SLFD PCR)是一种有效的合成长片段DNA的方法。采用一段已知的500～600bp碱基的DNA片段为PCR模板,根据所要合成的DNA序列可以设计一系列的PCR引物,这些引物都位于模板DNA的5’端,长度为50～60bp,且从5’到3’方向顺序重叠,重叠碱基数目为12～15,全部引物叠加所得到的DNA正是自己所要合成的DNA。这组引物中最3’端的一条含有一个BamH Ⅰ酶切位点,在该位点后面有15碱基与模板DNA5’端一致的序列。另外还设计一条与该模板匹配的下游引物,引物内也含有一个BamH Ⅰ酶切位点。首先采用5’端最右侧的引物与下游引物进行PCR,在PCR进行10个循环后,以此次PCR的产物为下一轮PCR的模板,该轮PCR采用右侧倒数第二个引物为上游引物,下游引物保持不变。采用类似的方法,完成所有的PCR循环,就可以得到所需要合成的DNA长片段。该方法尤其适合100～200碱基左右的长片段DNA的快速合成与克隆。     

SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2′-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)–containing RNA/DNA hybrids, their size (20–25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (M_r = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2′-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.     

Deoxyribonolactone Lesion in DNA: Synthesis of Fluorinated Analogues

Abstract

Mono- and difluorinated derivatives of 2-deoxyribonolactone were synthesized using diastereoselective Reformatski reaction as a key step.     

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07–0.2 (on a scale of 0–1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.     

Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes

Saccharomyces cerevisiae Pif1, an SF1B helicase, has been implicated in both mitochondrial and nuclear functions. Here we have characterized the preference of Pif1 for RNA:DNA heteroduplexes in vitro by investigating several kinetic parameters associated with unwinding. We show that the preferential unwinding of RNA:DNA hybrids is due to neither specific binding nor differences in the rate of strand separation. Instead, Pif1 is capable of unwinding RNA:DNA heteroduplexes with moderately greater processivity compared with its duplex DNA:DNA counterparts. This higher processivity of Pif1 is attributed to slower dissociation from RNA:DNA hybrids. Biologically, this preferential role of the helicase may contribute to its functions at both telomeric and nontelomeric sites.     

Mechanism of Intraparticle Synthesis of the Rotavirus Double-stranded RNA Genome

Rotaviruses perform the remarkable tasks of transcribing and replicating 11 distinct double-stranded RNA genome segments within the confines of a subviral particle. Multiple viral polymerases are tethered to the interior of a particle, each dedicated to a solitary genome segment but acting in synchrony to synthesize RNA. Although the rotavirus polymerase specifically recognizes RNA templates in the absence of other proteins, its enzymatic activity is contingent upon interaction with the viral capsid. This intraparticle strategy of RNA synthesis helps orchestrate the concerted packaging and replication of the viral genome. Here, we review our current understanding of rotavirus RNA synthetic mechanisms.     

Molecular recognition of a DNA:RNA hybrid: sub-nanomolar binding by a neomycin-methidium conjugate

A novel neomycin–methidium conjugate was synthesized. The covalent linkage of the aminoglycoside to an intercalator, a derivative of ethidium bromide, results in a new conjugate capable of selective recognition of the DNA:RNA hybrid duplex. Spectroscopic methods: UV, CD, fluorescence, and calorimetric techniques: DSC and ITC were used to characterize the sub-nanomolar binding displayed by the conjugate for the DNA:RNA hybrid duplex, poly(dA):poly(rU).     

Effects of Karenia brevis diet on RNA:DNA ratios and egg production of Acartia tonsa

Karenia brevis is a harmful alga associated with deleterious effects on zooplankton, but the exact cause (e.g. toxin, nutritional inadequacy or starvation) of these adverse effects is not clear. RNA:DNA ratios, fecundity and fecal pellet production of Acartia tonsa were measured on mono-algal and mixed-algal culture diets of K. brevis and Peridinium foliaceum to examine the usefulness of RNA:DNA ratios as an indicator of nutrition and to determine if adverse effects of K. brevis are due to the presence of toxins, poor nutritional quality or starvation. RNA:DNA ratios and egg production values were significantly higher for 100% P. foliaceum diet compared to 100% K. brevis diet. Significant differences in egg production, but not RNA:DNA ratios, were found between the various mixed diets, suggesting egg production is a more sensitive indicator of nutritional quality than RNA:DNA ratios. Changes in RNA:DNA ratios, fecundity and fecal pellet production of copepods fed two different toxic K. brevis strains were nearly identical, indicating that the presence of brevetoxins has little affect on A. tonsa. The similarity in RNA:DNA ratios, egg production, percent hatching and fecal production between the 100% K. brevis diet and starved copepods suggests that A. tonsa does not consume K. brevis when offered as its sole food source.     

迟钝爱德华氏菌RNA提取及痕量基因组DNA去除方法探究

迟钝爱德华氏菌EIB202是一类细胞壁结构特殊的革兰氏阴性菌,高质量RNA提取相对较难。为了从转录组水平研究这类致病菌的致病机理,需要摸索有效的RNA提取及RNA样品中痕量基因组DNA去除方法。对常规RNA提取步骤进行改进,增加PBS清洗、反复冻融及较高浓度溶菌酶处理等步骤;另外,利用小体系基因组DNA去除系统,Mg2+与Mn2+协同激活DNase I去除RNA样品中基因组DNA污染。利用优化方法提取的RNA在质量及浓度(1 740 ng/μL)方面均有了显著改善,并建立了一套完全去除RNA样品中痕量基因组DNA污染的程序。     

The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.     

Relative flexibility of DNA and RNA: a molecular dynamics study

State of the art molecular dynamics simulations are used to study the structure, dynamics, molecular interaction properties and flexibility of DNA and RNA duplexes in aqueous solution. Special attention is paid to the deformability of both types of structures, revisiting concepts on the relative flexibility of DNA and RNA duplexes. Our simulations strongly suggest that the concepts of flexibility, rigidity and deformability are much more complex than usually believed, and that it is not always true that DNA is more flexible than RNA.     

Qualitative and Quantitative Studies on DNA and RNA Synthesis in Loxodes striatus Nuclei*

SYNOPSIS. In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei are differentiated morphologically into a clearly recognizable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a ³H-labeled precursor ([³H]thymidine for DNA and [³H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [³H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [³H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macro-nucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components of the nuclear complex were investigated. (F) Contrary to the previously reported information our results suggest that DNA synthesis can occur in adult macronuclei. The possible explanation of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.     

The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.