Artificial ribozymes and deoxyribozymes.

RNA and DNA molecules with catalytic properties have been isolated by in vitro selection from combinatorial nucleic acid libraries. A broad range of chemical reactions is catalyzed and nucleic acids can accelerate bond formation between small organic substrates. The catalytic performance of nucleic acids can be enhanced by the incorporation of additional functional groups.     

功能核酸概念的内涵与外延

对功能核酸概念的分析需要建立在对功能核酸研究的基础上,从内涵和外延两个方面来进行探析。从内涵来看,它是对具有特殊结构、执行特定生物功能的核酸分子的统称;从外延来看,它包括适体、核酸核酶、核糖开关、发光核酸、修饰核酸、功能核酸裁剪、核酸自组装、功能核酸纳米材料、核酸纳米酶、核酸药物、核酸补充剂以及DNA存储技术等。目前功能核酸已成功地应用于生物传感、生物成像、生物医学等诸多领域。对功能核酸这一概念进行了探讨,并尝试对其范畴、特点进行归纳总结,以期梳理和完善功能核酸的基本概念,促进该领域的进一步发展。     

Generation and selection of ribozyme variants with potential application in protein engineering and synthetic biology

Over the past two decades, RNA catalysis has become a major topic of research. On the one hand, naturally occurring ribozymes have been extensively investigated concerning their structure and functional mechanisms. On the other hand, the knowledge gained from these studies has been used to engineer ribozyme variants with novel properties. In addition to RNA engineering by means of rational design, powerful techniques for selection of ribozymes from large pools of random sequences were developed and have been widely used for the generation of functional nucleic acids. RNA as catalyst has been accompanied by DNA, and nowadays a large number of ribozymes and deoxyribozymes are available. The field of ribozyme generation and selection has been extensively reviewed. With respect to the field of biotechnology, RNA and DNA catalysts working on peptides or proteins, or which are designed to control protein synthesis, are of utmost importance and interest. Therefore, in this review, we will focus on engineered nucleic acid catalysts for peptide synthesis and modification as well as for intracellular control of gene expression.     

A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes

CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection.     

The evolution of bat nucleic acid‐sensing Toll‐like receptors

We characterized the nucleic acid‐sensing Toll‐like receptors (TLR) of a New World bat species, the common vampire bat (Desmodus rotundus), and through a comparative molecular evolutionary approach searched for general adaptation patterns among the nucleic acid‐sensing TLRs of eight different bats species belonging to three families (Pteropodidae, Vespertilionidae and Phyllostomidae). We found that the bat TLRs are evolving slowly and mostly under purifying selection and that the divergence pattern of such receptors is overall congruent with the species tree, consistent with the evolution of many other mammalian nuclear genes. However, the chiropteran TLRs exhibited unique mutations fixed in ligand‐binding sites, some of which involved nonconservative amino acid changes and/or targets of positive selection. Such changes could potentially modify protein function and ligand‐binding properties, as some changes were predicted to alter nucleic acid binding motifs in TLR 9. Moreover, evidence for episodic diversifying selection acting specifically upon the bat lineage and sublineages was detected. Thus, the long‐term adaptation of chiropterans to a wide variety of environments and ecological niches with different pathogen profiles is likely to have shaped the evolution of the bat TLRs in an order‐specific manner. The observed evolutionary patterns provide evidence for potential functional differences between bat and other mammalian TLRs in terms of resistance to specific pathogens or recognition of nucleic acids in general.     

Evolutionary dynamics and population control during in vitro selection and amplification with multiple targets

Iterative cycles of in vitro selection and amplification allow rare functional nucleic acid molecules, aptamers, to be isolated from large sequence pools. Here we present an analysis of the progression of a selection experiment that simultaneously yielded two families of RNA aptamers against two disparate targets: the intended target protein (B52/SRp55) and the partitioning matrix. We tracked the sequence abundance and binding activity to reveal the enrichment of the aptamers through successive generations of selected pools. The two aptamer families showed distinct trajectories of evolution, as did members within a single family. We also developed a method to control the relative abundance of an aptamer family in selected pools. This method, involving specific ribonuclease digestion, can be used to reduce the background selection for aptamers that bind the matrix. Additionally, it can be used to isolate a full spectrum of aptamers in a sequential and exhaustive manner for all the different targets in a mixture.     

Nucleic acid carriers based on precise polymer conjugates

Polymer polydispersity, random conjugation of functional groups, and poorly understood structure-activity relationships have constantly hampered progress in the development of nucleic acid carriers. This review focuses on the synthetic concepts for the generation of precise polymers, site-specific conjugation strategies, and multifunctional conjugates for nucleic acid transport. Dendrimers, defined peptide carriers, sequence-defined polyamidoamines assembled by solid-phase supported synthesis, and precise lipopeptides or lipopolymers have been characterized for pDNA and siRNA delivery. Conjugation techniques such as click chemistries and peptide ligation are available for conjugating polymers with functional transport elements such as targeting or shielding domains and for direct covalent modification of therapeutic nucleic acids in a site-specific mode.     

功能核酸DNA水凝胶的制备与组装

功能核酸DNA水凝胶是一种以DNA为构建单元通过化学反应或物理缠结自组装而成的新型柔性材料,其构建单元中包含1种或多种能够形成功能核酸的特定序列。功能核酸是通过碱基修饰和DNA分子之间的相互作用力组合的一类特定核酸结构,包括核酸适配体、DNA核酶、G-四联体(G-quadruplex,G4)和i-motif结构等。传统上,高浓度的长DNA链是制备DNA水凝胶的必要条件,而核酸扩增方法的引入为DNA水凝胶的组装方式提供了新的可能。因此,对常用于制备DNA水凝胶的多种功能核酸以及核酸的提取、合成和扩增手段进行了详细的介绍。在此基础上,综述了通过化学或物理交联方式组装功能核酸DNA水凝胶的制备方法。最后,提出了DNA纳米材料的组装所面临的挑战和潜在的发展方向,以期为开发高效组装的功能核酸DNA水凝胶提供参考。     

Evidence that natural selection acts on silent mutation

Analysis of nucleic acid sequence data of mammalian hemoglobin, yeast cytochrome c, and human interferon reveals strong biases in favor of specific codons. These biases do not appear to dissipate over time, suggesting that an indirect form of selection acts on silent mutations. The data are compatible with the “bootstrapping” hypothesis that silent mutations which alter the rate of evolution can hitchhike with traits whose appearance they facilitate. Selection involving modulating effects of codon usage on gene expression may also be involved, but the data appear to exclude simple maximization of gene expression.     

Bioinformatic Analysis of the Contribution of Primer Sequences to Aptamer Structures

Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust.     

Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.     

Volumetric characterization of the hydration properties of heterocyclic bases and nucleosides

We have determined the partial molar volumes, expansibilities, and adiabatic compressibilities of six heterocyclic nucleic acid bases, five ribonucleosides, and six 2'-deoxyribonucleosides within the temperature range 18-55 degrees C. We interpret the resulting data in terms of the hydration of the component hydrophobic and polar atomic groups. From our temperature-dependent volumetric studies, we found that the total contraction of water caused by polar groups of each individual heterocyclic base and nucleoside depends on the proximity and chemical nature of other functional groups of the solute. In addition, the compressibility contributions of polar groups vary greatly in sign and magnitude depending on the surrounding functional groups. In agreement with previous studies, our results are suggestive of little or no interaction between the sugar and base moieties of a nucleoside. In general, our data shed light into the hydration properties of individual heterocyclic bases and nucleosides, which may have significant implications for the sequence-dependent hydration of nucleic acids. We discuss the potential importance of our results for developing an understanding of the role that solvent plays in the stabilization/destabilization of nucleic acid structures.     

Mutation hotspots in the p53 gene in tumors of different origin: correlation with evolutionary conservation and signs of positive selection

We present a classification analysis of the mutation spectra of the p53 gene and construct maps of hotspots for the germline (Li-Fraumein syndrome), different types of tumors and their derived cell lines. While spectra from solid tumors share common hotspots with the germline spectrum, they also contain unique sets of somatic hotspots that are not observed in the germline. All these hotspots correspond to amino acid replacements in the DNA-binding interface of p53. The mutation spectra of lymphomas and cell lines derived from lymphomas and lung cancers contained few hotspots compared to solid tumors. Thus, the distribution of hotspots in the p53 gene appears to depend on the tumor type and cell growth conditions; this specificity is missed by the bulk hotspot analysis. A negative correlation was detected between the amino acid replacement propensity in tumors and evolutionary variability: the hotspots are located in the positions that are highly conserved in p53 and its paralogs, p63 and p73. In all the mutation spectra, substitutions leading to amino acid replacements strongly dominate over silent substitutions, indicating that functional sites evolving under strong purifying selection are subject to intensive positive selection in p53-dependent tumors. These results are compatible with the gain-of-function concept of the role of p53 in tumorigenesis.     

Adding functional entities to plasmids

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.     

Heat capacity changes associated with nucleic acid folding

Whereas heat capacity changes (DeltaCPs) associated with folding transitions are commonplace in the literature of protein folding, they have long been considered a minor energetic contributor in nucleic acid folding. Recent advances in the understanding of nucleic acid folding and improved technology for measuring the energetics of folding transitions have allowed a greater experimental window for measuring these effects. We present in this review a survey of current literature that confronts the issue of DeltaCPs associated with nucleic acid folding transitions. This work helps to gather the molecular insights that can be gleaned from analysis of DeltaCPs and points toward the challenges that will need to be overcome if the energetic contribution of DeltaCP terms are to be put to use in improving free energy calculations for nucleic acid structure prediction.     

Nucleic Acid Content in Malformed Mango Inflorescence caused by Fusarium moniliforme var. subglutinans

The nucleic acid metabolism in obligate parasitism has although been quite frequently studied and elegantly reviewed (HEITEFUSS 1966), works on facultative parasitism are so far only a few. However, the recent demonstrations of inducing obligate parasites to grow on culture media has removed the sharp demarcation between the two groups of parasites to a great extent. Marked increase in the amount and activity of RNA in infected tissues was reported in a number of compatible combinations with susceptible hosts (QUICK and SHAW 1964, MUKHERJEE and SHAW 1962, TANI et al. 1975). As regards DNA content in infected tissues, an increase was reported on one hand by WILLIAMS et al. (1968) while on the other HEITEFUSS (1965, 1966 a) observed almost unchanged or only slight decrease in combinations having compatible reactions. The present investigation has been designed to study the nucleic acid contents in malformed inflorescence of two cultivars of mango (Mangi-fera indica L.) caused by Fusarium moniliforme var. subglutinans Wr. et Rg. Mango plants producing one of the principal fruit crops in the Indian subcontinent when infected by the fungus show development of abnormal in-florescence which fail to fruit and thus result in tremendous economic loss. The identification of the causal organism, isolated from a large number of inflorescences collected from different parts of Burdwan, West Bengal, India, was confirmed by IARI, New Delhi.     

Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers

The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced beta-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2'-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.     

Evolved RNA Secondary Structure and the Rooting of the Universal Tree of Life

The origin and diversification of RNA secondary structure were traced using cladistic methods. Structural components were coded as polarized and ordered multi-state characters, following a model of character state transformation outlined by considerations in statistical mechanics. Several classes of functional RNA were analyzed, including ribosomal RNA (rRNA). Considerable phylogenetic signal was present in their secondary structure. The intrinsically rooted phylogenies reconstructed from evolved RNA structure depicted those derived from nucleic acid sequence at all taxonomical levels, and grouped organisms in concordance with traditional classification, especially in the archaeal and eukaryal domains. Natural selection appears therefore to operate early in the information flow that originates in sequence and ends in an adapted phenotype. When examining the hierarchical classification of the living world, phylogenetic analysis of secondary structure of the small and large rRNA subunits reconstructed a universal tree of life that branched in three monophyletic groups corresponding to Eucarya, Archaea, and Bacteria, and was rooted in the eukaryotic branch. Ribosomal characters involved in the translational cycle could be easily traced and showed that transfer RNA (tRNA) binding domains in the large rRNA subunit evolved concurrently with the rest of the rRNA molecule. Results suggest it is equally parsimonious to consider that ancestral unicellular eukaryotes or prokaryotes gave rise to all extant life forms and provide a rare insight into the early evolution of nucleic acid and protein biosynthesis. Received: 13 September 2000 / Accepted: 27 August 2001