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。     

The conservation profile of a protein bears the imprint of the molecule that is evolutionarily coupled to the protein

The conservation profile of a protein is a curve of the conservation levels of amino acids along the sequence. Biologists are usually more interested in individual points on the curve (namely, the conserved amino acids) than the overall shape of the curve. Here, we show that the conservation curves of proteins bear the imprints of molecules that are evolutionarily coupled to the proteins. Our method is based on recent studies that a sequence conservation profile is quantitatively linked to its structural packing profile. We find that the conservation profiles of nucleic acid (NA) binding proteins are better correlated with the packing profiles of the protein–NA complexes than those of the proteins alone. This indicates that a nucleic acid binding protein evolves to accommodate the nucleic acid in such a way that the residues involved in binding have their conservation levels closely coupled with the specific nucleotides. Proteins 2015; 83:1407–1413. © 2015 Wiley Periodicals, Inc.     

G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K⁺ rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5′ with 3 nt and 3′ with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.     

Design,synthesis, and analysis of conformationally constrained nucleic acids

In this review I discuss straightforward and general methods to modify nucleic acid structure with disulfide cross-links. A motivating factor in developing this chemistry was the notion that disulfide bonds would be excellent tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross-links have been used to study proteins. The chemistry described has been used to synthesize disulfide cross-linked hairpins and duplexes, higher order structures like triplexes, nonground-state conformations, and tRNAs. Since the cross-links form quantitatively by mild air oxidation and do not perturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids. © 1998 John Wiley & Sons, Inc. Biopoly 48: 83–96, 1998     

A comparison of the folding kinetics of a small,artificially selected DNA aptamer with those of equivalently simple naturally occurring proteins

The folding of larger proteins generally differs from the folding of similarly large nucleic acids in the number and stability of the intermediates involved. To date, however, no similar comparison has been made between the folding of smaller proteins, which typically fold without well-populated intermediates, and the folding of small, simple nucleic acids. In response, in this study, we compare the folding of a 38-base DNA aptamer with the folding of a set of equivalently simple proteins. We find that, as is true for the large majority of simple, single domain proteins, the aptamer folds through a concerted, millisecond-scale process lacking well-populated intermediates. Perhaps surprisingly, the observed folding rate falls within error of a previously described relationship between the folding kinetics of single-domain proteins and their native state topology. Likewise, similarly to single-domain proteins, the aptamer exhibits a relatively low urea-derived Tanford β, suggesting that its folding transition state is modestly ordered. In contrast to this, however, and in contrast to the behavior of proteins, ϕ-value analysis suggests that the aptamer''s folding transition state is highly ordered, a discrepancy that presumably reflects the markedly more important role that secondary structure formation plays in the folding of nucleic acids. This difference notwithstanding, the similarities that we observe between the two-state folding of single-domain proteins and the two-state folding of this similarly simple DNA presumably reflect properties that are universal in the folding of all sufficiently cooperative heteropolymers irrespective of their chemical details.     

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。     

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。     

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Thiolated pyrrolidinyl peptide nucleic acids for the detection of DNA hybridization using surface plasmon resonance

Thiolated pyrrolidinyl peptide nucleic acids (HS-PNAs) bearing d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones with different lengths and types of thiol modifiers were synthesized and then characterized by MALDI–TOF mass spectrometry. These HS-PNAs were immobilized on gold-coated glass by self-assembled monolayer (SAM) formation via S atom linkage for the detection of DNA hybridization using surface plasmon resonance (SPR). The amount and the stability of the immobilized HS-PNAs, as well as the effects of spacer and blocking thiol on DNA hybridization efficiency, were determined. SPR results indicated that the hybridization efficiency was enhanced when the distance between the PNA portion and the thiol terminal was increased and/or when blocking thiol was used following the HS-PNA immobilization. The immobilized HS-PNA could discriminate between fully complementary DNA from one or two base mismatched DNA with a relatively high degree of mismatch discrimination (>45%) in PBS buffer at 25 °C. The lowest DNA concentration at which reliable discrimination between fully complementary and single mismatched DNA could still occur was at about 0.2 μM, which is equivalent to 10 pmol of DNA. This research demonstrates that using these novel thiolated PNAs in combination with the SPR technique offers a direct, rapid and non-label based method that could potentially be applied for the analysis of genomic or PCR-amplified DNA in the future.     

Combinatorial Chemistry of Nucleic Acids: SELEX

A method of irrational oligonucleotide design, SELEX, is considered. Individual SELEX products, aptamers, are small molecules (40–100 nt) that have a unique three-dimensional structure, which provides for their specific and high-affinity binding to targets varying from low-molecular-weight ligands to proteins. Thus, the sophisticated biosynthesis of recognizing protein elements, antibodies, can be emulated in vitro via selection and synthesis of principally new recognizing elements based on nucleic acids.     

Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’ for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by ‘hybrid’ phosphorothioate–LNA ODNs induces tumour necrosis factor-α production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.     

Calibration of ring-current effects in proteins and nucleic acids

Summary Density functional chemical shielding calculations are reported for methane molecules placed in a variety of positions near aromatic rings of the type found in proteins and nucleic acids. The results are compared to empirical formulas that relate these intermolecular shielding effects to magnetic anisotropy (ring-current) effects and to electrostatic polarization of the C–H bonds. Good agreement is found between the empirical formulas and the quantum chemistry results, allowing a reassessment of the ring-current intensity factors for aromatic amino acids and nucleic acid bases. Electrostatic interactions contribute significantly to the computed chemical shift dispersion. Prospects for using this information in the analysis of chemical shifts in proteins and nucleic acids are discussed.     

Determination of h2JNN and h1JHN coupling constants across Watson–Crick base pairs in the Antennapedia homeodomain–DNA complex using TROSY

This paper describes NMR measurements of ¹⁵N–¹⁵N and ¹H–¹⁵N scalar couplings across hydrogen bonds in Watson–Crick base pairs, ^h2 J _NN and ^h1 J _HN, in a 17 kDa Antennapedia homeodomain–DNA complex. A new NMR experiment is introduced which relies on zero-quantum coherence-based transverse relaxation-optimized spectroscopy (ZQ-TROSY) and enables measurements of ^h1 J _HN couplings in larger molecules. The ^h2 J _NN and ^h1 J _HN couplings open a new avenue for comparative studies of DNA duplexes and other forms of nucleic acids free in solution and in complexes with proteins, drugs or possibly other classes of compounds.     

The Biosynthesis of Thiol- and Thioether-containing Cofactors and Secondary Metabolites Catalyzed by Radical S-Adenosylmethionine Enzymes

Sulfur atoms are present as thiol and thioether functional groups in amino acids, coenzymes, cofactors, and various products of secondary metabolic pathways. The biosynthetic pathways for several sulfur-containing biomolecules require the substitution of sulfur for hydrogen at unreactive aliphatic or electron-rich aromatic carbon atoms. Examples discussed in this review include biotin, lipoic acid, methylthioether modifications found in some nucleic acids and proteins, and thioether cross-links found in peptide natural products. Radical S-adenosyl-l-methionine (SAM) enzymes use an iron-sulfur cluster to catalyze the reduction of SAM to methionine and a highly reactive 5′-deoxyadenosyl radical; this radical can abstract hydrogen atoms at unreactive positions, facilitating the introduction of a variety of functional groups. Radical SAM enzymes that catalyze sulfur insertion reactions contain a second iron-sulfur cluster that facilitates the chemistry, either by donating the cluster''s endogenous sulfide or by binding and activating exogenous sulfide or sulfur-containing substrates. The use of radical chemistry involving iron-sulfur clusters is an efficient anaerobic route to the generation of carbon-sulfur bonds in cofactors, secondary metabolites, and other natural products.     

FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.     

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se‐Met) strategy). This selenium derivatization strategy via MAD (multi‐wavelength anomalous dispersion) phasing has revolutionized protein X‐ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X‐ray crystal‐structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O‐atom at the positions 2′, 4′, 5′, and in nucleobases and non‐bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid‐phase chemical synthesis and enzymatic methods, and the Se‐derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein–nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2′‐position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom‐specific substitution of the nucleotide O‐atom, better stability under X‐ray radiation, and structure isomorphism. Therefore, our Se‐derivatization strategy has great potentials to provide rational solutions for both phase determination and high‐quality crystal growth in nucleic‐acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site‐specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se‐derivatization strategy in nucleic acid and protein research are also described in this review.