Molecular Glue for RNA: Regulating RNA Structure and Function through Synthetic RNA Binding Molecules

We introduce the concept of molecular glues for RNA, in which specific RNA-binding small molecules induce designed structural changes in target functional RNAs, resulting in modulation of the functions. (Z)-NCTS is an RNA-mismatch-binding small molecule that recognizes 5′-r(XGG)-3′/5′-r(XGG)-3′ sequences (X=U or A) and acts as a molecular glue for RNA. The binding of (Z)-NCTS brings two distinct 5′-r(XGG)-3′ domains into contact with each other, and this can result in higher-order structural changes of target RNAs. We applied (Z)-NCTS to induce the formation of a proposed tertiary structure of a ribozyme together with activation of RNA-cleaving ability. The concept of a molecular glue could inspire new small-molecule-based strategies for regulating biological functions: a synthetic small molecule targeting functional RNAs could regulate the RNA structure and function.     

差示扫描量热熔点分析法的应用性分析

选择不同温度段熔融的5个代表性品种、国内外不同来源的13个法定熔点标准物质作为研究对象,进行差示扫描量热法和毛细管法两种熔点分析方法的关联性研究,并进一步开展差示扫描量热熔点分析法关键参数的应用性分析。研究表明,毛细管法的熔点分析结果一般延后1~2℃;差示扫描量热熔点分析法的准确性和精密度更好,并且推荐采用10℃/min的升温速率参数,而升温起点、气体流量等方法参数影响较小。质量分析还证实,我国药典熔点标准物质已经具备国际竞争力。     

2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis,Fluorescent Properties,and Duplex Stability

The synthesis and properties two series of new 2′‐O‐methyl RNA probes, each containing a single insertion of a 2′‐bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21‐fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5′‐side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3′‐side are important: CC, CG, and UC dinucleotide units on the 3′‐side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2′‐bispyrene‐labeled 2′‐O‐methyl RNA probes might be useful tools for detection of RNAs.     

Malachite green derivatives for two-photon RNA detection

The design, preparation and characterisation of a library of malachite green (MG) derivatives for two-photon RNA labelling is described. Some of these MG derivatives exhibit an increased affinity for an MG-aptamer, as well as improved two-photon sensitivity when compared to the classical malachite green chloride. The underlying mechanisms and potential benefits for in vivo RNA visualisation are discussed.     

Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G‐Quadruplexes

We have developed fluorescent protein probes specific for parallel G‐quadruplexes by attaching cyan fluorescent protein to the G‐quadruplex‐binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G‐quadruplexes was characterized. The selective recognition and discrimination of G‐quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.     

Current Approaches for RNA Labeling in Vitro and in Cells Based on Click Reactions

Over recent years, click reactions have become recognized as valuable and flexible tools to label biomacromolecules such as proteins, nucleic acids, and glycans. Some of the developed strategies can be performed not only in aqueous solution but also in the presence of cellular components, as well as on (or even in) living cells. These labeling strategies require the initial, specific modification of the target molecule with a small, reactive moiety. In the second step, a click reaction is used to covalently couple a reporter molecule to the biomolecule. Depending on the type of reporter, labeling by the click reaction can be used in many different applications, ranging from isolation to functional studies of biomacromolecules. In this minireview, we focus on labeling strategies for RNA that rely on the click reaction. We first highlight click reactions that have been used successfully to label modified RNA, and then describe different strategies to introduce the required reactive groups into target RNA. The benefits and potential limitations of the strategies are critically discussed with regard to possible future developments.     

Controlling the Fluorescence of Benzofuran‐Modified Uracil Residues in Oligonucleotides by Triple‐Helix Formation

We developed fluorescent turn‐on probes containing a fluorescent nucleoside, 5‐(benzofuran‐2‐yl)deoxyuridine (dU^BF) or 5‐(3‐methylbenzofuran‐2‐yl)deoxyuridine (dU^MBF), for the detection of single‐stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dU^MBF achieved superior fluorescence enhancement than that containing dU^BF. NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3‐methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3‐methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex‐induced fluorescence enhancement of the oligonucleotide probe containing dU^MBF.     

Ultrasensitive Molecular Beacon Designed with Totally Serinol Nucleic Acid (SNA) for Monitoring mRNA in Cells

An artificial nucleic acid based on acyclic serinol building blocks and termed “serinol nucleic acid” (SNA) was used to construct a fluorescent probe for RNA visualization in cells. The molecular beacon (MB) composed of only SNA with a fluorophore at one terminus and a quencher at the other was resistant to enzymatic digestion, due to its unnatural acyclic scaffold. The SNA‐MB could detect its complementary RNA with extremely high sensitivity; the signal‐to‐background (S/B) ratio was as high as 930 when perylene and anthraquinone were used as the fluorophore and quencher pair. A high S/B ratio was also achieved with SNA‐MB tethering the conventional Cy3 fluorophore, and this probe enabled selective visualization of target mRNA in fixed cells. Thus, SNA‐MB has potential for use as a biological tool capable of visualizing RNA in living cells.     

差示扫描量热分析技术在粉末涂料中的应用

介绍了热分析技术及差示扫描量热分析技术（简称DSC）的发展。结合实际应用,阐述了粉末涂料中几个常用技术指标（Tg、熔点、软化点、结晶度）的物理意义及测定方法,详细探讨了差示扫描量热分析技术在粉末涂料固化行为方面的应用研究,分析讨论了影响检测结果的几个主要因素。     

Differential Scanning Calorimetry of Stressed Polymers

Stresses which have been frozen into a polymer by cooling to below its glass transition temperature (Tg) are relieved when the polymer is reheated above its Tg. Polymers which have been elongated will contract above Tg. This sudden contraction just above Tg is detected as a sharp drop in the differential scanning calorimetric curve. This is the reverse of the well known superheating endotherm accompanying the expansion of annealed polymers. The decrease in the differential scanning calorimetric curve immediately after the Tg inflection can be quantitatively related to the residual strain in poly(vinyl chloride) and polycarbonate.     

两种聚酯纤维的DSC定量分析方法

利用差示扫描量热仪(DSC),分别对两种聚酯纤维——聚对苯二甲酸乙二酯(PET)和聚对苯二甲酸丙二酯(PTT)的热性能进行了测试,通过研究PET和PTT在不同升温速率下的热焓变化规律确定了升温速率,研究了PET的质量与热焓的线性关系,确定了PET-PTT混纺面料产品中各组分的定量分析方法。     

DSC加速老化试验方法在PP寿命估算中的应用

利用差示扫描量热(DSC)加速老化试验方法,以不同温度下测得的氧化诱导时间(OIT)为基础,建立温度–寿命相关的阿伦尼乌斯方程,并以此对不同温度下聚丙烯的寿命进行快速估算。结果表明,其估算结果有效,是筛选抗氧剂配方的一种较好的方法。     

Bivalent Display of Dicysteine on Peptide Nucleic Acids for Homogenous DNA/RNA Detection through in Situ Fluorescence Labelling

Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached through covalent bonding to an oligonucleotide probe. However, the additional conjugation step increases costs. Here we introduce a method that avoids the requirement for the preparation of fluorescence‐labelled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine‐containing peptide nucleic acid (PNA) probes to form a bipartite tetracysteine motif that binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases in fluorescence emission (with response factors of up to 80‐fold and high brightness up to 50 mL mol^?1 cm^?1). The detection system provides sub‐nanomolar limits of detection and allows discrimination of single nucleotide variations through more than 20‐fold changes in fluorescence intensity. To demonstrate its usefulness, the FIAsH‐based readout of the bivalent CysCys‐PNA display was interfaced with a rolling‐circle amplification (RCA) assay used to detect disease‐associated microRNA let‐7a.     

Differential Scanning Calorimetry Cure Studies of Tetra-N-glycidyldiaminodiphenylmethane Epoxy Resins 3 - Reaction with Dicyandiamide

Continuation of work on the curing characteristics of an epoxy resin which is widely used as a composite matrix is reported. Results are given of study of the cure of the resin with dicyandiamide as hardener and diuron as accelerator, using differential scanning calorimetry to monitor the cure. The effect of diuron on the cure of the resin with an aromatic amine curing agent is also described. Distinctive effects of composition are found and values of the apparent activation energy of the curing reaction are derived.     

Design and synthesis of caged fluorescent nucleotides and application to live-cell RNA imaging

A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization-sensitive thiazole orange units has been designed for area-specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization-sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex-formation ability. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area-specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.