End Modification Research Articles

A T7 exonuclease assisted dual-cycle signal amplification assay of miRNA using nanospheres-enhanced fluorescence polarization

Based on streptavidin coated nanospheres and T7 exonuclease assisted dual-cycle signal amplification, we developed a novel sensitive fluorescence polarization detection method for miRNA. When target miRNA was present in the system, hairpin probe hybridized with miRNA, forming a double-stranded structure. The 5′ end of hairpin probe was then recognized and digested by T7 exonuclease, releasing the non-degraded single strand DNA fragments and miRNA. The released target miRNA could trigger the next cycle of hybridization and digestion, releasing more non-degraded fragments from hairpin probe. The fragments could hybridize with a signal probe (with carboxyfluorescein modification at 5′-end and biotin modification at 3′-end). The formed blunt 5′-end of signal probe was then recognized and degraded by T7 exonuclease, releasing the fragments and the fluorophore carboxyfluorescein. The next cycle of hybridization and digestion of signal probe was triggered by the released fragment at the same time. The free carboxyfluorescein cannot connect with streptavidin coated nanospheres which were used as the fluorescence polarization signal amplifier. So, there was a big change of fluorescence polarization signal after adding miRNA into the detection system, due to the different fluorescence polarization signal between nanospheres-captured intact signal probe and free carboxyfluorescein. The detection limit of this method is about 0.001 nM, and it has a good selectivity. In addition, it was also applicable to determine target miRNA in total miRNA extracts and compare the expression level of target miRNA in different cells. Consequently, the proposed method is expected to be used for the potential cancer diagnosis and the related biomedical research.

Writing a wrong: Coupled RNA polymerase II transcription and RNA quality control.

Processing and maturation of precursor RNA species is coupled to RNA polymerase II transcription. Co‐transcriptional RNA processing helps to ensure efficient and proper capping, splicing, and 3′ end processing of different RNA species to help ensure quality control of the transcriptome. Many improperly processed transcripts are not exported from the nucleus, are restricted to the site of transcription, and are in some cases degraded, which helps to limit any possibility of aberrant RNA causing harm to cellular health. These critical quality control pathways are regulated by the highly dynamic protein–protein interaction network at the site of transcription. Recent work has further revealed the extent to which the processes of transcription and RNA processing and quality control are integrated, and how critically their coupling relies upon the dynamic protein interactions that take place co‐transcriptionally. This review focuses specifically on the intricate balance between 3′ end processing and RNA decay during transcription termination.This article is categorized under:RNA Turnover and Surveillance > Turnover/Surveillance MechanismsRNA Processing > 3' End ProcessingRNA Processing > Splicing MechanismsRNA Processing > Capping and 5' End Modifications

Cytoplasmic ends of tetraspanin 7 harbour epitopes recognised by autoantibodies in type 1 diabetes.

The beta cell protein tetraspanin 7 is a target of autoantibodies in individuals with type 1 diabetes. The aim of this study was to identify autoantibody epitope-containing regions and key residues for autoantibody binding. Autoantibody epitope regions were identified by immunoprecipitation of luciferase-tagged single or multiple tetraspanin 7 domains using tetraspanin 7 antibody-positive sera. Subsequently, amino acids (AAs) relevant for autoantibody binding were identified by single AA mutations. In tetraspanin 7 antibody-positive sera, antibody binding was most frequent to tetraspanin 7 proteins that contained the NH2-terminal cytoplasmic domain 1 (C1; up to 39%) or COOH-terminal C3 (up to 22%). Binding to C3 was more frequent when the domain was expressed along with the flanking transmembrane domain, suggesting that conformation is likely to be important. Binding to external domains was not observed. Single AA mutations of C3 identified residues Y246, E247 and R239 as critical for COOH-terminal binding of 9/10, 10/10 and 8/10 sera tested, respectively. Mutation of cysteines adjacent to the transmembrane domain at either residues C235 or C236 resulted in both decreased (8/178 and 15/178 individuals, respectively; >twofold decrease) and increased (30/178 and 13/178 individuals, respectively; >twofold increase) binding in participant sera vs wild-type protein. We hypothesise that conformation and, potentially, modification of protein terminal ends of tetraspanin 7 may be important for autoantibody binding in type 1 diabetes.

Bias-minimized quantification of microRNA reveals widespread alternative processing and 3' end modification.

MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification of miRNAs has been limited owing to the severe ligation bias in conventional sRNA-seq methods. Here, we quantify miRNAs and their variants (known as isomiRs) by an improved sRNA-seq protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which minimize the ligation bias during library preparation. Measurement using AQ-seq allows us to correct the previously misannotated 5′ end usage and strand preference in public databases. Importantly, the analysis of 5′ terminal heterogeneity reveals widespread alternative processing events which have been underestimated. We also identify highly uridylated miRNAs originating from the 3p strands, indicating regulations mediated by terminal uridylyl transferases at the pre-miRNA stage. Taken together, our study reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).

Myriad RNAs and RNA-Binding Proteins Control Cell Functions, Explain Diseases, and Guide New Therapies.

This summary of the 84th Cold Spring Harbor Laboratory (CSHL) Symposium on Quantitative Biology: RNA Control and Regulation, held in May 2019, highlights key emerging themes in this field, which now impacts nearly every aspect of biology and medicine. Recent discoveries accelerated by technological developments reveal enormous diversity of RNAs and RNA-binding proteins (RBPs) with ever-increasing roles in eukaryotes. Atomic structures and live-cell imaging of transcription, RNA splicing, 3'-end processing, modifications, and degradation machineries provide mechanistic insights, explaining hundreds of diseases caused by their perturbations. This great progress uncovered numerous targets for therapies, some of which have already been successfully exploited, and many opportunities for pharmacological intervention and RNA-guided genome engineering. Myriad unexplained RNAs and RBPs leave the RNA field open for many more exciting discoveries.

Unraveling 3'-end RNA uridylation at nucleotide resolution.

Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.

Pathologic Replication-Independent Endogenous DNA Double-Strand Breaks Repair Defect in Chronological Aging Yeast.

Reduction of physiologic replication-independent endogenous DNA double strand breaks (Phy-RIND-EDSBs) in chronological aging yeast increases pathologic RIND-EDSBs (Path-RIND-EDSBs). Path-RIND-EDSBs can occur spontaneously in non-dividing cells without any inductive agents, and they must be repaired immediately otherwise their accumulation can lead to senescence. If yeasts have DSB repair defect, retention of Path-RIND-EDSBs can be found. Previously, we found that Path-RIND-EDSBs are not only produced but also retained in chronological aging yeast. Here, we evaluated if chronological aging yeasts have a DSB repair defect. We found a significant accumulation of Path-RIND-EDSBs around the same level in aging cells and caffeine treated cells and at a much higher level in the DSB repair mutant cells. Especially in the mutant, some unknown sequence was found inserted at the breaks. In addition, % difference of cell viability between HO induced and non-induced cells was significantly greater in aging cells. Our results suggested that RIND-EDSBs repair efficiency declines, but is not absent, in chronological aging yeast which might promote senescence phenotype. When a repair protein is deficient, an alternative pathway might be employed or an end modification process might occur as inserted sequences at the breaks were observed. Restoring repair defects might slow down the deterioration of cells from chronological aging.

Noncanonical RNA-capping: Discovery, mechanism, and physiological role debate.

Recently a new type of 5'-RNA cap was discovered. In contrast to the specialized eukaryotic m7 G cap, the novel caps are abundant cellular cofactors like NAD+ . RNAs capped with cofactors are found in prokaryotes and eukaryotes. Unlike m7 G cap, installed by specialized enzymes, cofactors are attached by main enzyme of transcription, RNA polymerase (RNAP). Cofactors act as noncanonical initiating substrates, provided cofactor's nucleoside base-pairs with template DNA at the transcription start site. Adenosine-containing NAD(H), flavin adenine dinucleotide (FAD), and CoA modify transcripts on promoters starting with +1A. Similarly, uridine-containing cell wall precursors, for example, uridine diphosphate-N-acetylglucosamine were shown to cap RNA in vitro on +1U promoters. Noncanonical capping is a universal feature of evolutionary unrelated RNAPs-multisubunit bacterial and eukaryotic RNAPs, and single-subunit mitochondrial RNAP. Cellular concentrations of cofactors, for example, NAD(H) are significantly higher than their Km in transcription. Yet, only a small proportion of a given cellular RNA is noncanonically capped (if at all). This proportion is a net balance between capping, seemingly stochastic, and decapping, possibly determined by RNA folding, protein binding and transcription rate. NUDIX hydrolases in bacteria and eukaryotes, and DXO family proteins eukaryotes act as decapping enzymes for noncanonical caps. The physiological role of noncanonical RNA capping is only starting to emerge. It was demonstrated to affect RNA stability in vivo in bacteria and eukaryotes and to stimulate RNAP promoter escape in vitro in Escherichia coli. NAD+ /NADH capping ratio may connect transcription to cellular redox state. Potentially, noncanonical capping affects mRNA translation, RNA-protein binding and RNA localization. This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Export and Localization > RNA Localization RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry.

The complex enzymology of mRNA decapping: Enzymes of four classes cleave pyrophosphate bonds.

The 5' ends of most RNAs are chemically modified to enable protection from nucleases. In bacteria, this is often achieved by keeping the triphosphate terminus originating from transcriptional initiation, while most eukaryotic mRNAs and small nuclear RNAs have a 5'→5' linked N7 -methyl guanosine (m7 G) cap added. Several other chemical modifications have been described at RNA 5' ends. Common to all modifications is the presence of at least one pyrophosphate bond. To enable RNA turnover, these chemical modifications at the RNA 5' end need to be reversible. Dependent on the direction of the RNA decay pathway (5'→3' or 3'→5'), some enzymes cleave the 5'→5' cap linkage of intact RNAs to initiate decay, while others act as scavengers and hydrolyse the cap element of the remnants of the 3'→5' decay pathway. In eukaryotes, there is also a cap quality control pathway. Most enzymes involved in the cleavage of the RNA 5' ends are pyrophosphohydrolases, with only a few having (additional) 5' triphosphonucleotide hydrolase activities. Despite the identity of their enzyme activities, the enzymes belong to four different enzyme classes. Nudix hydrolases decap intact RNAs as part of the 5'→3' decay pathway, DXO family members mainly degrade faulty RNAs, members of the histidine triad (HIT) family are scavenger proteins, while an ApaH-like phosphatase is the major mRNA decay enzyme of trypanosomes, whose RNAs have a unique cap structure. Many novel cap structures and decapping enzymes have only recently been discovered, indicating that we are only beginning to understand the mechanisms of RNA decapping. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability RNA Processing > Capping and 5' End Modifications.

Noncanonical features and modifications on the 5'-end of bacterial sRNAs and mRNAs.

Although many eukaryotic transcripts contain cap structures, it has been long thought that bacterial RNAs do not carry any special modifications on their 5'-ends. In bacteria, primary transcripts are produced by transcription initiated with a nucleoside triphosphate and are therefore triphosphorylated on 5'-ends. Some transcripts are then processed by nucleases that yield monophosphorylated RNAs for specific cellular activities. Many primary transcripts are also converted to monophosphorylated species by removal of the terminal pyrophosphate for 5'-end-dependent degradation. Recent studies surprisingly revealed an expanded repertoire of chemical groups on 5'-ends of bacterial RNAs. In addition to mono- and triphosphorylated moieties, some mRNAs and sRNAs contain cap-like structures and diphosphates on their 5'-ends. Although incorporation and removal of these groups have become better understood in recent years, the physiological significance of these modifications remain obscure. This review highlights recent studies aimed at identification and elucidation of novel modifications on the 5'-ends of bacterial RNAs and discusses possible physiological applications of the modified RNAs. This article is categorized under: RNA Turnover and Surveillance > Regulation of RNA Stability RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Processing > Capping and 5' End Modifications.

A recap of RNA recapping.

The N7-methylguanosine cap is a hallmark of the 5' end of eukaryotic mRNAs and is required for gene expression. Loss of the cap was believed to lead irreversibly to decay. However, nearly a decade ago, it was discovered that mammalian cells contain enzymes in the cytoplasm that are capable of restoring caps onto uncapped RNAs. In this review, we summarize recent advances in our understanding of cytoplasmic RNA recapping and discuss the biochemistry of this process and its impact on regulating and diversifying the transcriptome. Although most studies focus on mammalian RNA recapping, we also highlight new observations for recapping in disparate eukaryotic organisms, with the trypanosome recapping system appearing to be a fascinating example of convergent evolution. We conclude with emerging insights into the biological significance of RNA recapping and prospects for the future of this evolving area of study. This article is categorized under: RNA Processing > RNA Editing and Modification Translation > Translation Regulation RNA Processing > Capping and 5' End Modifications RNA Turnover and Surveillance > Regulation of RNA Stability.

Effects of Tooth Modifications on the Mesh and Dynamic Characteristics of Differential Gearbox Used in Electric Vehicle

In order to reduce the vibration and noise for the differential gearbox used in electric vehicle, a coupled gear–shaft–bearing–housing dynamic model was developed considering the elastic support by shaft, bearing and housing. Three tooth modification schemes were proposed to investigate the effects of modification type on the mesh behaviors. Results show that the tooth modification scheme with a combined drum–tooth end modification for lead direction and linear addendum modification for profile direction can make the contact load distribution better. The contact pattern was located in the middle of the tooth, and the maximum contact pressure was decreased by 19% to 937 MPa. And the peak–peak value of transmission error was decreased by 33% for high-speed stage and 26% for intermediate stage. The tooth modifications tend to decrease the acceleration responses obviously, especially for the high-frequency range. The structure noise was reduced about 3.5–4.5 dB in x, y and z directions for the selected bearing locations. The analysis results can be applied to guide the design of electric vehicle differential gearbox with low noise level.

LARP1 on TOP of ribosome production.

The ribosome is an essential unit of all living organisms that commands protein synthesis, ultimately fuelling cell growth (accumulation of cell mass) and cell proliferation (increase in cell number). The eukaryotic ribosome consists of 4 ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs). Despite its fundamental role in every living organism, our present understanding of how higher eukaryotes produce the various ribosome components is incomplete. Uncovering the mechanisms utilized by human cells to generate functional ribosomes will likely have far-reaching implications in human disease. Recent biochemical and structural studies revealed La-related protein 1 (LARP1) as a key new player in RP production. LARP1 is an RNA-binding protein that belongs to the LARP superfamily; it controls the translation and stability of the mRNAs that encode RPs and translation factors, which are characterized by a 5' terminal oligopyrimidine (5'TOP) motif and are thus known as TOP mRNAs. The activity of LARP1 is regulated by the mammalian target of rapamycin complex 1 (mTORC1): a eukaryotic protein kinase complex that integrates nutrient sensing with mRNA translation, particularly that of TOP mRNAs. In this review, we provide an overview of the role of LARP1 in the control of ribosome production in multicellular eukaryotes. This article is categorized under: Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Processing > Capping and 5' End Modifications.

Genetic and Epigenetic Regulation in Nonalcoholic Fatty Liver Disease (NAFLD).

Genetics and epigenetics play a key role in the development of several diseases, including nonalcoholic fatty liver disease (NAFLD). Family studies demonstrate that first degree relatives of patients with NAFLD are at a much higher risk of the disease than the general population. The development of the Genome Wide Association Study (GWAS) technology has allowed the identification of numerous genetic polymorphisms involved in the evolution of diseases (e.g., PNPLA3, MBOAT7). On the other hand, epigenetic changes interact with inherited risk factors to determine an individual’s susceptibility to NAFLD. Modifications of the histones amino-terminal ends are key factors in the maintenance of chromatin structure and gene expression (cAMP-responsive element binding protein H (CREBH) or SIRT1). Activation of SIRT1 showed potential against the physiological mechanisms related to NAFLD. Abnormal DNA methylation represents a starting point for cancer development in NAFLD patients. Besides, the evaluation of circulating miRNA profiles represents a promising approach to assess and non-invasively monitor liver disease severity. To date, there is no approved pharmacologic therapy for NAFLD and the current treatment remains weight loss with lifestyle modification and exercise. In this review, the status of research into relevant genetic and epigenetic modifiers of NAFLD progression will be discussed.

Lipoyl Ester Terminated Star PLGA as a Simple and Smart Material for Controlled Drug Delivery Application

PLGA, a copolymer of lactide and glycolide, is one of the most used biodegradable polymers that find a wide range of biomedical applications including drug delivery and tissue engineering. However, in spite of remarkable advancement, nanotherapeutics based on PLGA might have drawbacks of inadequate stability, drug leakage, and slow drug release at the tumor site, which reduces its targeting ability and therapeutic efficacy. Here, we report that direct modification of star PLGA ends with lipoic acid, a natural antioxidant present in our human body, affords a smart material (sPLGA-LA) that forms reversibly crosslinked and bioresponsive multifunctional nanoparticles (sPLGA XNPs). Interestingly, sPLGA XNPs obtained in the presence of 23.0 wt % PEG-PDLLA displayed a small hydrodynamic size of 73 ± 1.2 nm, high stability against dilution and 10% serum, while fast destabilization under a reductive environment. Moreover, sPLGA XNPs achieved efficient loading of lipophilic anticancer drug model, doxorubicin (DOX), at a theoretical drug loading content of 13.3 wt %, giving DOX-loaded sPLGA XNPs with reduced drug leakage under physiological conditions as well as significantly accelerated drug release under 10 mM glutathione condition compared with both linear and star PLGA controls (denoted as lPLGA NPs and sPLGA NPs, respectively). Confocal microscopy and flow cytometry displayed obviously stronger DOX fluorescence in B16F10 melanoma cells treated with DOX-loaded sPLGA XNPs than with lPLGA and sPLGA counterparts. MTT assays revealed that DOX-sPLGA XNPs caused 2.4- and 4.2-fold higher antitumor activity toward B16F10 cells than DOX-sPLGA NPs and DOX-lPLGA NPs, respectively. Notably, in vivo pharmacokinetics studies showed prolonged circulation time and significantly improved AUC for DOX-sPLGA XNPs over lPLGA NPs control. Hence, lipoyl ester terminated star PLGA emerges as a simple and smart material for better-controlled anticancer drug delivery.

Synthesis of Poly(N-H benzamide)-b-poly(lauryl methacrylate)-b-poly(N-H benzamide) symmetrical triblock copolymers by combinations of CGCP, SARA ATRP, and SA ATRC

We design a novel ABA type triblock copolymer (triBCP) by performing efficient synthetic methods of chain-growth condensation polymerization (CGCP), supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP), and styrenics-assisted atom transfer radical coupling (SA ATRC). A well-defined poly(3-(N-(octyloxy)benzyl benzamide) having a 2-bromo-2-phenylacetate effective ATRP initiating site (i.e., POOBBA-BrPA) through CGCP following quantitative chain end modifications, was firstly synthesized. The POOBBA-BrPA macroinitiator then conducted chain extension with lauryl methacrylate (LMA) via SARA ATRP to obtain the POOBBA-b-PLMA-Br diBCP (Mn = 8800 and Mw/Mn = 1.15). This was followed by SA ATRC of POOBBA-b-PLMA-Br with a high extent of coupling (xc = 0.85, Mn = 15300, and Mw/Mn = 1.26). The obtained triBCP was further purified and the selective removal of the protecting group yielded poly(N-H benzamide)-b-poly(lauryl methacrylate)-b-poly(N-H benzamide) (i.e., PNHBA-b-PLMA-b-PNHBA) was conducted. The characterization of these copolymers was performed using 1H NMR, GPC, FT-IR, TGA, DSC, AFM and SAXS.

Analysis of 3' End Modifications in microRNAs by High-Throughput Sequencing.

MicroRNAs are ~22nt small, non-coding RNAs that direct posttranscriptional silencing of gene expression to regulate animal development, physiology, and disease. An emerging mechanism that controls the biogenesis of microRNAs is the addition of non-templated nucleotides, predominantly uridine, to the 3' end of precursor-microRNAs, in a process that is commonly referred to as tailing. Here, we describe methods that enable the systematic characterization of tailing events in mature microRNAs and their precursors. We report protocols for untargeted and targeted cDNA library preparation procedures, as exemplified in the context of the model organism Drosophila melanogaster and focusing on precursor-microRNAs. We also refer to a dedicated computational framework for the subsequent analysis of untemplated nucleotide additions in cDNA libraries. The described methods for the systematic characterization of posttranscriptional modifications in gene regulatory small RNAs and their precursors will be instrumental in clarifying regulatory concepts that control posttranscriptional gene silencing.

Early outcomes of modification of end to side repair of coarctation of aorta with arch hypoplasia in neonates and infants.

Background:In coarctation of aorta associated with proximal arch hypoplasia, extended end-to-end anastomosis through a thoracotomy would result in a residual gradient between the origins of the innominate and the left common carotid arteries. To eliminate this, we modified the surgical technique.Patients and Methods:Between March 2012 and May 2017, 50 patients (14 neonates) underwent repair of coarctation of aorta through a thoracotomy. The age ranged from 6 days to 2 years (median 2 months) and the weight from 1.8 to 8.0 kg (median 4.3 kg). A total of 15 patients (Group A) underwent repair by the extended end-to-end anastomosis. Among them, two patients developed early restenosis at the proximal arch requiring surgical reintervention. Hence, in the second half of the study, 35 patients (Group B) who were identified to have significant hypoplasia of the proximal arch underwent a modified end-to-side anastomosis of the descending aorta to the proximal arch incorporating the distal ascending aorta in the anastomosis and leaving the left subclavian artery end of the isthmus as an end-on vessel.Results:One neonate in Group B died due to a cause not related to the repair. All the other patients in Group B are doing well without a residual gradient during a median follow-up of 23 months. There were no airway issues related to extensive mobilization of the aorta.Conclusion:End-to-side anastomosis of the descending aorta to the proximal arch and side of the ascending aorta is possible through a thoracotomy and can be achieved with good outcome in neonates and infants.

Exclusive Synthesis of Poly(3-hexylthiophene) with an Ethynyl Group at Only One End for Effective Block Copolymerization.

Well-controlled synthesis of ethynyl-functionalized poly(3-hexylthiophene) (P3HT) is crucial for preparation of block copolymers containing the P3HT segment by means of click coupling reaction. A well-known chain end modification method, in which Kumada-Tamao catalyst-transfer polymerization is quenched with ethynylmagnesium chloride, under various conditions is re-examined, but in all cases not only P3HT with an ethynyl group at one end but also P3HT di-ethynylated at both ends is obtained. Accordingly, Sonogashira coupling reaction of P3HT having H/Br ends with trimethylsilylacetylene is tried, followed by removal of the trimethylsilyl group, and it is found that this protocol affords exclusively P3HT with an ethynyl group at one end. This post end-modification method is applied to the synthesis of an amphiphilic diblock copolymer of P3HT and poly(2-ethyl-2-oxazoline) (PEtOx) by means of click reaction between ethynylated P3HT and PEtOx with an azide group at one end, and the product is confirmed to be free from contamination with triblock copolymer. Micellization of this block copolymer is confirmed in tetrahydrofuran (THF)/water and THF/methanol mixtures.

Amphiphilic Polysaccharide Block Copolymers for pH-Responsive Micellar Nanoparticles.

A full polysaccharide amphiphilic block copolymer was prepared from end group-functionalized dextrans using copper-mediated azide-alkyne click chemistry. Sufficient modification of the reducing end in both blocks was achieved by microwave-enhanced reductive amination in a borate-buffer/methanol solvent system. The combination of a hydrophilic dextran block with a hydrophobic acetalated dextran block results in an amphiphilic structure that turns water-soluble upon acid treatment. The material has a low critical micelle concentration and self-assembles in water to spherical micellar nanoparticles. The formed nanoparticles have a narrow size distribution below 70 nm in diameter and disassemble in slightly acidic conditions. The amphiphilic polysaccharide system shows low toxicity and can stabilize the hydrophobic model drug curcumin in aqueous solutions over extended time periods.