利用DNA Assembly方法构建重组腺病毒载体

目的:建立一种基于DNA Assembly方法的重组腺病毒载体构建方法。方法:首先,通过设计合适的酶切位点及同源臂,采用了传统的限制性内切核酸酶连接方法和DNA Assembly方法获得63型猩猩腺病毒(Chimpanzee adenovirus serotype 63,ChAd63)的骨架质粒pChAd63。随后,采用Sca I单酶切携带EGFP基因的穿梭pShuttle63/EGFP,Hpa I单酶切pChAd63,以DNA Assembly方法,获得重组腺病毒载体pChAd63/EGFP。最后,在293细胞中拯救获得重组腺病毒rChAd63/EGFP。结果:采用DNA Assembly方法成功构建了重组腺病毒载体pChAd63/EGFP,并拯救出重组腺病毒rChAd63/EGFP。结论:DNA Assembly可用于重组腺病毒载体的构建,有利于提高重组腺病毒载体的构建效率。

Efficient de novo assembly and modification of large DNA fragments

Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes.The construction of the large,gigabase(Gb)-sized genomes of higher organisms will deepen our understanding of genetic blueprints significantly.But for the synthesis and assembly of such large-scale genomes,the development of new or expanded methods is required.In this study,we develop an efficient pipeline for the construction of large DNA fragments sized 100 kilobases(kb)or above from scratches and describe an efficient method for“scar-free”engineering of the assembled sequences.Our method,therefore,should provide a standard framework for producing long DNA molecules,which are critical materials for synthetic genomics and metabolic engineering.

Gel electrophoresis as a nanoseparation tool serving DNA nanotechnology

The past years have witnessed a rapid development of DNA nanotechnology in nanomaterials science with a central focus on programmable material construction on the nanoscale. An efficient method is therefore highly desirable（but challenging） for analytical/preparative purification of DNA-conjugated nano-objects and their DNA-assemblies. In this regard, agarose gel electrophoresis, a traditional technique that has been invented for biomacromolecule separation, has found many innovative uses.This includes shape, size, charge, and ligand-valence separations of nanoparticle building blocks as well as monitoring a self-assembly process towards product identification and purification.

RNA nanostructure transformation into DNA ones

RNAs and their assemblies can form diverse nano-structures and nano-shapes,offering various biological functions.However,by simply mimicking RNA sequences,DNAs cannot normally form the corresponding nanostructures,which makes the natureinspired transformations and designs challenging.To understand the possible designs and connections between related RNA and DNA nano-shapes,herein we have reported several DNA squares transformed and derived from an RNA assembled square,by gradually replacing RNA with DNA nucleotides.We have found that there were key RNA nucleotides:Their presences maintained the square,while their absences disrupted it.Interestingly,we have revealed that as long as the conditions of higher ionic strengths or longer duplexes were included,the square RNA assembly could be completely replaced with DNA nucleotides,still offering the stable DNA nano-shapes.Our experimental results have demonstrated that similar RNA nanostructure can be easily transformed into DNA ones via designing and increasing ionic strengths or duplex lengths.

The formation of fibers via complementary base pairing of DNA-conjugated bovine serum albumin

Assembled protein-based substances are emerging and promising classes of materials that provide unique properties for various applications in biotechnology and nanotechnolegy. Self-assembly is an effective way to immobilize protein. In this study, DNAs-conjugated bovine serum albumin （BSA） assembled into fibers via DNA hybridization is demonstrated. The morphology of fibers was observed by optical microscopy, scanning electron microscopy （SEM）, and atomic force microscopy （AFM）, and the assembly mechanism was then analysed and discussed. BSA molecules were first linked by DNA molecule and formed linear chains. These chains then were parallelly linked through additional DNA hybridization. Finally, several BSA chains further assembled into fibers by layering lamellae in a parallel manner. This work perhaps will provide a guide to the immobilization of enzyme, which could be applied to increase its catalytic efficiency in biomedicine and nanotechnology.

RPA functions as a platform for coupling of nucleosome assembly to DNA replication

Subject Code:C05 With the support by the National Natural Science Foundation of China,the research team led by Dr.Li Qing(李晴)at the State Key Laboratory of Protein and Plant Gene Research,School of Life Sciences and Peking-Tsinghua Center for Life Sciences,Peking University,Beijing,recently reported that

Rapid Shape Memory and p H-modulated Spontaneous Actuation of Dopamine Containing Hydrogels

The dopamine containing hydrogels with rapid responsive shape memory capability were synthesized by a one-pot method. The temporary shape of hydrogel was fixed within 20 s in Na OH solution by the tris-complex crosslinking of metalligand complex between Fe3＋ ions and catechol groups, while the permanent shape was recovered completely in HCl solution within 60 s upon the change from tris-complex to mono-complex. The hydrogel showed unique spontaneous actuation behavior. It could curl spontaneously without further external force deformation when immersed in Na OH solution again after the first shape recovery in HCl solution. This might be attributed to the competitive result of swelling and additional tris-complex crosslinking formation when immersed in Na OH solution. In addition, the hydrogels also had proper modulus, elongation ratio and tensile strength. Such hydrogel provides a new candidate material for designing soft actuators and robots modulated with spontaneous actuating.