Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs

Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed “toolkits” utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.     

噬菌体与宿主菌间的角逐

噬菌体和它们的宿主菌组成了地球上教目最庞大的微生物种群,噬菌体靠寄生宿主菌来扩增繁衍。但在漫长的进化中,噬菌体与宿主菌间不单是捕食关系,它们间还形成了复杂的相互对抗机制,其中抵抗防御机制的抗防御机制也会促使抗．抗防御机制的产生。从生态学角度来看,噬菌体和宿主菌间的共进化保持着动态平衡。本文综述近年来这一领域的研究,为更清楚地了解噬菌体与宿主菌间的关系和应用提供了参考。     

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Bacteriophage–host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.     

Propagation of fluorescent viruses in growing plaques

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.     

Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia

Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.