Isolation of MLL1 Inhibitory RNA Aptamers

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (Systemic Evolution of Ligands by Exponential enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers—APT1 and APT2—represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5′-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3′. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.

Inhibitory effects of TLR7 on Mycobacterium tuberculosis / 中国人兽共患病学报

ABSTRACT:The aim of the present study was to investigate the inhibitory effects of TLR7 on Mycobacterium tuberculosis . TLR7 on infected RAW264 .7 cells was activated by chemical synthesis of TLR7 activation motif ssRNA .Activated RAW264 .7 cells were inoculated with Mycobacterium tuberculosis ,quantitative PCR method was applied to detect the phagocytosis rate of cell to bacteria at different time after infection .Cytokine production was measured by ELISA from cell supernatant .Cells were cultured on Roche medium and counted after sterile cracked with TritonX‐100 and diluted with PBS .Scanning electronic micro‐scope ( SEM ) was applied to detect the morphological changes of cells treated with TLR7 activation motif ssRNA .The highest phagocytosis rate of bacteria of RAW264 .7 cells was at 3 hours post infection (P>0 .05) .Compared with that of the control group ,treatment after 36 hours intracellular bacterial quantity in ssRNA treated group was lower (P<0 .05) ,levels of IL‐12 (P<0 .05) and IL‐4 (P<0 .05) were increased .For treatment after 48 hours ,level of IL‐4 (P<0 .05) was decreased ,and TNF‐α (P<0 .05) was increased .For treatment after 3 hours ,cell morphology of the ssRNA group was obviously better than the control group and appeared lots of phagosomes .Results suggested that TLR7 could enhance macrophages in killing Myco‐bacterium tuberculosis by forming phagosomes and regulating cytokines production ,and TLR7 activation motif ssRNA could be used in the treatment of tuberculosis .

Interactions of 3' terminal and 5' terminal regions of physalis mottle virus genomic RNA with its replication complex.

Physalis mottle virus (PhMV) belongs to the tymogroup of positive-strand RNA viruses with a genome size of 6 kb. Crude membrane preparations from PhMV-infected Nicotiana glutinosa plants catalyzed the synthesis of PhMV genomic RNA from endogenously bound template. Addition of exogenous gnomic RNA enhanced the synthesis which was specifically inhibited by the addition of sense and antisense transcripts corresponding to 3' terminal 242 nucleotides as well as the 5' terminal 458 nucleotides of PhMV genomic RNA while yeast tRNA or ribosomal RNA failed to inhibit the synthesis. This specific inhibition suggested that the 5' and 3' non-coding regions of PhMV RNA might play an important role in viral replication.