核酸化学特性课件.ppt

* (1) Self Defense Defense against virus and exogenous DNA invasion Function： (2)Used as a gene knockdown tool * RNA干扰机制 1、启动阶段： dsRNA被Dicer酶逐步切割成siRNA （ short interfering RNA ） 。 2、效应阶段: ① siRNA与酶聚集形成RNA诱导沉默复合物Risc（RNA-induced silencing complex）。 ② Risc的激活： siRNA双链解开。 ③ 激活的Risc定位到同源mRNA上，降解该mRNA。 Dicer RNase III家族成员－－核酸内切酶，特异性识别并切割dsRNA * mechanism of RNAi RISC：RNA-induced silencing complex （RISC） Genome has genes that code for microRNAs Regulate 30% of mammalian gene products Have been described in invertebrates and vertebrates: worms, fungi, plants, and mammals Many are conserved between vertebrates and invertebrates 7. microRNA(miRNA） Definiton： a noncoding RNA with stem-loop structure, typically 20-24nucleotides in length. Paired with target mRNA, arise mRNA cleavage or translational repression. * Esquela-Kerscher, A., and Slack, F.J. (2006). Nature Reviews 6, p. 263 Biogenesis -Primary-miRNA is transcribed in the nucleus, and is usually several kilobases long; posses 5’ cap and a poly-A tail. -Cleaved in the nucleus by Drocha enzyme to 70nt hairpin transcript (pre-miRNA). -Transported to the cytoplasm by Exportin 5 through nuclear pores. -Cleaved by Dicer into 20-24nt ds-transcripts. -one strand miRNA is incorporated into the RNA-induced silencing complex (RISC) -paired with target mRNA and lead to mRNA cleavage or translational repression。 * siRNA miRNA * * (I) General Properties 1.Macromolecule：Mammals 3×109bp Unit of Nucleic acid size （1）Dalton （2）base pair（bp） （3）chain length（?m） （4）sedimentation coefficient（S） 3000bp=2×106 Dal=1 ?m Methods to analyze the molecule size： Gel Electrophoresis，Ultracentrifugation， Sequencing IV. Physiologic chemical Properties * Sanger Sequencing: key reagent: dideoxynucleotides triphosphates (ddNTP) When a ddNTP is incorporated elongation of the primer is terminated The ddNTP is specifically incorporated opposite its complementary nucleotide ba