Preparation and preliminary biological evaluation of DNA bipyramid fluorescent molecular probes
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摘要:
目的 构建基于DNA三角双锥纳米结构（DBN）的荧光分子探针，于活体动物水平探索探针的生物学性质。 方法 采用一步退火法，制备携带侧臂链的DBN；与DyLight 755偶联的寡聚核苷酸单链A20（DyLight 755-A20）混匀，制备荧光分子探针DyLight 755-DBN；将DyLight 755-DBN通过尾静脉注射实验鼠体内，于不同时间点处死，取感兴趣脏器测定荧光计数，探索分子探针的体内分布情况；将DyLight 755-DBN通过尾静脉注射体内，于不同时间点行小动物活体成像研究。 结果 成功制备携带侧臂链的DBN，利用聚丙烯酰胺凝胶电泳表征；通过与DyLight 755-A20等摩尔量杂交，成功制备荧光分子探针DyLight 755-DBN。体内分布实验显示DyLight 755-DBN进入体内后，荧光信号主要集中在肾脏、肝脏、脾脏、胃；注射分子探针5~15 min，在实验鼠的肺部有一定的荧光信号，但15 min后，荧光信号几乎没有。活体成像结果显示DyLight 755-DBN进入实验鼠体内主要集中在肝脏和胃，膀胱中维持较强的荧光信号。 结论 荧光分子探针DyLight 755-DBN是一种性质优良的分子影像探针。 -
关键词:
- DNA三角双锥纳米结构 /
- 荧光分子探针 /
- 体内分布 /
- 活体成像
Abstract:Objective To construct a kind of fluorescent molecular probes based on DNA Bipyramid Nanostructure (DBN), and to explore the biological properties of the probes at the level of living animals. Methods DBN with side arm chains, which was prepared through the one-step annealing method, was mixed with single-stranded oligonucleotide A20 coupled with DyLight 755 (DyLight 755-A20) to prepare fluorescent molecular probe DyLight 755-DBN. DyLight 755-DBN was injected into the mice through the tail vein, then the mice were executed at different time points, and fluorescence counts were taken from organs of interest to explore the in-vivo distribution of the molecular probes. DyLight 755-DBN was injected into the mouse through the tail vein, and in-vivo imaging studies of small animals were performed at different time points. Results The DBN with side arm chains was successfully prepared and characterized by polyacrylamide gel electrophoresis. The fluorescent molecular probe DyLight 755-DBN was successfully prepared by the equimolar hybridization of DBN with side arm chains and DyLight 755-A20. In vivo distribution experiments showed that after DyLight 755-DBN was entering the body, the fluorescent signal was mainly concentrated in the kidney, liver, spleen and stomach. There was a certain fluorescent signal in the lungs of experimental mouse during 5-15 min, but the fluorescence signal was almost absent after 15 min. In vivo imaging results showed that the fluorescent signal of DyLight 755-DBN was mainly concentrated in the liver and stomach, and a strong fluorescent signal in the bladder. Conclusion The fluorescent molecular probe DyLight 755-DBN is a kind of molecular imaging probe with excellent properties. -
图 1 方案一：DBN及荧光分子探针Dylight755-DBN的制备
Figure 1. Option 1: Preparation of DBN and fluorescent molecular probe Dylight755-DBN
图 2 利用聚丙烯酰胺凝胶电泳技术表征T20-DBN
Figure 2. Characterization of T20-DBN by Polyacrylamide Gel Electrophoresis
图 3 DyLight 755-DBN在实验鼠体内的分布情况
Figure 3. Distribution of DyLight 755-DBN in experimental mice (n=3)
图 4 DyLight 755-DBN注射后不同时间点的实验鼠活体显像图
注: 上排: 俯卧位; 下排: 仰卧位.
Figure 4. In vivo imaging of experimental mice at different time points after injection of DyLight 755-DBN
表 1 DNA寡聚核苷酸单链的序列信息
Table 1. Sequence information of DNA oligonucleotide single strand
ssDNA 序列信息 a AGGCAGTTGAGACGAACATTCCTAAGTCTGAAATTTATCACCCGCCATAGTAGACGTATCACC b CTTGCTACACGATTCAGACTTAGGAATGTTCGACATGCGAGGGTCCAATACCGACGATTACAG c GGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCCACTACTATGGCG A CTGCGCGGATGACTCAACTGCCTGGTGATACGATCTAGTCTCTACGTCAAGTAAGAACCTTAG B CCTCGCATGACATCCGCGCAGCTAAGGTTCAAAGTTCCTGCCGCTTCACGGACGGTATTGGAC C CTCTTCCCGACCGTGAAGCGGCAGGAACTTATACTTGACGTAGAGACTAGAAGGATGGGCATG T20-a* TTTTTTTTTTTTTTTTTTTTCTAAGTCTGAAATTTATCACCCGCCATAGTA GACGTATCACCAGGCAGTTGAGACGAACATTC Dylight755-A20 Dylight755-AAAAAAAAAAAAAAAAAAAA 
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