MOLECULAR STUDIES OF GENE TARGETED OLIGONUCLEOTIDES
基因靶向寡核苷酸的分子研究
基本信息
- 批准号:6151246
- 负责人:
- 金额:$ 33.95万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1999
- 资助国家:美国
- 起止时间:1999-02-01 至 2003-01-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
The antisense technology in theory provides a superb method for
validating gene function. However, significant problems exist in the use
of the all-phosphorothioate backbone oligonucleotides. These problems
result from charge, sulfur content, and non-sequence specificity. We
hypothesize that reducing charge density can ameliorate these.
Therefore, in Specific Aim 1 we will synthesize oligos which contain
partially charged, alternating methyklphosphonate (MP)/phosphodiester
(PO) and phosphorothioate (PS) backbones (alt-OMPs). Antisense alt-OMPs
contain 2'-O-methylribonucleosides (alt-mr-OMPs) and are designed to
interact with single-stranded RNA targets. Methods will be developed to
prepare alt-mr-OMPs with MP linkages of defined configuration. Chimeric
oligomers (chi-OMPs) having MP linkages at the 3' and 5'-ends of the
oligomer and internal PO or PS linages will also be examined. In order
to quantitate non sequence specificity, we will determine a rank order
of oligo non-sequence specificity by their ability to bind to Mac-1.
Comparisons will be made between two oligos, antisense c-myb, which is
delivered naked, and antisense PKC-alpha (Isis 3521) which is delivered
with a novel vehicle, a cationic porphyrin. We will then, in Specific
Aim 2, discriminate "true" antisense at the mRNA level from toxicity by:
1) using stereoregular PS oligos because of differential nuclease
cleavage of the 3' terminal PS to a nucleotide monothiophosphate: MP
linkages of defined stereochemistry will also be examined; 2) We will
develop a functional method to determine of alt-mr-OMPs interact with
their mRNA targets in living cells. The procedure will use backbone-
optimized, biotinylated, psoralen-derivatized alt-mr-OMPs which are
designed to form a covalent adduct with mRNA. The resulting restriction
fragment which is attached to the alt-mr-OMP will be captured on
streptavidin beads. The captured fragment will be amplified by PCR using
a set of specific primers. 3) Determining if the c-myb oligo has higher
order structure, and leads to alterations in the rate of endosomal
efflux. We will correlate this with the ability of Specific Aim 1 oligos
to escape from endosomes. These experiments will be performed by a
confocal microscopic technique. Finally, in Specific Aim 3, we will
amplify the delivery of alt-OMPs and chi-OMPs to cells by condensing
them with a novel delivery agent, tetra (N-methylpyridyl) porphine.
理论上的反义技术提供了一种极好的方法
验证基因功能。然而,在使用中存在着重大问题。
全硫代主链寡核苷酸。这些问题
由电荷、硫含量和非序列特异性所致。我们
假设降低电荷密度可以改善这些。
因此,在特定的目标1中,我们将合成包含
部分带电的交替甲基磷酸盐(MP)/磷酸二酯
(PO)和(PS)主干(ALT-OMPS)。反义ALT-OMPS
含有2‘-O-甲基核糖核苷(ALT-MR-OMPS),旨在
与单链RNA靶标相互作用。方法将被开发为
准备具有定义配置的MP链接的ALT-MR-OMPS。嵌合体
在3‘和5’端具有MP键的齐聚物(CHI-OMPS)
还将检查齐聚物和内部PO或PS衬里。按顺序
为了量化非序列特异性,我们将确定一个等级顺序
通过它们与Mac-1结合的能力而具有寡聚非序列特异性。
将对两种寡核苷酸进行比较,反义c-myb,它是
裸体和反义PKC-α(ISIS 3521)
用一种新的载体,阳离子卟啉。然后,我们将具体地
目的2,通过以下方法在信使核糖核酸水平上区分“真正的”反义和毒性:
1)由于核酸酶的差异,使用立体规则的PS寡聚糖
3‘端PS裂解成核苷酸一硫代磷酸:MP
还将审查已定义的立体化学的联系;2)我们将
建立测定ALT-MR-OMPS相互作用的功能方法
它们的信使核糖核酸以活细胞为靶标。该程序将使用主干-
优化的、生物素化的、补骨脂素衍生化的ALT-MR-OMPS
被设计成与信使核糖核酸形成共价加合物。由此产生的限制
附加到ALT-MR-OMP的片段将在
链霉亲和素珠子。捕获的片段将通过使用
一套特定的引子。3)确定c-myb寡核苷酸是否具有更高的
有序结构,并导致内体速率的变化
外流。我们会将其与特定目标1寡核苷酸的能力相关联
从内体中逃脱。这些实验将由一名
共聚焦显微镜技术。最后,在具体目标3中,我们将
通过浓缩扩增ALT-OMPS和CHI-OMPS到细胞的递送
它们与一种新型的递送剂,四(N-甲基吡啶)卟啉。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('Cy A STEIN', 18)}}的其他基金
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6620770 - 财政年份:2002
- 资助金额:
$ 33.95万 - 项目类别:
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6703068 - 财政年份:2002
- 资助金额:
$ 33.95万 - 项目类别:
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6868959 - 财政年份:2002
- 资助金额:
$ 33.95万 - 项目类别:
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6421638 - 财政年份:2002
- 资助金额:
$ 33.95万 - 项目类别:
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6823670 - 财政年份:2002
- 资助金额:
$ 33.95万 - 项目类别:
MOLECULAR STUDIES OF GENE TARGETED OLIGONUCLEOTIDES
基因靶向寡核苷酸的分子研究
- 批准号:
2740016 - 财政年份:1999
- 资助金额:
$ 33.95万 - 项目类别:
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