Cell-Penetrating Aptamers Targeting Sub-Cellular Compartments

靶向亚细胞室的细胞穿透适配子 摘要：现代纳米医学中一个尚未得到满足的需求是一种有效地fi输送核酸的方法 和相关的货物进入活组织中的相关亚细胞隔间。当细胞暴露在 核酸或核酸/阳离子脂类络合物，常规方法会导致膜摄取- 结合的小泡(仍在细胞外的拓扑学上)或不加区别的膜融合。此外，许多人 载体脂质制剂是有毒的，在体内的使用有限。而体外选择(SELEX)的力量 以前被应用于选择结合细胞或获得优先囊泡摄取的核酸序列 进入特定的fic细胞或组织，通过细胞内靶向的囊泡逃逸还没有被设想。我们有 为实现这一目标开发了一种新的方法。我们应用体外选择来鉴定核酸适配子 EFfi通过选择经过酶处理的序列来进入特殊的fic亚细胞室 Modifi阳离子依赖于fic胞内酶活性。我们证明了这种“奖励”方法 可以识别裸露的DNA适配子，大大改善了对细胞核的递送(“亲核” 适配子)。该方法将扩展到识别不同组织的细胞穿透适配子fic。 活着的老鼠。能够使囊泡逃逸和亚细胞室递送的奖励适配子序列 开启了一个充满机遇的新fi领域。在未来，可能会扩展这种奖励方法，以确定 以其他亚细胞室为目标的序列。四个特定的fic目标被用来检验假设。 靶向特定fic亚细胞室的细胞穿透性dna适配子可通过fi鉴定 选择由细胞器特有的fic酶活性调节的分子，以及这种归巢适配子 可以有效地将fi的货物运送到细胞和组织。目标1将继续选择亲核(核- 寻的)DNA适配子，为靶向其他亚细胞室奠定了基础。目标2将寻求 了解亲核DNA适配子的核递送机制。目标3将探索以下能力 亲核DNA适配子将货物直接输送到细胞内。Aim 4将在体内将这种奖励方法扩展到 建立小鼠组织特异性fic亲核DNA适配子文库。

Cell-penetrating aptamers targeting sub-cellular compartments ABSTRACT: An unmet need in modern nanomedicine is a method for efficient delivery of nucleic acids and related cargo into relevant sub-cellular compartments in living tissues. Upon exposure of cells to nucleic acids or nucleic acid/cationic lipid complexes, conventional methods result in uptake into membrane- bound vesicles (still topologically outside of the cell) or indiscriminate membrane fusion. Moreover, many carrier lipid formulations are toxic and of limited use in vivo. While the power of in vitro selection (SELEX) has been previously applied to select nucleic acid sequences that bind cells or gain preferential vesicular uptake into specific cells or tissues, vesicular escape with intracellular targeting has not been envisioned. We have developed a novel approach to this goal. We apply in vitro selection to identify nucleic acid aptamers that efficiently enter specific sub-cellular compartments by selecting for sequences that undergo enzymatic modification dependent on specific intracellular enzyme activities. We show that this "reward" approach can identify naked DNA aptamers with substantially improved delivery to the cell nucleus (“karyophilic” aptamers). The method will be extended to identify cell-penetrating aptamers specific for different tissues in living mice. Rewarding aptamer sequences capable of vesicle escape and sub-cellular compartment delivery opens a new field of opportunities. In the future it may be possible to extend this reward approach to identify sequences that target other sub-cellular compartments. Four specific aims are proposed to test the hypotheses that cell-penetrating DNA aptamers targeting specific sub-cellular compartments can be identified by selecting molecules modified by organelle-specific enzyme activities, and that such homing aptamers can efficiently deliver cargo to cells and tissues. Aim 1 will continue our selection of karyophilic (nucleus- homing) DNA aptamers, setting the stage for the targeting of other sub-cellular compartments. Aim 2 will seek to understand the mechanism of nuclear delivery of karyophilic DNA aptamers. Aim 3 will explore the ability of karyophilic DNA aptamers to direct cargo delivery into cells. Aim 4 will extend this reward approach in vivo to develop a library of tissue-specific karyophilic DNA aptamers in mice.