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首页> 外文会议>BioMEMS and Nanotechnology II; Progress in Biomedical Optics and Imaging; vol.6 no.40 >Porous Silicon Microparticles as an Alternative Support for Solid Phase DNA Synthesis
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Porous Silicon Microparticles as an Alternative Support for Solid Phase DNA Synthesis

机译：多孔硅微粒作为固相DNA合成的替代支持

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摘要

Current methods to produce short DNA strands (oligonucleotides) involve the stepwise coupling of phosphoramidites onto a solid support, typically controlled pore glass. The full-length oligonucleotide is then cleaved from the solid support using a suitable aqueous or organic base and the oligonucleotide is subsequently separated from the spent support. This final step, albeit seemingly easy, invariably leads to increased production costs due to increased synthesis time and reduced yields. This paper describes the preparation of a dissolvable support for DNA synthesis based on porous silicon (pSi). Initially it was thought that the pSi support would undergo dissolution by hydrolysis upon cleavage of the freshly synthesised oligonucleotide strands with ammonium hydroxide. The ability to dissolve the solid support after completion of the synthesis cycle would eliminate the separation step required in current DNA synthesis protocols, leading to simpler and faster synthesis as well as increased yields, however it was found that the functionalisation of the pSi imparted a stability that impeded the dissolution. This strategy may also find applications for drug delivery where the controlled release of carrier-immobilised short antisense DNA is desired. The approach taken involves the fabrication of porous silicon (pSi) microparticles and films. Subsequently, the pSi is oxidised and functionalised with a dimethoxytrityl protected propanediol to facilitate the stepwise solid phase synthesis of DNA oligonucleotides. The functionalisation of the pSi is monitored by diffuse reflectance infrared spectroscopy and the successful trityl labelling of the pSi is detected by UV-Vis spectroscopy after release of the dimethoxytrityl cation in the presence of trichloroacetic acid (TCA). Oligonucleotide yields can be quantified by UV-Vis spectroscopy.
机译：当前产生短DNA链（寡核苷酸）的方法包括将亚磷酰胺逐步偶联到固体载体上，该载体通常是可控的孔玻璃。然后使用合适的水性或有机碱从固体支持物上切割全长寡核苷酸，随后将寡核苷酸与用过的支持物分离。尽管看起来很容易，但是最后的步骤由于合成时间的增加和产率的降低而总是导致生产成本的增加。本文介绍了基于多孔硅（pSi）的DNA合成可溶性载体的制备。最初认为，在用氢氧化铵裂解新合成的寡核苷酸链时，pSi载体将通过水解而溶解。合成循环完成后溶解固相支持物的能力将消除当前DNA合成规程中所需的分离步骤，从而导致更简单，更快的合成以及提高的产率，但是发现pSi的功能化赋予了稳定性阻止了解散。在需要控制释放固定有载体的短反义DNA的情况下，该策略还可用于药物递送。所采用的方法涉及多孔硅（pSi）微粒和薄膜的制造。随后，用二甲氧基三苯甲基保护的丙二醇氧化和官能化pSi，以促进DNA寡核苷酸的逐步固相合成。通过扩散反射红外光谱法监测pSi的功能化，并在三氯乙酸（TCA）存在下释放二甲氧基三苯甲基阳离子后，通过UV-Vis光谱法检测pSi的成功三苯甲基标记。寡核苷酸产率可以通过UV-Vis光谱法定量。

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