Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation
Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation
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DOI：
10.1016/j.bioeng.2005.07.003
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发表时间：
2005-12-01
期刊：
影响因子：
--
通讯作者：
Smith, CIE
中科院分区：
文献类型：
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作者：
Lundin, KE;Hasan, M;Smith, CIE
低细胞摄取和差的核转移阻碍了非病毒载体在基因治疗中的使用。使用Bioplex技术向质粒中添加功能实体具有显著提高转移效率的潜力。我们研究了通过将肽核酸（PNA）和锁核酸（LNA）低聚物作为“开放器”与相对DNA链上的部分重叠位点杂交来稳定肽核酸（PNA）锚定功能肽与质粒DNA的序列特异性结合的可能性。PNA“开放器”稳定了“线性”PNA锚与盐水中的混合碱基超螺旋DNA的结合。为了在生理条件下获得更高的稳定性，使用了bisPNA锚。为了在与高度阳离子构建体杂交时减少非特异性相互作用，并且为了在分子不带电荷或带负电荷时适应对增加量的bisPNA的需要，我们使用PNA和LNA寡聚体作为“开放剂”以增加结合动力学。据我们所知，这是LNA首次与PNA一起用于促进链侵入。该程序允许在降低的PNA与质粒比率下杂交，即使在低至2：1的比率下也允许大于80%的杂交。使用显著较低量的PNA-肽结合较短的孵育时间减少了非特异性结合并促进纯化。(C)2005 Elsevier B. V.保留所有权利。
Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2: 1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification. (C) 2005 Elsevier B.V. All rights reserved.