LNA-containing oligonucleotides bind DNA more tightly than standard DNA, so they can interact with targeted sequences and affect multiple processes. When a desired DNA is present at low concentrations relative to nearly identical undesired DNAs, LNAs can block amplification of unwanted DNAs. Using a short rAAV and synthetic DNA sequence as a model, we studied the length, number, and positioning of LNA bases to improve blocker effectiveness. Oligonucleotides 18–24 bases long with LNAs at every other position were most effective. Highly degenerate targets were used to characterize the impact of mismatches on blocking. Mismatches at LNA ends had little impact on blocking activity. Single and double mismatches were tolerated with longer blockers, especially if the mismatches were near LNA ends. Shorter LNAs were more selective, with > 1 mismatch preventing effective blocking. Neither the strand to which a blocker bound nor the distance between the blocker and priming sites greatly impacted blocking efficiency. We used these findings to design blockers of wild-type DNA versus the single-base A1AT PiZ allele. Blockers are most specific when the mismatch is located away from the LNA 5′ end. Pairs of partially overlapping blockers on opposite strands with a centrally-located mismatch have maximal activity and specificity.

含有 LNA 的寡核苷酸比标准 DNA 更紧密地结合 DNA，因此它们可以与目标序列相互作用并影响多个过程。当所需的 DNA 相对于几乎相同的不需要的 DNA 以低浓度存在时，LNA 可以阻止不需要的 DNA 的扩增。我们使用短 rAAV 和合成 DNA 序列作为模型，研究了 LNA 碱基的长度、数量和定位，以提高阻断剂的有效性。18-24 个碱基长的寡核苷酸在每个其他位置都有 LNA 是最有效的。高度退化的目标被用来描述不匹配对阻塞的影响。LNA 末端的不匹配对阻断活动几乎没有影响。较长的阻滞剂可以容忍单错配和双错配，尤其是当错配靠近 LNA 末端时。较短的 LNA 更具选择性，具有 > 1 不匹配阻止有效阻塞。阻断剂结合的链以及阻断剂和启动位点之间的距离都不会显着影响阻断效率。我们使用这些发现来设计野生型 DNA 与单碱基的阻断剂A1AT PiZ 等位基因。当错配位于远离 LNA 5' 端时，阻滞剂最为特异。位于中心位置的错配的相反链上的成对部分重叠的阻断剂具有最大的活性和特异性。