LNA blockers for improved amplification selectivity

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.