LNA blockers for improved amplification selectivity

Abstract

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.

Figure 1

Potential interactions among intended reactants during PCR blockage. Potential intra- and inter-molecular interactions are shown for the PCR primer (black), the LNA blocker (red), the target DNA (blue), and the DNA polymerase (green). The DNAs/LNA can undergo folding into interfering secondary structures (1). The primer/blocker can potentially interact in a non-productive complex (2) with each other or in a productive or non-productive complex with the target DNA (3). LNAs have a slower off-rate versus DNAs. The DNA polymerase can then bind the primer/blocker/target complex (4) and either extend the primer to generate an Amplified Target (5) or, if polymerization is halted by the LNA when the LNA is bound to the target DNA, a Blocked Target (6).

Figure 2

Template_0 target DNA and primer/LNA binding locations. The target oligonucleotide to be blocked (Template_0) includes a left primer region (1–43), a multiple cloning site (MCS) with SbfI, XbaI, and SnaBI sites (44–59), AAV2 sequence (4495–4558 in http://www.ncbi.nlm.nih.gov/nuccore/AF043303.1, 60–124, shown in red) and a right primer region (125–175). Two forward primer sequences on the left, F1 and F3, and three reverse primers on the right, R1, R3A, and R3B (blue arrows) were used to amplify the sequence with or without LNA blockers. Each LNA blocker (green double arrows) was made in four versions, two corresponding to the top strand (F, forward) and two corresponding to the bottom strand (R, reverse). The pair of F LNAs and the pair of R LNAs were synthesized starting with LNAs at either position 1 or position 2. These are listed individually in Table 2 and Table S1.

Figure 3

Predicted and observed mismatch read frequencies. (A) The predicted mismatch frequency (solid fill) and observed variant read frequency for unblocked samples (hatched fill) as a percent of total reads normalized by sample for 0–10 variants is shown for degenerate DNA lengths of 18 (blue), 20 (gray), and 21 (red) nt. These predictions were made using the combination equation in the text where R = 0.79, L = 18 or 20 or 21 and MM = 0–10. > 99% of all molecules with these degeneracy lengths and 79% reference sequence should have 10 or fewer variants so higher variant values are not shown. Even though the 24D blockers are 24 nt long, a length of 21 is used for its calculations because only 21 of the 24 positions on the target DNA are degenerate. The calculated frequencies are nearly identical to the observed variant frequencies in DNA reads when the amplification is not blocked by LNAs (Table S2). (B) For forward (F2) or reverse (R2) blockers covering 18, 20, or 21 degenerate positions, the percent of reads for each sample versus no LNA present is shown for each mismatch value. The number of mismatches allowed while still retaining blocking activity varies by length.

Figure 4

Blocking as function of position. (A) The relative read frequency for amplification with blockers versus amplification with no LNAs is shown as a function of position. In panel A, the frequency of reads with single mismatches is shown for 18 (red) and 20 (blue) nt LNA blockers. Blockers identical to the forward strand are shown with solid lines/symbols, and blockers identical to the reverse strand are shown with dashed lines/open symbols. The overall shape for all forward primers and for all reverse primers is similar. (B) LNAs blockers that cover 21 degenerate positions are shown with data for reads for one mismatch (1MM, solid lines/symbols) and for reads with two mismatches (2MM, dashed lines/open symbols). Blockers identical to the forward strand are shown in blue, and the reverse strand in red. Blocking in the presence of mismatches is not necessarily desirable as it indicates a lack of selectivity.

Figure 5

Blocking A1AT. (A) The sequence surrounding the PiZ allele in A1AT is shown (central base, R/Y). G/C is the wild-type sequence and A/T is the PiZ allele. LNAs are named with an F if identical to the top strand and R if identical to the bottom strand. The number adjacent to F/R indicates where the PiZ allele is in the LNA. Individual LNAs and how they overlap are shaded. Because each LNA varies by two in length, the alternating LNAs are positioned identically in these series. Based on potency in initial studies at 1 and 10 µM, concentrations were titrated to obtain good but not complete blocking at the lowest concentrations used. The lowest concentration for A1AT_R1-2 was 0.1 µM, followed by A1AT_F3-2, A1AT_R3-2, and A1AT_R5-2 at 0.2 µM, A1AT_F5-2 and A1AT_R7-2 at 0.5 µM, A1AT_F7-2 at 1 µM, and A1AT_F1-2 at 3.3 µM. (B) Blocking of wild-type DNA with LNA concentrations listed in (A). (C) Blocking of PiZ DNA with LNA concentrations listed in (A). (D) Blocking of PiZ DNA with LNA concentrations 16-fold higher than listed in (A). Data is not shown for the 16 × wild-type DNA because all LNAs completely blocked amplification.