PMC

Calculated ΔT_m dependency on oligonucleotide length by single- and two-point mutations excluding the three end points. Filled diamonds represent single-point mutations, filled squares two-point mutations, and open diamonds represent 2 × ΔT_m by single-point mutations. Dotted lines are fitted graphs.

Specific sequence changes correlated with significantly different calculated ΔT_m of 20mer oligonucleotides in the distance between mutation sites at fixed position 10. The distances from position 10 are one (filled diamond), two (filled square), three (open triangle) and four (open circle).

Correlation between theoretical T_m and experimental T_d from Urakawa et al. (). (A) T_m was calculated using OMP software. (B) T_m was calculated using equation 2 and the parameters in the table in Supplementary Material. Filled diamonds are from spm (oligo1) and its mismatched pairs and open squares are from npm (oligo2) and its mismatched pairs in Urakawa et al. ().

Calculated ΔT_m dependency on the reference position and the distance between two mutation sites by two-point mutations of 20mer oligonucleotide. Open blue diamonds represent reference position 1, open red squares reference position 2, open green triangle position 3, filled red squares position 4, filled blue diamonds position 5, filled green squares position 6, filled orange triangles position 7, filled pink circles position 8, filled red diamonds position 9 and filled black square position 10. Only reference positions 6 and 10 display standard deviations. Certain reference position and distances, explained in the main text, are annotated.

Hybridization T_m dependency (A) and T_m variance (ΔT_m) dependency (B) on oligonucleotide length due to single-point mutation based on OMP calculation. Open diamonds represent data obtained from oligo sequences of EphB1 mRNA. Filled squares represent data obtained from IgG Fc binding protein mRNA. Shorter oligos are subsets of longer ones. Dotted lines in B are fitted graphs providing the exponent α in equation 5 in the main text. One original oligo pair in (A) produces (3 × oligo length) mutated pairs in (B).

Schematic diagrams of oligonucleotides. (A) Original T_m is defined as the hybridization T_m of the perfect match pair. The point-mutated site is described in × and the mutated T_m is defined as their hybridization T_m. Three possible mutations occur at each site. (B) The mutation site is defined as the position from the end of the oligos, ignoring 3′ and 5′ direction. A single-point mutation is performed at position 5. (C) In the two-point mutations, the reference position is defined as a smaller position number between two mutation sites. The distance is defined as 1 plus the number of nucleotides between them.

Calculated ΔT_m dependency on nucleotide mutation and position in 20mer oligonucleotides. (A) Average ΔT_m values of all sequence changes at each position are shown with standard deviations. (B) Average ΔT_m values of each sequence change are shown. Data point colors represent the original sequences: blue, A; pink, T; green, C; and orange, G, while shapes represent the mutated sequences: square, A; diamond, T; circle, C; and triangle, G (blue diamonds, from A to T; blue circles, A to C; blue triangles, A to G; pink squares, T to A; pink circles, T to C; pink triangles, T to G; green squares, C to A; green diamonds, C to T; green triangles, C to G; orange squares, G to A; orange diamonds, G to T; and orange circles, G to C). (C) ΔT_m dependency on nucleotide in 20mer oligonucleotides excluding the three end points. (D) All ΔT_d values of each sequence change from Urakawa et al. () are shown at each position from the 5′-end excluding the three end positions. Data from oligo 1 and oligo 2 in Figure are not distinguished. Color and shape representations are as in (B). A data point indicated with an arrow is a [G, C] mismatch case, which will be ignored in the comparison.