A rule of seven in Watson-Crick base-pairing of mismatched sequences

Abstract

Sequence recognition through base-pairing is essential for DNA repair and gene regulation, but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized, despite its potential importance in nucleic acid-based biotechnologies and gene silencing. Here we use single-molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to precisely quantify the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement for seven nucleotides of complementarity to seed gene silencing by small noncoding RNA and may help guide performance improvement in DNA- and RNA-based bio- and nanotechnologies, in which off-target effects can be detrimental.

Figure 1

Single-molecule porous vesicle encapsulation assay. (a) Schemes for the 9 bp DNA duplex and (b) DNA encapsulation; (c–l) Salt dependent dynamics for 9 bp DNA in 200 nm vesicles: single molecule time traces (c, d, e, f), and histograms from over 100 vesicles (g, h, i), at 5 mM (c, d, g), 20 mM (e, h), and 50 mM Na⁺ (f, i). K_d, k_off, and k_on vs [Na⁺] (j, k, l); 541 vesicles were used to calculate the rates in j, k and l. All error bars represent standard error from triplicate experiments at 23 °C. In c, green curve denotes the donor intensity and red the acceptor intensity.

Figure 2

The effect of terminal base pair mismatch. (a) K_d, (b) k_off, and (c) k_on at 10 mM Na⁺; A total of 309 vesicles were used to calculate the rates in this figure.

Figure 3

The effect of DNA mismatch position. (a, e) Schemes of mismatched constructs; (b) K_d, (c) k_off, and (d) k_on were measured at 23 °C, 10 mM Na⁺; (e) For mismatch positions 5 through 9, k_on was additionally measured at 33 °C, 150 mM Na⁺ and (f) at 37 °C, 200 mM Na⁺; A total of 723 vesicles were used for b, c, and d, 937 vesicles in f, 511 vesicles in g, and 697 vesicles in i.

Figure 4

The effect of mismatch position on RNA. (a) K_d, (b) k_off, and (c) k_on for 8 bp RNA constructs with 7 and 6 contiguous base pairs at 23 °C, 5 mM Na⁺; A total of 228 vesicles were used to calculate the rates in this figure.