DNA base pair resolution by single molecule force spectroscopy

Abstract

The forces that hold complementary strands of DNA together in a double helix, and the role of base mismatches in these, are examined by single molecule force spectroscopy using an atomic force microscope (AFM). These forces are important when considering the binding of proteins to DNA, since these proteins often mechanically stretch the DNA during their action. In AFM measurement of forces, there is an inherent instrumental limitation that makes it difficult to compare results from different experimental runs. This is circumvented by using an oligonucleotide microarray, which allowed a direct comparison of the forces between perfectly matched short oligonucleotides and those containing a single or double mismatch. Through this greatly increased sensitivity, the force contribution of a single AT base pair was derived. The results indicate that the contribution to forces from the stacking interactions is more important than that from hydrogen bonding.

Figure 1

The design. (A) The ODNs used have a hexacarbon thiol-terminated linker attached to the 3′ end. (B) The TIP sequence is attached to the AFM probe tip. The MATCH, 1MIS, 2MIS and TIP (control) are arrayed on the gold substrate. (C) In FS, depiction of three possible overlaps between the TIP and the ODNs: 60, 80 or 100%.

Figure 2

Typical force curves corresponding to none to three rupture events (top to bottom). The final rupture event, due to breaking a single pair interaction, is the only event counted.

Figure 3

The rupture force for MATCH (square), 1MIS (circle) and 2MIS (triangle) against (A) the number of stacked bases in the helix at the time of the rupture event, and (B) the number of H-bonds present. Inset of (A), typical error bars. The box labeled ‘20’ shows the rupture force for 20 stacked bases over the three data sets. The box labeled ‘48’ shows the rupture force for 48 H-bonds in the TIP/MATCH and TIP/1MIS data sets.

Figure 4

(A) Absolute and (B) relative force cost of H-bonding for AT (square), AC/TG (circle) and GC (triangle) depending on the helical length.

Figure 5

(A) Absolute and (B) relative force cost of base stacking for AT (square), AC/TG (circle) and GC (triangle), depending on the length of DNA duplex.

Figure 6

Positional force cost of total loss of base pairing. Difference between consecutive datapoints of the same data set (Figure 4). Solid bars represent AT base pair, hashed bars represent GC base pair, white for TIP/MATCH, gray for TIP/1MIS and black for TIP/2MIS.

Figure 7

Average cost of base pairing. The average rupture force of AT (meshed) and GC (hashed) base pairs as a function of helical length.