Figure 2d illustrates the formation of the eight-helix bundle from the arched four-helix bundle.
Blunt molecules are used to avoid aggregation. Lane 1 contains the arched four-helix bundle.
Note the clean band visible there, indicating proper target formation. Lane 2 contains the arched
four-helix bundle capable of binding to itself only at Point A (See Figure 1a), and lane 3 contains
the same material, except that now it is capable of binding to itself only at Point B. In both
cases, the lanes contain smears and breakdown products below the main bands. The most
prominent of these other features are of greater molecular weight than the 4HB monomer and
may indicate species that are dynamically closed (particularly for lane 2) or dissociated from
one or both of the linkages. Lane 4 contains the arched four-helix bundle, now capable of
binding to itself at both Points A and B. The band is clean, and there is no extraneous material
in the lane. This is an important finding, because it indicates that both connection points are
necessary to make a well-defined product. The significantly higher mobility than the linear
duplex markers of equivalent molecular weight is likely a consequence of the lowered surface
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
area of the circular 8HB motif, as observed with 6HB previously.10 The difference in the
mobilities between the two open 8HB species is probably because of irregular inner angles of
each edge at the helices. Consistently, open 8HB molecules connected at the self-assembly
points with wider inner angles (point A with 171°, lane 2) migrate more slowly than the
molecules with a narrower inner angle (point B with 120°, lane 3).