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Polyadenine helix-coil kinetics

Under physiological conditions, polyadenylic acid [poly(A)] molecules form a secondary structure containing both helical and coiled domains, where the 20–50 base long helical portions are stabilized by stacking interactions of the adenine side groups. The biological functions of poly(A) involves interactions with various proteins, which are expected to significantly alter the kinetics of the poly(A) helix-coil transition as compared to that for free poly(A). To better understand the dynamical properties of poly(A) in the proximity of proteins, we have studied the spontaneous fluctuations of individual poly(A) strands threaded through a narrow protein channel (alpha Hemolysin or a-HL).

To observe poly(A) kinetics, we rely upon the fact that duplex nucleic acids (~2.2 nm) cannot be threaded through the a-HL’s smallest constriction. Therefore, when duplex nucleic acids with a single-stranded overhang are in the cis chamber, the single-stranded portion may be threaded through the pore, but the molecule remains effectively arrested for an extended period of time until the trailing duplex region is completely unzipped (see Figure 1). The left panel of Figure 2 displays representative for three molecules: (i) duplex-dA50, serving as a control (a DNA duplex with a 50- base poly(dA) overhang), (ii) duplex-C25, also serving as a control (a RNA duplex with a 25-base poly(C) overhang), and (iii) duplex A25, a RNA duplex with a 25-base poly(A) overhang. The duplex region in all molecules has the same 10-bp sequence. Notabely, only the ion current signal associated with duplex A25 displays large discrete fluctuations between two current states (8.4 and 21 pA) as seen from the current histogram on the right.

Further analysis of the current fluctuations caused by the poly(A) molecule reveals that the current jumps between two discrete states (see Figure 3a). These two levels are associated with the coiled state and helical state of poly(A), since the more compact helical state is expected to give rise to a lower blocakde level of the ion current. Analyzing >2000 transitions, we find that both distributions can be
fitted using mono-exponentials (see Figure 3b), which yield the characteristic lifetimes of the helix and the coil state, (helix and coil, respectively). The millisecond time scale here is nearly 3 orders of magnitude longer than that observed for free poly(A) in bulk.

References:

  1. Lin J, Kolomeisky A, and A. Meller (2010) Helix-coil kinetics of individual polyadenylic acid molecules in a protein channel. Phys Rev Lett. 104, 158101-4.
Figure 1
poly(A)
Figure 2

2 states poly(A)