Book of Abstracts: Albany 2005
Pseudocomplementary PNAs as Artificial Sequence-specific DNA-bending Agents
DNA bending is of importance for various DNA functions in the cell. In particular, a number of transcription factors are known to induce DNA bending and to provide the necessary DNA architecture for formation of the transcriptional activation complex. Artificial agents that induce DNA bending in a site-specific manner therefore may find use, for instance, as transcriptional regulators of specific genes. We show that pseudocomplementary peptide nucleic acids (pcPNAs) represent a new class of sequence-specific DNA-bending agents.
Figure 1: (A) Chemical structure of pcPNA. In pcPNA, the nucleobases A and T are substituted by the modified nucleobases 2,6-diaminopurine (D) and 2-thiouracil (sU), respectively. (B) The modified nucleobases prevent pcPNAs from forming a stable PNA-PNA duplex due to steric hindrance between D and sU bases, whereas they do not prevent pcPNAs from forming stable PNA-DNA heteroduplexes with complementary DNA strands (C) Double-duplex invasion complex formed at binding of a pair of pcPNAs to the target sequence in double-stranded (ds) DNA.
In the first examples studied so far, strand invasion of pcPNAs into one duplex DNA target site results in moderate anisotropic DNA bending with mean values of 40-70°, as determined by circular permutation assay and by electron microscopy. We also demonstrate that DNA bending by pcPNAs can be modulated by targeting two closely located DNA sites. In so doing, an enhanced DNA bend with a mean value close to 90o is obtained, when the two induced bends are approximately located in the same plane and direction. We have further validated the occurrence of such a sharp bend within the DNA double helix through efficient formation of 170-bp-long DNA minicircles via dimerization of two bent DNA fragments.
We conclude that pcPNAs offer two main advantages over previously designed classes of non-natural DNA-bending agents: (i) they allow DNA targeting with very mild sequence restrictions and (ii) they can easily be designed for a chosen target sequence, since their binding obeys the principle of complementarity.
This work was supported by National Institutes of Health Grant GM59173 (to MDF-K) and by a Boston University Special Program for Research Initiation Grant (to VVD).
References and Footnotes
1Center for Advanced Biotechnology