Sequence Specific Targeting Duplex DNA

When: Thursday, October 20, 2011 at 4:00 pm
Where: DA 114
Speaker: Maxim D. Frank-Kamenetskii
Organization: Department of Biomedical Engineering, Boston University
Sponsor: CIRCS

Although many proteins are capable of targeting duplex DNA (dsDNA) in a sequence-specific manner,  our ability to design de novo proteins with desired sequence specificity are very limited, at best. That is why the ability of the Peptide Nucleic Acid (PNA) to sequence specifically recognize dsDNA has attracted such considerable interest. During the past years, the basic understanding of the process of dsDNA invasion by pyrimidine bis-PNAs and various applications of the phenomenon have been elucidated. As a result, novel approaches for detecting short (about 20-bp-long) signature sites on genomes have been developed in our laboratory.  In particular, a PD-loop-based method of pathogen detection has been developed, which makes it possible to distinguish not only different bacterial species but also to discriminate drug sensitive versus drug resistant strains. The next step would be to extend the approach to human cells, which would open the way for even more promising applications. Some encouraging data in this direction will be presented. Other promising applications of bis-PNAs include their use in DNA detection with solid-state nanopores.

A serious  limitation of generic PNAs with respect to targeting duplex DNA consists in requirement for the homopurine nature of the DNA strand, which is captured by bis- PNA via the triplex formation. Therefore, various chemical modifications of the backbone and the bases have been tested in recent years, which could allow to lift this sequence limitation. Two modifications are of greatest interest: pseudocomplementary PNAs and g-PNAs. Chiral g-PNAs with some cytosines replaced with G-clamp bases have proved to be especially promising.   The capability of g-PNA to invade dsDNA in a sequence-unrestricted manned has been demonstrated.  The g-PNA makes it possible the exceedingly specific DNA capturing in the duplex form. This opens up a very interesting opportunity of capturing the chromatin in its native form for further investigation of higher-level chromatin structures.

Over the years, the ability of synthetic DNA analogs to invade dsDNA has been considered as something purely artificial, without any parallels in vivo.  The situation has radically changed recently after discovery of the CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeat), a bacterial immune system.  It  has been shown that in the CRISPR pathway small (about 40-60-nt-long) single-stranded RNAs recognize the complementary DNA strand within dsDNA via a still unknown mechanism with the help of special proteins of the CRISPR system.  Advancements in the field of dsDNA recognition by artificial DNA analogs not only provides with new tools for DNA analysis but also can prove to be of great help in understanding new important biological mechanisms.