Novel biophysical method to characterize drug candidates for anti-gene therapy

Triplex-forming oligonucleotides (TFOs) are simple sequence-specific DNA drugs designed to target and bend the stiff structure of DNA, resulting in gene expression inhibition in a process known as anti-gene strategy.

HFSP Program Grant holder Electra Gizeli and colleagues
authored on Fri, 15 January 2010

TFOs have received special attention since recent studies, using bioinformatics tools, revealed the existence of sequences in the majority of human genes which can interact with TFOs and become candidate targets for anti-gene therapies. The design of therapeutical TFO agents requires TFOs’ evaluation in relation to factors associated with the triplex stability and specificity as well as the mechanical properties (flexibility and bending) of DNA segments. Traditional gel based assays and fluorescent methods can only provide semi-quantitative evaluation of TFOs’ properties and are not applicable to rapid analysis and high-throughput screening.
 
Recently, an exciting new biophysical approach, developed in our lab, showed that it is possible to use acoustic wave sensors to detect structural features of surface attached DNA molecules [1, 2]. Specifically, acoustic measurements, in combination with a suitable mathematical treatment, were used to provide quantitative information on the conformation of surface-tethered DNA molecules, distinguishing between DNAs of same mass but different shape and measuring the bending angle of the molecules. This novel detection method is based on the wave’s sensitivity to the viscosity of the sample at the device/liquid interface; binding of DNA molecules alters the viscosity of the interface in a manner that is related to the DNA intrinsic viscosity; the latter can be further related to its geometrical characteristics and internal structure.
 
The above approach was applied to the measurement of the degree of DNA bending upon the formation of short triple helical molecules [3]. A sequence-specific DNA ligand, known to induce a bending angle of 119±3o, was applied. The DNA bending angle, quantified through our acoustic measurements was found to be 124±6o, in excellent agreement with the published value. The simplicity of acoustic measurements in relation to the quantitative detection of DNA bending suggests that acoustic biosensors can emerge as a powerful technique for the biophysical study of DNA flexibility as well as the high-throughput screening for drugs that alter the conformation of DNA and affect transcription.
 
The technique can be expanded to the study of other biological processes in which changes in the conformation of biomolecules or biostructures are known to take place; examples of such processes include protein structural transitions, amyloid fiber formation and peptide-membrane interactions, which can occur via different mechanisms and are related to various human diseases.
 

Figure: A double stranded DNA fragment can be converted into a triplex DNA molecule adopting a “straight” or “bent” conformation depending on the properties of the triplex forming oligo (TFO1 or TFO2, respectively) that interacts with the specific DNA target sequence. Both procedures are monitored by the acoustic wave biosensor.
 

Reference
Triple-helix DNA structural studies using a Love wave acoustic biosensor. Papadakis, G., Tsortos, A. and Gizeli, E. Biosens. Bioelectron. 25, 702-707 (2009).

Additional References
[1] Quantitative determination of size and shape of surface-bound DNA using an acoustic wave sensor. Tsortos, A., Papadakis, G., Mitsakakis, K., Melzak, A.K. and Gizeli, E.. Biophys. J. 94, 2706-2715 (2008).
 
[2] Shear acoustic wave biosensor for detecting DNA intrinsic viscosity and conformation: A study with QCM-D. Tsortos, A., Papadakis, G. and Gizeli, E. Biosens. Bioelectron. 24, 836-841 (2008).


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