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The Idea


Relevance of DNA - small molecule interactions


Containing the blueprint for every function and structure of life, DNA plays an essential role in most fields of the biosciences. Because of this reason, detailed knowledge of compounds that interact with DNA is of great importance. For instance, small DNA binding molecules including intercalators and minor/ major groove binders can cause changes in the geometry of double-helical DNA domains. Such changes can affect the dynamics of transcriptional regulation and the activity of nucleases. To name an example, the minor binder Ciprofloxacin is a potent inhibitor of bacterial DNA gyrases with a broad range of activity, including the notorious nosocomial pathogen Pseudomonas aeruginosa.
Hence, small DNA binding molecules can represent candidate compounds with antibiotic or tumor-repressing activity.  Elucidating the microscopic structural changes caused by small molecules can thus help to select candidate molecules as well as shed light on functional mechanisms.

Why DNA origami?

The working principle of DNA origami is based on the highly specific interaction behavior of DNA. Because of this specificity, the binding behavior of two strands can be encoded in the base sequence of said strands. By including sequences complementary to two strands on another, a trimolecular system can be created where one strand binds two different ones together. By including more and more complementary sequences on a single strand, increasingly complex configurations can be created. DNA Origami uses this modular behavior to encode the shape of the planned structure in the sequences of the used DNA strands. Designing a new structure is generally done according to the following steps. During the computer-aided design of the structure, a long single stranded DNA called the 'scaffold' is first laid out such that it fills out the shape of the planned structure. Next, short DNA strands called 'staples' are chosen such that they are complementary to partial sequences of the scaffold. By binding to those complementary sequences, the staples force the scaffold into a set conformation, removing degrees of freedom. With each bound staple, more and more helices with fixed relative positions are created. Once all bases of the scaffold are hybridized, the scaffold is forced into one specific shape.
If the design of an origami contains certain targeted insertions and deletions of base pairs, it is possible to create a built-in twist or curvature [Dietz et. al., 2009]. This work was truly inspiring because it implies the general idea of an origami's overall shape to be liable to distortions of it's microstructure. According to this concept, the above mentioned constraints within the DNA Origami allow the effects of multiple DNA binders to be transported along the structure so that they sum up, amplifying otherwise immeasurable influences. Furthermore, the unparalleled positional control of DNA Origami allows us to place probe molecules on the structure with high accuracy, permitting the observation of minute changes.

Project specification

Our goals were to develop a simple and fast assay for
a) identifying whether or not a target compound binds DNA in a structure-altering fashion and
b) provide means for quantifying how the target molecule changes helical pitch and length of double-helical DNA domains.
To this end we set out to develop a self-assembled DNA origami device that amplifies microscopic structure changes imposed on its constituent double-helical DNA domains by a large global conformational change. The global structure alteration becomes detectable either by direct imaging with transmission electron microscopy (TEM) but ideally also in a simple fluorometric assay with equipment that is available to many laboratories in the world.
We have focussed on changes in helical pitch and tested our device with a set of different DNA binders from the three major classes, including spermine (major groove binding), ethidium bromide (intercalating), and DAPI (minor groove binding) in different concentrations by direct imaging with TEM. In accordance to computational models that we made for our device, the local deformations led to observable global structural changes. In the light of the time constraints of this project we therefore consider the goals  a) and b) accomplished by our proof-of-principle experiments with TEM alone.

However, since access to TEM microscopes is limited, for practical application of the device it is desirable that it can function in simple fluorometric assays. We have therefore also taken first steps toward using fluorescent signals to report on structural changes that our device undergoes upon binding of small molecules. We present some promising preliminary results obtained with single-molecule FRET microscopy and bulk photometry.