DNA as a Functional Polymer

By Professor Juewen Liu

Sixty years ago, the famous structure of the DNA double helix was solved, bringing about the birth of modern molecular biology. Since then, DNA has been extensively studied as a genetic material. In the 1970s, solid-phase DNA synthesis was invented, allowing one to obtain arbitrary oligonucleotide sequences. In 1986, the invention of polymerase chain reaction (PCR) allowed an infinite number of DNA copies to be amplified from even a single molecule. It is these two techniques that have made it possible to explore new functions of DNA.

DNA has highly programmable structures that can be designed based on a simple base pairing rule. For example, a field called structural DNA nanotechnology has experienced rapid developments, manifested by the many published sophisticated 2D and 3D nanostructures.[1] Upon these structures, various nanoparticles have been deposited to offer other functions. Another interesting advancement was the discovery of DNA as a catalyst (catalytic DNA) and for molecular recognition (aptamer), making DNA a functional substitute for proteins. Compared to proteins, DNA is much more stable, easier to perform site-specific labeling, and easier for conjugation to various materials, popularizing DNA as the molecule of choice in constructing functional nano- and biomaterials.

Nanomaterials

Nanomaterials are attractive because they possess unique size and distance-dependent physical properties. While particle size can be well controlled through chemical synthesis, the control of inter-particle distance with sub-nm precision and the organization of different types of particles remained difficult. DNA provides a unique solution to solving these problems. On the other hand, the molecular recognition property of DNA and DNA aptamers allows these nanomaterials to be used for biosensing and biomedical applications.[2,3] My lab is interested in exploring the biophysical interface between DNA and various nanomaterials to guide the design of better biosensors, biomaterials, and drug delivery systems.

Fundamental understandings

Within a persistent length of ~50 nm, double-stranded DNA can be considered as a rigid rod with a diameter of just 2 nm. Each additional base pair contributes to a length increase of 0.34 nm. Therefore, sub-nm control of distance can be achieved using DNA. With the availability of a diverse range of attachment chemistry, DNA can be linked to almost all known nanomaterials. We are interested in studying the distance-dependent properties of various nanomaterials, including gold nanoparticles, liposomes, magnetic nanoparticles, quantum dots, and graphene using DNA as a linker. For example, Figure 1A shows the assembly of DNA-functionalized gold nanoparticles using a linker DNA with a subsequent color change from red to purple.[4] The same idea can be applied to the assembly of soft liposome nanoparticles (Figure 1B),[5] as well as gold-liposome hybrid (Figure 1C).[6] The inter-particle distance can be precisely controlled by changing the DNA sequence. Studying these systems can provide insights into the DNA-surface interaction as well as the coupling of physical events among the particles.

Figure 1. Schematics of DNA-directed assembly of gold nanoparticles (A), liposomes (B), and their hybrids (C).
(D) A representative TEM micrograph of the structure shown in (C).

Environmental monitoring

Beyond a simple structural molecule, DNA can recognize a wide range of ions, molecules, and even cells with high specificity. In the case of detecting the highly toxic mercury, a thymine rich DNA is used. As shown in Figure 2, this DNA can fold into a hairpin upon mercury binding where upon addition of a DNA binding dye called SYBR Green I (SG), a green fluorescence is obtained. Immobilizing the mercury detecting DNA to a hydrogel has a number of advantages. The gel allows sensor drying and regeneration. More importantly, the gel can actively adsorb mercury, increasing its concentration within the gel. Via immobilization, we have achieved a highly selective and sensitive detection of mercury without the use of any analytical instrument.[7,8]

Figure 2. A DNA-based biosensor immobilized on a hydrogel for mercury detection where
SG becomes highly fluorescent upon binding to the double-stranded region in the DNA.

Biomedical diagnosis

Apart from the recognition of heavy metal ions, aptamers can be selected to bind other molecules such as proteins and metabolites. This aptamer selection process starts with a huge library of random DNA molecules where only the sequences that bind the metabolite are retained. In Figure 3, the sequence that can bind to ATP is shown.[9] While this aptamer can detect ATP effectively in pure buffer, its performance is strongly interfered by the presence of blood serum. For blood samples, it is important to achieve detection in a very small sample volume. We found that by attaching the aptamer-based sensor onto a magnetic microparticle (MMP), it is possible to achieve detection in just 10 mL of human blood serum. Because of the MMP, we could separate the ATP binding step from the fluorescence signal detection step, allowing us to dilute the effect of blood serum.[10]

Figure 3. Sequence of the ATP binding aptamer and its immobilization on
a MMP allowing effective ATP detection in human blood serum.

Drug delivery applications

The same DNA selection method can also be used to target cancer cells.[11] Many DNA aptamers have already been isolated to target many different tumor cell lines. For example, a guanine rich sequence has entered clinical trials. Taking advantage of nanomaterials for drug loading and imaging, DNA-functionalized biomaterials can allow sophisticated functions to be realized including targeted delivery and diagnosis.

In summary, DNA is a very versatile molecule with both structural and functional properties. Interfacing DNA with various nano- and biomaterials can significantly improve the performance of these DNA molecules in various applications. At the same time, the structural property of DNA allows precise assembly of nanomaterials with high precision, allowing fundamental biophysical understandings that can fuel the further development of various applications.

Reference

1. Seeman NC. DNA in a material world. Nature 2003; 421: 427-31.
2. Storhoff JJ, Mirkin CA. Programmed Materials Synthesis with DNA. Chem. Rev. 1999; 99: 1849-62.
3. Liu J, Cao Z, Lu Y. Functional Nucleic Acid Sensors. Chem. Rev. 2009; 109: 1948–98.
4. Smith BD, Liu J. Assembly of DNA-Functionalized Nanoparticles in Alcoholic Solvents Reveals Opposite Thermodynamic and Kinetic Trends for DNA Hybridization. J. Am. Chem. Soc. 2010; 132: 6300–1.
5. Dave N, Liu J. Programmable Assembly of DNA-Functionalized Liposomes by DNA. Acs Nano 2011; 5: 1304–12.
6. Dave N, Liu J. Protection and Promotion of UV Radiation-Induced Liposome Leakage via DNA-Directed Assembly with Gold Nanoparticles. Adv. Mater. 2011; in press.
7. Dave N, Huang P-JJ, Chan MY, Smith BD, Liu J. Regenerable DNA-Functionalized Hydrogels for Ultrasensitive, Instrument-Free Mercury(II) Detection and Removal in Water. J. Am. Chem. Soc. 2010; 132: 12668–73.
8. Joseph KA, Dave N, Liu J. Electrostatically Directed Visual Fluorescence Response of DNA-Functionalized Monolithic Hydrogels for Highly Sensitive Hg2+ Detection. ACS Appl. Mater. Inter. 2011; 3: 733–9.
9. Huizenga DE, Szostak JW. A DNA Aptamer That Binds Adenosine and ATP. Biochemistry 1995; 34: 656-65.
10. Huang PJJ, Liu JW. Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor. Anal. Chem. 2010; 82: 4020-6.
11. Fang XH, Tan WH. Aptamers Generated from Cell-SELEX for Molecular Medicine: A Chemical Biology Approach. Acc. Chem. Res. 2010; 43: 48-57.