Self-assembly of oligonucleotides

Transcription

1 Self-assembly of oligonucleotides Dr. K. Uma Maheswari Professor, School of Chemical & Biotechnology SASTRA University Joint Initiative of IITs and IISc Funded by MHRD Page 1 of 9

2 Table of Contents 1 APPLICATIONS OF SELF-ASSEMBLED DNA NANOSTRUCTURES MOLECULAR BEACON CANCER THERAPY SENSORS DNA NANOMECHANICAL DEVICES MOLECULAR SWITCHES INFORMATION STORAGE MOLECULAR ELECTRONICS PLASMONICS CONCLUSION REFERENCE ADDITIONAL READING...9 Joint Initiative of IITs and IISc Funded by MHRD Page 2 of 9

3 In the previous lectures, we had seen some of the devices that could be constructed using oligonucleotides. In this lecture, we will attempt to explore the possible applications of these structures in different fields. Many of the applications are yet to be translated into commercial products due to a host of challenges. However, none of these challenges are related to the function of these structures. 1 Applications of self-assembled DNA nanostructures 1.1 Molecular beacon A beacon literally means a device that helps to indicate the location of an object. Example: a lighthouse, tail lights in a car etc. Similarly, molecular beacons are made up of oligonucleotides that have been used to indicate the presence of a particular nucleic acid sequence in a sample. Just as conventional beacons indicate the location by glowing, these molecular beacons are also designed to indicate the presence of a specific nucleic acid sequence by emission of electromagnetic radiation in the visible range. So what is the principle of these molecular beacons? The spontaneous process of hybridization between two oligonucleotide sequences based on the complementary base pairing is the underlying principle of this molecular beacon. The molecular beacon consists of a single stranded DNA known as a probe strand, which folds in to a hairpin loop because of some complementary base pairing between the two ends of the strand. This is known as self-complementary pairing. One end of the strand is modified to covalently attach a fluorescent molecule while the other end is linked to a quencher (like gold nanoparticle). When the probe strand is in this conformation, the emission from the fluorescent probe is quenched by the quencher because of their close proximity. The sequence of the probe strand in the molecular beacon is chosen such that it is exactly complementary to the nucleic acid sequence it is to detect. The strand can be modified at the terminals with short sequences to ensure formation of the hairpin loop structure spontaneously. Now, let us add the sample. If the sample contains the nucleic acid sequence of interest, it will hybridize with the probe strand by complementary base pairing. This will lead to straightening of the hairpin loop thereby separating the fluorescent molecule and the quencher. This will cause appearance of the emission by the fluorescent molecule. Why should the hairpin structure change? The number of complementary base pairs formed between the nucleic acid sequence in the sample and the probe strand are greater than those that existed between the two terminals of the same probe strand. Greater complementary base pairing means greater number of hydrogen bonds between the base pairs and thus greater stability. This drives the straightening of the hairpin loop structure of the probe DNA strand. Such types of molecular beacons are now commonly used in the RT-PCR technique to denote the Joint Initiative of IITs and IISc Funded by MHRD Page 3 of 9

4 progress of the amplification reaction. The following animation (Figure 1) depicts the functioning of a molecular beacon. Fig.1: Functioning of molecular beacon Note: Can be viewed only in Acrobat Reader 9.0 and above 1.2 Cancer therapy The molecular beacon concept can be exploited for killing cancer cells. How? Gold nanoparticles can be used as the quencher and the sequence in the molecular beacon could be designed to specifically pair with a oligonucleotide sequence unique to the cancer cell. Now gold nanoparticles can also serve as an antenna and can be heated by an inductively coupled radio frequency magnetic field from outside. This local increase in temperature can bring about denaturation of DNA in the cancer cell thus leading to its death. This concept can be highly effective if applied. However, the challenges involved in realizing this concept practically are: (i) Identification of unique cancer DNA signatures still to be accomplished and (ii) methods to deliver these beacons into the cancer cell selectively, enable their escape from the endosome and guide their entry into the nucleus of cancer cells are yet to be developed. The following animation (Figure 2) describes the concept involved in the treatment of cancer employing molecular beacons. Joint Initiative of IITs and IISc Funded by MHRD Page 4 of 9

5 Fig. 2: Molecular beacons for cancer therapy Note: Can be viewed only in Acrobat Reader 9.0 and above 1.3 Sensors The ability of the oligonucleotides to be modified at the 5 and 3 terminals coupled with their ability to hybridize with complementary bases through hydrogen bonds can be exploited for biosensing applications. For example, the molecular beacon concept can be extended to identify specific gene sequences in a sample by observing the intensity of fluorescence. Another interesting oligonucleotide structure that has been developed recently as a ph sensor involves formation of unnatural base pairs by hydrogen bonding a phenomenon known as Hoogsteen pairing. The DUTE device had highlighted the formation of G-quartets through hydrogen bonds under specific Joint Initiative of IITs and IISc Funded by MHRD Page 5 of 9

6 ionic concentrations. Similarly, an A-motif has been recently developed using a gold nanoparticle-adenosine rich oligonucleotide hybrid structure. The principle of this sensor is very simple. Gold-oligonucleotide hybrids were formed by thiolating one end of the oligonucleotides to form a thiol link with the gold nanoparticle. Each gold nanoparticle was linked with eight oligonucleotides. Four of these nucleotides were rich in adenosine residues while the other four oligonucleotides were short spacer strands. When the gold nanoparticle-oligonucleotide hybrid is at physiological ph, the emission due to the surface plasmon resonance of the individual gold nanoparticles is observed. When the ph is lowered, the emission wavelength shifts indicating a change in the size of the gold nanoparticles. Why is this shift observed? At acidic ph, the adenosine residues become protonated thereby increasing the number of hydrogen donor groups and hence the ability to hybridize. Formation of hydrogen bonds with adjacent adenosine residues is known as Hoogsteen base pairing. This hydridization will cause association of neighbouring oligonucleotide strands and hence result in aggregation of the gold nanoparticles. This leads to the shift in the emission wavelength. The following animation (Figure 3) represents the principle of a goldoligonucleotide hybrid. Fig. 3: Principle of gold-oligonucleotide hybrid Note: Can be viewed only in Acrobat Reader 9.0 and above This aggregation is ph dependent and hence can be used as a ph sensor. What advantages does this DNA hybrid sensor possess over conventional fluorescent ph sensors? The fluorescent sensors do not function well below ph 5 but the DNA hybrid Joint Initiative of IITs and IISc Funded by MHRD Page 6 of 9

7 sensor can function effectively in low ph. The active ph range can be tuned by altering the length of the oligonucleotide and substituting certain A residues with T residues. 1.4 DNA Nanomechanical devices The forces generated during the conformation changes of the active DNA structures are very high and can be harnessed to push or pull nanostructured elements in a device. For example, it has been estimated that the transition from the open state to the closed state in a DNA tweezer involves a force of about 15 pn. Similarly, in the case of a DUTE device, the force involved to fold the structure is about 2.2 pn while the force generated during its extension is about 20.7 pn. This property can be exploited in the design and development of nanomechanical devices. 1.5 Molecular switches Another interesting possibility of these active structures could be as molecular switches that can turn on or off a chemical interaction by alternately exposing or masking a target group attached to one of the terminals of the probe strand as it toggles from one state to another in response to the introduction of the stimulus. This concept can be exploited for highly efficient organic synthesis in response to environmental cues. This same concept can be built upon to provide an effective platform for synthetic biology. What is synthetic biology? It involves use of engineered biological systems natural or biomimetic, to understand complex biological phenomena or to use these systems for production of commercially useful products such as pharmaceuticals, fuels or food. How can DNA nanostructures be useful in synthetic biology? The nucleic acid self-assembled structures can serve as scaffolds that mimic natural compartments to bring about selective interaction of enzymes and substrates. Though the practical realization of this concept is still far off, the development of such structures offers new directions in the field synthetic biology. 1.6 Information storage The self-assembled DNA active device can be used to store information just like a computer that stores information in the form of binary digits 0 and 1. Each conformation state can be equated to either a 0 or a 1. A DNA array could be constructed by immobilizing the strands on a substrate. All strands could be at the same conformation. The addition of fuel strand to specific sites in the array could transfer the information by bringing about the conformation change at the specific sites. Addition of anti-fuel strands can reprogram the array where now fresh information could be stored! The same concept can be extended to development of molecular logic gates that can form the basis of an exciting new era of DNA computing. The following animation (Figure 4) explains this concept. Joint Initiative of IITs and IISc Funded by MHRD Page 7 of 9

8 Fig. 4: Use of DNA structures for information storage Note: Can be viewed only in Acrobat Reader 9.0 and above 1.7 Molecular electronics The DNA wires can find extensive use in molecular electronics where high aspect ratios can be achieved without compromising the electrical conductivity. Also design of ultrafine conducting pathways has always been a challenge in miniaturization of electronic devices. Such conducting pathways are difficult to create reproducibly and in a defect-free manner by currently available nanofabrication techniques. Use of selfassembled oligonucleotide wires offers a viable alternative for creating conducting pathways in miniaturized electronic structures. Recently, scientists have developed a bi-fi or biological internet, that can enable transmit messages to cells over long distances. They have employed a virus known as M13 that can effectively pack the DNA introduced by the scientists in a protein casing and send it to cells even in remote location. This bi-fi has enormous potential in cell reprogramming and molecular therapy! Joint Initiative of IITs and IISc Funded by MHRD Page 8 of 9

9 1.8 Plasmonics When metal nanoparticles such as gold or silver are exposed to electromagnetic radiations, the electrons in their conduction band exhibit a phenomenon known as surface plasmon resonance. An enhancement in the field can be achieved if the nanoparticles are closely spaced due to coupling between the neighbouring particles. However, the size and shape of the nanoparticle array is a major determinant in realizing optimal field enhancements. In this regard, self-assembled oligonucleotide arrays can be useful to enable precise positioning of the nanoparticles and control the spacing between them. Only hitch in exploiting the nucleic acid arrays is the limitations imposed by the length of the oligonucleotides used. For effective transmission, longer lengths are required and hence optimization and control of the self-assembly involving long nucleotide chains need to be achieved. 2 Conclusion The scope of DNA nanotechnology is infinite and this lecture attempted to provide a glimpse into the interesting possibilities that exist in manipulating this unique biomolecule for applications that can be applied in diverse fields. In future, it is likely that DNA-based devices become a commercial reality. 3 Reference Encyclopedia of Nanoscience & Nanotechnology, Volumes 2 & 9. Edited by: H.S. Nalwa, American Scientific Publishers, Additional Reading 1. Tunable, colorimetric DNA-based ph sensors mediated by A-motif formation, Sonali Saha, Kasturi Chakraborty and Yamuna Krishnan, Chem. Commun., 2012, 48, Nanomechanical Devices Based on DNA, Christof M. Niemeyer and Michael Adler, Angew. Chem. Int. Ed. 2002, 41, No. 20, Joint Initiative of IITs and IISc Funded by MHRD Page 9 of 9