Biotechnology

Paper No. : 11 Nanobiotechnology Module : 09 Design of Nanostructures from Nucleic Acids Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi Paper Coordinator: Dr Minni Singh, Associate Professor, Punjabi University, Patiala, Punjab Patiala-147002 Punjab" Content Writer: Dr Asish Pal, Scientist - E, Associate Professor, Institute of Nano Science & Technology, Mohali, Punjab Content Reviewer: Dr Minni Singh, Associate Professor, Punjabi University, Patiala, Punjab Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids Description of Module Subject Name Biotechnology Paper Name Nanobiotechnology Module Name/Title Design of Nanostructures from Nucleic Acids Module Id 09 Pre-requisites Objectives Keywords DNA-2D lattice, DNA origami, Holliday Junction, RNA techtosquare Table of Contents 1. Learning objectives 2. Chemical toolbox for processing nucleic acids 3. Nanostructures from DNA 4. Nanostructures from RNA 5. Summary 1. Learning Objectives: This module is intended to apprise students about strategy to form nanostructures from nucleic acids. Students will also learn about various processing enzyme such as restriction enzymes, ligages, polymerase for cutting, joining nucleic acids at specific position. These strategies can generate linear or branch nucleic acids with sticky and blunt ends. Such motifs will be used for making various nanostructures such as DNA-2D lattice, DNA polyhedra, DNA origami and RNA techtosquare. Eventually, the module will demonstrate some applications of the defined DNA nanostructures towards drug delivery. 2. Chemical toolbox for processing nucleic acids: In bottom-up DNA nanotechnology, the base sequence of the strands are rationally designed in order to cause perfect hydrogen bonding mediated base-paring interaction and self- Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids assembly of the strands in desired conformation. As the matched base pairing is energetically favorable, the sequence of bases thus determines the pattern of binding making the overall structure easily controllable. In structural DNA nanotechnology, it is important to cut the double stranded DNA at a specific Cutting DNA double strands Ligation of sticky ends Figure 1. DNA processing strategies: Cutting DNA double strands using restriction enzymes (left) and Ligation of the join sticky ends of DNA (right). Figure 2. Palindromic sequences and cutting of DNA at specific position with various restriction enzymes. Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids sequence, which can be done by restriction enzymes (Figure 1). These sites are known as recognition sites and have palindromic sequences i.e., it reads the same on the reverse and forward strands, if they are read in the same direction. Figure 2 shows palindromic sequence [1-2] GAATTC which is identified by restriction enzyme EcoRI and it cuts the DNA strand (hydrolysis of phosphodiester bond) after guanine base. Similarly, there are different palindromic sequences, recognized by different restriction enzymes and cuts DNA strands at different locations. The sticky ends of the DNA fragments can be joined by reformation of hydrogen bonding interaction between opposite strands followed by formation of the phosphodiester bonds as marked in Figure 2 (right). The enzyme facilitating the process is known as DNA ligase. DNA fragments bind to the template strand through basepairing interaction and hydroxyl group of the sugar from 3’ end of one strand form the ester bond with the phosphate group from 5’ end of another strand in presence of ligase (Figure 3). [3-4] Figure 3. DNA ligase joining two single strands in double stranded DNA through formation of phophodiester bonds. Polymerase chain reaction is an important technique which has revolutionized the DNA nanotechnology because of its relative ease to copy and multiply a specific DNA fragment and is facilitated by DNA polymerase enzyme. It has three definite cycles and Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids mononucleotides, primers and DNA polymerase are integral part of the temperature variable process (Figure 4). In the first step, DNA duplex is denatured at temperature more than DNA melting point. In the second step, DNA sequence to be copied is made to form DNA duplex with a carefully Figure 4. Cycles of polymerase chain reaction (PCR) to amplify DNA fragments. Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids designed primar, which has complementary base sequence at a lower annealing temperature. In the final step of elongation, the temperature was elevated enough to facilitate DNA polymerase binding and elongation of the primer length with attachment of mononucleotides. The cycles are repeated and the number of product (daughter DNA strands) increases exponentially. Thus, after 2nd, 5th and 10th cycles, the number of DNA fragments are 22, 25 and 210. [5-6] 3. Nanostructures from DNA: In order to design nanostructures from DNA, one need to combine DNA motifs with sticky ends with base pairing interaction, that is the principle of structural DNA nanotechnology. The strategy to produce predefined and well structured DNA materials is an active and evolving research area since 1980s. The sticky ended cohesion is depicted in Figure 5, where two duplex DNAs have overhangs which are complementary. [7] Under ambient condition, the sticky ends can be ligated in a sequence–specific manner. DNA nanotechnologist depicts a DNA as a line motif. However, the line can also have kink and cohering such lines results branched DNA nanostructures. Thus, four double stranded DNA form branched structures, known as Holliday Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids Figure 5. Two duplex DNAs with complimentary sticky ends to form base pair and make continuous double stranded DNA. (Source: N. A. Seeman, Chemistry & Biology, 2003, 10, 1151-1159). junction named after molecular biologist, Robin Holliday. [8] As depicted in Figure 6, Holliday junction may exist in different conformational forms based on the base pairing Figure 6. Holiday junction from DNA sticky ends from four DNA strands (left), one of the possible conformational isomers among unstacked and coaxial stacked isomers. interaction in the arms of the strands. One of the conformations of the Holliday junction is demonstrated which lacks basepairing in the central part of the double helical region. Similarly, there are other conformers with different patterns of coaxial stacking among the four DNA strands. The Holliday junction is an important intermediate in genetic recombination process to increase diversity by shifting the genes between chromosomes. The modified Holliday junction of four strands of DNA, each having sticky ends can be programmed to design 4 x 4 tiles as depicted in Figure 7A. The complimentary sticky ends form DNA duplex and are depicted as rectangle, while the Holliday junction has four such rectangles to form extended 2-D lattice like DNA nanogrids (Figure 7B), as evident from the AFM images. Such nanogrid structures can further be programmed to design template for array of proteins or quantum dots for molecular device applications. [9] Highly selective binding in the complementary strands makes DNA motifs amenable for designing much complex 3-dimentional hierarchical nanostructures. Three types of DNA strands as depicted in Figure 8 can be combined to form three-point-star motif, which are Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids considered to be the primary building blocks for a concentration driven self-assembly to form DNA polyhedra. [10] Thus, 4, 20 and 60 three-point-star-tiles self-assemble to form tetrahedral (10 nm), dodecahedra (24 nm) and much larger buckyball (42 nm) respectively at A Figure 7. DNA nanostructures from (A) four strands of DNA with sticky ends to form 2-dimensional self-assembly, (B) design of extended lattice and AFM images of the lattice in amplitude mode and height mode. (Source: H. Yan Science 2003, 301, 1882-1884) different concentration regime of the building blocks. Single particle 3D construction from the TEM images shows a perfect matching of the calculated sizes with that from DLS, AFM and cryo-TEM. Thus, DNA can be programmed into well-defined synthetic 3D nanocontainer, which paved way for useful technology known as DNA origami. The basic principle is folding of DNA to create complex 2D and 3D shapes at nanoscale in the Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids following 5 steps. [11] Firstly, a geometrical model of DNA in the desired shape of the origami needs to be built with double helices considered as cylinder (Figure 9, Top panel). Secondly, single long DNA scaffold needs to be folded back and forth in raster progression involving cross-over involving Figure 8. Hierarchical self-assembly of DNA in various symmetric DNA polyhedra via combination of three-point-star motifs, which are constructed from seven DNA strands as denoted S, M and L/L’. AFM images of the polyhedral at different concentration regime of the three-point-star motifs with single particle 3D reconstruction molecular modeling based on cryo-TEM. (Source: Y. He Nature, 2008, 452,198-202) Nanobiotechnology Biotechnology Design of Nanostructures

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