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 from Nucleic Acids

DNA twist. The designed geometrical model and folding path are fed into computer program as input of DNA length and offset of half-turns, which gives rise to staple strands with Watson-Crick complementary base pairing shown in colored DNA strands. Thus, different geometries

Figure 9. Design of DNA origami in different steps (a) geometrical model construction (b) folding path design involving raster progression from single polynucleotide folding with DNA crossovers and (c) the stape strands with complementary base paring. Intended shapes such as square, start and disk with three holes in the design of origami structures and the corresponding AFM images of the resulting origami structures. (Source: P. W.K. Rothemund Nature 2006, 440, 297-302)

Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids

namely square, start and disk with three holes can be created in stepwise manner. Figure 9 depicts the intended geometrical model construction with cylindrical DNA helices and helix bend at and away from crossover regions. AFM images show formation of desired origami structures. Such scaffolded origami structures is useful for investigating various complex biological process such as protein or virus self-assembly due to the special organization.

Figure 10. DNA origami mediated doxorubicin delivery to cancer cells. (Source: Q. Jiang et al. J. Am. Chem. Soc. 2012, 134, 13396-13403)

Owing to high negative charge it is difficult for the nucleic acids to penetrate the cell membrane and transfection agents are necessary to compensate the charge. However, a number of following features make DNA nanostructures as interesting cargo. They are: Predictable and well-defined structures, high capacity of cargo loading, ability to be internalized by cells, structural stability and biocompatibility. Such origami structures can be used as efficient carrier with high loading efficiency for anti-cancer drugs to tumor sites. [12] A ssDNA M13mp18 was folded in triangular and tubular origami structures with rational design of staple stands (Figure 10). Doxorubicin molecules noncovalently binds with the DNA basepairs through intercalation and can have as high as 50-60% loading of drugs. The drug-loaded origami structures exhibited remarkable cytotoxicity to regular human breast adenocarcinoma cancer cells (MCF-7) and enhances the cellular internalization of doxorubicin.

Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids

4. Nanostructures from RNA: Since RNA is chemically more labile than DNA, dynamic and modular supramolecular engineering to RNA nanostructures is possible akin to jigsaw puzzle game. RNA techtonics are short modular RNA segments, which can be self-assembled in presence of cation such as Mg2+ with well controlled size, shape and pattern to programmable stable 2D architectures known as tectosquares, RNA supramolecules (Figure 11). [13] These tectosquares with sticky

Figure 11. RNA nanostructures: (A) Tectosquares formation from tectoRNA, (B) primary sequence of techtoRNA, (C) front and side view of the tectosquare model, (D) the modifcations in 3’ end of the tectoRNA, (E) possible cis and trans configuration of the tectosqure self-assembly and (F) different types of possible tectosquares. (Sources: L. Jaeger et al. Science, 2004, 306, 2068-2071).

Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids

ends can further be assembled in well defined lattice-like structures. Thus, choosing the RNA connectors with complimentary sticky end is very important as it leads to either 1D ladder- like pattern or 2D fishnet like pattern as shown by the AFM images in Figure 12.

Figure 12. RNA nanostructures from jigsaw puzzle tectosquares: AFM images of tectosquare assembly in ladder (left) and fish net pattern (right). (Source: L. Jaeger et al. Science, 2004, 306, 2068-2071)

5. Summary: This module demonstrates the rational strategy to design nanostructures from nucleic acid motif. A number of important and relevant enzymes and processes e.g. restriction enzymes and palindromic sequence, ligases, DNA polymerase and PCR have been discussed for tailoring designed motifs with sticky ends with line or branched DNA motifs. Such motifs can further be assembled to form well defined 2D lattice, 3D polyhedral etc. The basic principle and simple steps to form DNA origami has also been introduced with an application in the field of drug delivery and cancer therapeutics. Lastly, tectoRNA and RNA tectosquares were introduced to understand the stepwise formation of modular RNA nanostructures. Thus, we discussed from basics of DNA line motifs and branched junction to increasing complex DNA 2D lattice, polyhedra and finally DNA origami structures, which also followed the

Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids

buildup of the DNA nanotechnology field (Figure 13). [14] Presently, much importance is being given to the

Figure 13. Timeline and progress of DNA nanotechnology since its inception. (Source: D. S. Lee et al. Chem. Soc. Rev., 2016, 45, 4199-4225)

applicability of such DNA nanostructures in designing nano devices or understanding cellular internalization process of such nanostructures in order to develop next generation bio nanotechnologies.

Nanobiotechnology Biotechnology Design of Nanostructures from Nucleic Acids