ScienceWeek

CHEMISTRY: ON ENGINEERED NUCLEIC ACIDS

The following points are made by Ronald R. Breaker (Nature 2004 432:838):

1) Exploring the full complexity of cells at the molecular level will require the fashioning of new tools that allow researchers to manipulate complex biological processes in unique ways. Small organic molecules that block or otherwise perturb the normal functions of the cellular machinery have long served as powerful tools for exploring biochemical processes. Similarly, new tools that take advantage of the natural functions of proteins and nucleic acids are proving to be enormously useful as researchers continue to probe the details of complex biochemical systems.

2) Living systems have been expanding and diversifying their natural collection of biochemical tools for billions of years. For example, enzymes build RNA, DNA and proteins with high fidelity and with impressive speed; in some cases more than 100 monomeric units are added to the polymer per second. Many other enzymes are known to selectively cut or join nucleic acids or proteins, and still others catalyze chemical reactions with great speed and accuracy. This provides us with a large set of verified technologies which, if harnessed by researchers, can be applied to understand and manipulate biological processes at their most fundamental level. Indeed, there is a considerable history of scientists taking bits and pieces of proteins and nucleic acids from natural sources, tailoring them by purposefully mutating or splicing them in different ways, and using them as reagents for biological study or for therapeutic applications.

3) More recently, researchers have begun to harness darwinian evolution to optimize existing functions of proteins[1,2] and nucleic acids[3,4], and to create new ones. In combination with rational design methods, these techniques for directing the evolution of biopolymers allow researchers to become a creative force for molecular change and invention. In many instances, we no longer need to be limited to using a less-than-optimal protein or nucleic acid molecule from natural sources. Some natural proteins and nucleic acids can be enhanced by using directed evolution or entirely new functions can be derived using similar engineering strategies.

4) Simple, engineered nucleic acids already provide us with useful tools for detecting and manipulating other nucleic acids. For example, the selective amplification of genomic fragments by the polymerase chain reaction (PCR)[5] or by related techniques requires the use of designed synthetic DNA primers. Similarly, the targeted inactivation of gene expression by using short synthetic oligonucleotides or small interfering RNAs (siRNAs) is becoming increasingly routine. These applications are greatly aided by efficient methods for the sequence-specific chemical and enzymatic synthesis of RNA and DNA. In addition, the design of nucleic acids that bind to other nucleic acids with high affinity and specificity follows the simple and long-established rules of Watson-Crick base pairing.

5) However, it is becoming increasingly clear that nucleic acids can have far greater use than that shown by simple base-paired structures. For example, the hammerhead ribozyme consists of just over 30 nucleotides and can catalyze RNA-strand scission at a rate that is millions of times faster than spontaneous RNA cleavage. At the opposite end of the spectrum is the ribosome, which at its core carries a staggeringly complex ribozyme structure that catalyses peptide-bond formation.

References (abridged):

1. Kolkman, J. A. & Stemmer, P. C. Directed evolution of proteins by exon shuffling. Nature Biotechnol. 19, 423-428 (2001)

2. Zhao, H., Chockalingam, K. & Chen, Z. Directed evolution of enzymes and pathways for industrial biocatalysis. Curr. Opin. Biotechnol. 13, 104-110 (2002)

3. Joyce, G. F. Directed evolution of nucleic acid enzymes. Annu. Rev. Biochem. 73, 791-836 (2004)

4. Wilson, D. S. & Szostak, J. W. In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68, 611-647 (1999)

5. McPherson, M. J. & Møller, S. G. PCR (Springer, New York, 2000)

Nature http://www.nature.com/nature

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MATERIALS SCIENCE: ON NUCLEIC ACID NANOTECHNOLOGY

The following points are made by Hao Yan (Science 2004 306:2048):

1) Nucleic acids are best known as the carriers of genetic information, but they are also a versatile material for designing nanometer-scale structures, because nucleic acid sequences can be designed such that the strands fold into well-defined secondary arrangements. In 1982, Seeman [1] first proposed using branched DNA building blocks to construct ordered arrays. In recent years, DNA has been shown to be an ideal molecule for building micrometer-scale arrays [2,3] with nanometer-scale features. DNA can also be used to make nanometer-scale materials with moving parts, such as nanotweezers [4].

2) Currently, two major challenges face nucleic acid-based nanotechnology: (a) to produce complex superstructures from simple molecular building blocks, and (b) to perform controlled mechanical movements in molecular devices. Liao and Seeman [5] have presented a DNA device that can program the synthesis of linear polymers through positional alignment of reactants. Chworos et al [6] have used rationally designed RNA building blocks as jigsaw puzzle pieces that direct pattern formation. The two studies demonstrate that it will be feasible to build functional materials and devices from "designer" nucleic acids.

3) Nanotechnology researchers have sought to mimic nature's biological motors to create nanometer-scale machines that can function in an engineered environment. Liao and Seeman [5] take an important step in this direction with a device that mimics the translational capabilities of the ribosome. The device consists of two subsections, each with two structural states. Different pairs of DNA "set strands" can be added or removed to bring the device into any one of four states. Each state allows the positional alignment of a specific pair of DNA motifs that are selected from a pool. The pairs bear polymer components that can then be fused in a specific order.

4) As proof of principle, Liao and Seeman [5] used DNA as the polymer that is aligned, and enzymatic ligation to fuse the polymers. Positional synthesis with the prototype device thus results in four different DNA strands, each containing a defined sequence. In this ribosome-like DNA device, there is no complementary relationship between the signal sequence and the products. Furthermore, all polymer reactants exist simultaneously in one solution. These features make the device appealing for building nanometer-scale machines that control massively parallel chemical synthesis. Kanan et al (2004) have shown that DNA-templated organic synthesis can be used to discover new bond-forming chemical reactions.

References (abridged):

1. N. C. Seeman, J. Theor. Biol. 99, 237 (1982)

2. E. Winfree, F. Liu, L. A. Wenzler, N. C. Seeman, Nature 394, 539 (1998)

3. H. Yan, S. H. Park, G. Finkelstein, J. H. Reif, T. H. LaBean, Science 301, 1882 (2003)

4. B. Yurke, A. J. Turberfield, A. P. Mills Jr., F. C. Simmel, J. E. Neumann, Nature 406, 605 (2000)

5. S. Liao, N. C. Seeman, Science 306, 2072 (2004)

Science http://www.sciencemag.org

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ON SUPRAMOLECULAR ASSEMBLY ON SURFACES

The following points are made by G.M. Credo et al (J. Am. Chem. Soc. 2002 124:9036):

1) Metal-molecule-metal junctions have been used to elucidate single-molecule properties in organic monolayers that are applicable in molecular electronics. For example, pore-based sandwich structures(1,2), mechanical break junctions(3), and Hg drop electrode top contacts(4,5) have been used to characterize the current-voltage properties of organic monolayers. Studies have also employed scanning tunneling microscopy (STM) and conducting atomic force microscopy (cAFM) to probe the current-voltage properties of molecular junctions with more limited contact areas. In particular, reports of STM studies on redox-active molecular monolayers have described the use of electroactive moieties in molecular junctions to facilitate nonlinear current-voltage behavior. In a recent example, the nonlinear current-voltage phenomenon of negative differential resistance (NDR) was observed in an electroactive, ferrocene-terminated self-assembled monolayer (SAM). The identification of nonlinear current-voltage properties such as NDR for individual molecules expands the potential applicability of molecule-scale components from use as conductive wires to multistate molecular switches.

2) Chemical self-assembly is an attractive method for reversibly constructing well-defined supramolecular systems with properties defined by their molecular components. In particular, hydrogen bonding is a familiar construction motif in natural systems and has been used to assemble functional nanostructures, such as metal nanoparticle-based networks. Applying these concepts, noncovalent self-assembly provides a potential method to install and subsequently remove electroactive functionality in molecular electronics systems. To explore this possibility, the authors report they patterned a footprint region for molecular assembly on a surface featuring a recognition-element-terminated thiol. The authors then used moieties featuring complementary recognition to tune the current-voltage properties of the patterned region. In the current work, the authors used an STM tip to pattern and probe molecular assemblies and independently verified the hydrogen bond-mediated assembly process using bulk electrochemical and spectroscopic techniques.

3) In summary: The authors report that molecules capable of complementary hydrogen bonding were used to control the noncovalent self-assembly and electronic properties of a chemically well-defined surface mesostructure. In this work, the authors patterned a footprint region for molecular assembly on a surface and used moieties featuring complementary recognition to tune the current-voltage properties of the patterned region. With the appropriate functionalities on the complementary moieties, the authors were able to increase and decrease the observed conductance in surface-bound mesoscale structures imaged by scanning tunneling microscopy (STM).

References (abridged):

1. Chen, J.; Wang, W.; Reed, M. A.; Rawlett, A. M.; Price, D. W.; Tour, J. M. Appl. Phys. Lett. 2000, 77, 1224-1226.

2. Chen, J.; Reed, M. A.; Rawlett, A. M.; Tour, J. M. Science 1999, 286, 1550-1552.

3. Reed, M. A.; Zhou, C.; Muller, C. J.; Burgin, T. P.; Tour, J. M. Science 1997, 278, 252-254.

4. (a) Holmlin, E. E.; Haag, R.; Chabinyc, M. L.; Ismagilov, R. F.; Cohen, A. E.; Rampi, M. A.; Terfort, A.; Whitesides, G. M. J. Am. Chem. Soc. 2001, 123, 5075-5085. (b) Holmlin, R. E.; Ismagilov, R. F.; Haag, R.; Mujica, V.; Ratner, M. A.; Rampi, M. A.; Whitesides, G. M. Angew. Chem., Intl. Ed. 2001, 2316-2320.

5. Selzer, Y.; Salomon, A.; Ghabboun, J.; Cahen, D. Angew. Chem., Int. Ed. 2002, 41, 827-830.

J. Am. Chem. Soc. http://pubs.acs.org/JACS

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