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An Investigation of Nucleic Acid/DNA-Based Manufacturing by Frank Boehm Aug/2004 Research contribution to the Center for Responsible Nanotechnology: Thirty

Essential Nanotechnology Studies- #10 (What will be required to develop nucleic acid manufacturing and products?)

Considering that the information encoded in DNA, combined with its translational capability and a protein fabricating infrastructure, has given rise to such a wide array of richly diverse and complex biological life forms, it seems quite apparent that it stands out as a strong molecular manufacturing contender. Dr. Eric Drexler mentions that I am confident that systems that could reasonably be said to perform DNA-based molecular manufacturing are physically possible. [89]

Speculatively, it may someday enable even atomic fabrication processes. Its ability to store vast amounts of information in such a minute physical space, and its potential for conveyance of elaborate programmable building instructions (via deliberately designed base-pair linkages) are attractive features when one has visions of extrapolating its manufacturing effectiveness into a massively parallel DNA manufacturing complex.

This study will explore various single strand and hybridized nucleic acid structures and how their specific kinetics may be applied to the potential construction of purposefully designed and useful end products fabricated at the molecular level. A further investigation will look at how these entities might be configured in a stable manner to perform robust and repeatable work

together as parallel units.

DNA- Structure and Function: The most pervasive form of the DNA molecule in biological organisms is B-DNA (righthanded) having its native configuration in the form of the double-helix (duplex). It is ~2nm in diameter, having 0.34nm between bases, with 10 base pairs (bp) per double helical turn. BDNA is a stable structure due to water molecules that bind along the length of the molecule. [55] Other primary species include A-DNA (right-handed) 11 bp per double helical turn, and ZDNA (left-handed) 8 bp per double helical turn (this form is more likely to appear where there exists a high concentration of salt). There is also a less common H-DNA (triple helix). [2] Nonstandard bonding enables nucleic acids to form many novel and complicated structures. It is assumed that B and Z DNA have varied electronic properties due to their differences in stacking ( electron overlap in base pairs). This may provide a one-dimensional conduit for electron flow through the central axis of the helix. [41]

It is, in single-strand form, a linkage of nucleotides comprising a linear polymer. The nucleotides are made of the 2-deoxyribose sugar tethered to one of four different bases (A,C,T,G) adenine, cytosine, thymine, and guanine. They are also chiral and are held together by a phosphodiester link spanning the 5 carbon atom in one nucleotides sugar and the 3 carbon atom of its neighbour. One end of the molecule has a contrasting chemical makeup compared to its other end.

Complementary base-pairs (A-T) (C-G) are held together by weak hydrogen bonds. A hydrogen atom when bound to an electronegative atom has a portion of its charge density transferred into 2

the bond. This will give rise to an area of positive charge on the side of the hydrogen away from the bond. This domain will subsequently interact weakly with a lone pair of electrons existing on another atom. [1] The force required to pull apart two DNA strands is ~70-75 pN/bond and the DNA backbone can tolerate ~10nN in tension. [58] About twelve base pairs must associate to form a duplex that is stable at room temperature. [223]

The external geometry consists of successive sugar-phosphate backbones. These are separated by two grooves (major and minor) and are defined by the positions of the base-pairs. There are exposed atoms (nitrogen and carbon) that face into the major groove that might be used as attachment points for building up pre-designed structures.[2]

Experimentation has revealed that DNA can be stretched to about 2.1 times its original length without breaking. [101] Its tensile strength of DNA has been determined to be ~270 pN (lower bound), [106] and the amount of force necessary to break a strand of DNA has been estimated at ~476 +/- 84 pN. [102] For comparison, the smallest diameter spider silk is ~20nm with a nominal tensile strength of ~1.3 GPa [104,105] The average tensile strength of polysilicon is 1.45 0.19 Gpa. [107] Single and multi-walled nanotubes have a tensile strength estimated at ~130 Gpa. [108]

Survey of Nanodevices Based on DNA: Within the cells of living organisms, chemical energy converted into mechanical actuation via 3

microscopic machines is widespread. [3,4] Extrapolations can be derived from these entities, and lessons learned as to how one might design analogous nanometric synthetic devices capable of similar functionality. In the biologic realm, molecular motors are predominant purveyors of motion (e.g. dynein activating cilia and flagella, and actin, myosin, kinesin transfer cellular components).

The replication of the molecule DNA can be construed as a complex assembly mechanism, which makes copies of itself almost flawlessly, performs error corrections, and proof-reading. [5] Human designed nanometric actuation devices may acquire the ability of movement by the application of synthetic molecular motors. Chemical energy to power these machines might be in the forms of ATP (adenosine triphosphate), GTP (guanosine triphosphate), via glucose oxidation/reduction, or hydrolysis, among other sources.

In the bacterial flagella, protons are transited through the motor and their energy is extracted as they pass through the electrically charged cell wall. [6] It is conceivable that nanoscale motors may use energy sources quite apart from what is found in natural systems. To date, several molecular-level actuating entities have been devised, one being a light-driven rotary motor. [7,8] In addition, catalytically active electrodes may be employed to drive redox reactions. [109]

Looking at DNA-based physical actuation mechanisms, several devices have been constructed using the motive energy forces inherent to base-pair matching resulting in the formation of the well-recognised double-helix. Although it has been demonstrated that this can be accomplished,

these devices currently represent very preliminary steps although they represent a pathway to enabling a full-fledged nucleic-acid fabrication technology. In the not-too-distant future, however, similar mechanisms may form components of the infrastructure required for molecular manufacturing systems. Dr. Nadrian Seeman, (New York University) has pioneered the design and construction of many unique DNA structures and viable actuating devices, and continues to break new ground toward the advent of a true and robust DNA molecular manufacturing capability. [43]

Due to its powerful molecular recognition savvy, exquisite versatility, and relative stiffness (in its double-helix form), DNA is an extraordinary molecule with which to construct a myriad of novel nanometric tools. The discipline of biotechnology furnishes the expertise and technologies for its predictable formation by having the capability for designing in virtually any sequence. Interactions between two single strands can in this way be manipulated to create structural elements that may be manifested in a predetermined way. Sequences (~100 basepairs) long can be custom-made using todays technology, including molecule attachment nodes and many other modifications. This allows for the attachment of dye molecules (for example) at specific locals along the length of the DNA molecule, and the addition of catalytic regions to enable bonding to a substrate. [9]

Various enzymes can reconfigure DNA in specific ways. Polymerase enzymes can amplify DNA strands, the ligase enzymes promote covalent bonding of DNA strands, and the restriction enzymes cleave DNA strands very accurately as directed by the sequence of the bases. Facilitation for construction of the most elaborate nanoscale mechanisms ever attempted has

been so far made possible by these means. [9]

Complimentary base-pair bonding (e.g. A-T, C-G) orients two single strands in an anti-parallel fashion. An unstable DNA molecule will result when base-pairs are mismatched, as adhesive forces are much weaker than when they are bound to their natural complement. If these mismatches are significant enough in number then the double-helix will not form. When immersed in an aqueous environment, two strands in such a situation will separate. This powerful recognition trait is employed when constructing DNA-based structures, as the two complementary strands will only associate in a precise way. They gravitate toward each other and will self assemble due to the random effects of Brownian motion. This automatic process of matching of strands is termed hybridization.[9]

Hybridization of complementary base pairs is considered a second order chemical reaction, (a second-order reaction has a rate proportional to the concentration of the square of a single reactant or the product of the concentration of two reactants) [110] whereas the unzipping of DNA strands into separate entities is a first order reaction. Temperature and duplex length are primary factors for dissociation of the duplex into single strands, however they have less influence on their association. [10] A negligible rate of dissociation becomes manifest at low temperatures, whereas hybridization rates for strands having enough length can be held at acceptable levels. DNA requires a salty environment to hybridize; instant dissociation will ensue if the salt is removed. [57]

A DNA duplex section 20 bases in length in a 1M salt buffer at 20C is reasonably stable, and

the estimate for dissociation is ~105 years via data extrapolation. [10] The hybridization rate for single strands present at a 1M concentration is several minutes. The average assembly time required for the construction of a nanostructure, or to actuate a nanometric motor operated by the action of nucleic acid base-pairing, can be indicated by these studies.

The immense number of possible base combinations translates to the potential for a vast array of self-assembled complex structures. These include two-dimensional sheets of DNA and wireframe polyhedra [11-14]. DNA-based computers can also be fabricated. [15,19] The relative stiffness (in comparison to other biopolymers) of this double-helical polymeric molecule makes it a good candidate as a nanometric construction material when employed in multi-strand structural configurations.

In solution the single-strand polymer is unceasingly bumped around by Brownian motion affinities. A short section of polymer will be bumped about and rotated by these constant encounters, whereas long segments will react by bending. Its persistence length defines this bending effect over a certain length, and can give an indication as to its stiffness, and hence its propensity for remaining stable. [20]

The persistence length of a single strand of DNA is ~3 bases (1nm) analogous to a flexible section of spaghetti. [21] This parameter in duplex form is ~50nm, which exhibits a greater stiffness, however, it is still quite floppy as a chain 5nm long will bend by 25, and 1nm will bend by 11. [22,111] This variance of stiffness might be exploited to provide motion within structures made of DNA. Available free energy (via the matching of base-pairs) can be tapped

to move molecular objects. This parameter is specified by the species of base-pairs in the process and that of its adjoining set. Each base-pair has an approximate free-energy value of ~73meV (1.8 Kcal mol-1). [23,24] An object may be displaced (see Fig.-1) by twice the 0.34nm nucleotide spacing (in a single strand of DNA), by virtue of the hybridization process.

Fig.-1: Hypothetical scenario depicting DNA initiated displacement of a weight pulled against the force of gravity F. The hybridizing free energy is changed into work when a base pair is produced and the weight is displaced by twice the distance x between bases in single-stranded DNA, h = 2x. (from F.C. Simmel, B. Yurke) [9]

AFM measurements, optical traps, as well as other tools, verify that a force of 15pN is required to stall a DNA motor. This is based on forces required to pull complementary strands of DNA apart, and are equivalent to those generated by biological nanoscale motors. [25-27] The force required to stall a walking kinesin on a microtubule is ~5pN. [28] The kinetics of DNA hybridization is the driving force behind the assembly of nanometric structures comprised of nucleic acids, and the functioning of DNA-based molecular motors. [9] There appear to be many ongoing efforts which may give rise to a wide array of tools applicable to DNA-base

manufacturing. [152]

A phenomenon termed branch migration can displace specific DNA strands within a nanostructure. This happens when two single strands having the same sequence of bases attempt binding simultaneously with one or two complementary strands. Slight deviations in thermal parameters can cleave base-pairs at the locations where they compete to bind with their mates. The creation of subsequent base-pairs can initiate the movement of the branch point, one basepair to the right or left of the original position. This process can also be applied to threestranded branch migration. When the concentration of magnesium is high enough, it can slow down or even halt this process. [9]

In nature, branch point migration is inherent feature of DNA repair activities and DNA recombination. A four-way mobile junction, called a Holliday intermediate structure, is created by strand exchange between two sequences that are homologous. [29] Four-way junctions in non-homologous DNA duplexes can give rise to a static structure, as the branch point is immobilised. These might serve as good candidates for fundamental structural units in the fabrication of nanometric devices, having both double (DX) and triple multiple crossover points. [14,30]

A Toolbox of Nucleic Acid Devices: B-Z DNA Torsion Device: The first nanomechanical device to be covered makes use of the configurative changes from the

B-form of DNA (right-handed), to the Z-form of DNA (left-handed). This is called the B-Z transition, and can be utilised to initiate motion at the nanometric scale. [31] In this design, changing chemical conditions alter an active region in combination within stiff and level DX members, to give rise to the B-Z transition. A DNA strand containing methylated cytosine residues (which are more prone to B-Z transition) are connected to the two stiff DX structures. By altering the concentration of specific chemical groups the B-Z device can be actuated (the two level members rotate one-half turn with respect to each others original orientation) repeatedly.

Indirect verification of this cycling can be accomplished by a common biophysical method called fluorescence resonance transfer (FRET). This is used for verifying motion and measuring nanoscale distances, and is derived from differences in the energy transfer efficiency between two fluorescent dye molecules as the distance between them is varied. The energy transfer distance (Frster distance) between typical dye combinations is usually ~ 2-6nm. [9]

One drawback to this albeit robust device is that when many such mechanisms are bound together, the entire construct would still have only two states B or Z. Bestowing individuality upon each singular unit would require that separate and unique activating strands be designed to trigger each entity.[43]

DNA Actuation Devices: This device is induced to movement by hybridization using external fuel strands which are engineered; they are single strands of DNA that combine with exposed flexible complementary

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sections within stiff members, which may be anchored to a substrate. [9] Anchoring is an important consideration when one wishes to control movement in a certain manner while achieving accuracy of component/product placement. Accomplishing this control would be exponentially more problematic when free-floating in an aqueous environment.

Repeatable actuation of the device is dependent on re-establishing its original state through displacement of the fuel strands with their complements. This can be done using the DNA branch migration process. For example, a strand X (the fuel strand) can be peeled off its complement Y (displaced) by another strand Z (the removal strand) if X and Z are complementary, and X and Y are only partially complementary so that a "toehold" of X remains dangling, unattached to Y. Z binds to the toehold and proceeds to unzip X from Y. At the end of the process, X and Z are bound together, and Y is free. [112]

A number of additional DNA-based actuation devices have been developed: [134] - silicon cantilevers can be bent using DNA hybridization. [135-139] - a piston demonstrates linear motor type movement of 5nm. [140] - a single DNA-molecule inchworm motor has been constructed. [141] - a rotary motor has been fabricated having a configuration of six strands of RNA surrounding a central DNA strand, and activated by ATP generating 50-60pN of force. [142] - a grasp and release entity made of DNA can bind to and release specific types of molecular proteins and might possibly be endowed with the capacity for binding and releasing almost any type of protein. [143] - a continuously running DNA motor, and sliding strut actuator have been proposed. [144]

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- X-shaped DNA tiles can be linked to make up a grid-work that can be expanded and retracted by chemical manipulation. [145-148] - RNA polymerase positioning motor (15-20pN) can be made to walk along a DNA duplex with nanometric precision. [149] - designs have been put forward for autonomous DNA nanomechanical devices that can actuate without external environmental changes. [150-151]

DNA Tweezers: This device set a precedent by being the first to incorporate branch migration as a vital part of its functionality. [32] Starting with three single strands (A, B, and C) creates DNA tweezers. Engineering of B and C sequences ensures that they hybridise with strand A for a 18bp length each. The final mechanism is comprised of two 18bp duplex sections spanned by a 4bp long section that is single stranded. Extending from the two double-helical arms are 24bp long single strands (unbonded sections of B and C). Because the persistence length of the duplexed DNA is large, the 18bp long members can be regarded as stiff entities while single-stranded sections behave as interconnects with flexibility. [9]

The addition of a fuel strand (F1) to the open state can bring the two arms together to close the device. The F1 fuel strand is made of 48 bases and is comprised of two 20 base long sections. These are complements to the unhybridized sections of B and C, with the inclusion of a 8 base toehold overhang section to facilitate the strands removal by a removal strand F2. F2 can remove F1 via branch migration to return the tweezers to their open state. In this way the tweezers can be repeatably cycled between the two conformations. Again, the movement of

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the tweezers can be verified by the FRET technique. [9]

There are two inherent disadvantages with this device. Firstly, there is a deficient flexibility in the open configuration. Secondly, the accumulation of waste product after each cycle, when F2 dissociates F1 from the device, can stall the mechanism. Occasional removal of the waste strands would remedy this problem. A disequilibrium is sustained in some biological models. For example the dissociation of the inorganic phosphate in ATP to ADP conversion is restored as the phosphate in these molecular entities is tied to metabolic functions. [9]

DNA Scissors: In this device, hybridization drives the transduction of the closing action of one side of the device, and transfers it to another section which is 13nm distant on the other side, actuating a scissor-like motion.(see Fig.-2) This action is accomplished by adding short sections that act as links. These links provide the freedom of rotation by functioning as rigid hinges. One drawback in the force transferral is that the handles side closes more efficiently than the jaws side.[33]

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Fig. 2: DNA scissors: The double helical segments of the device are coupled by C3 spacers. When the right pair of arms handles of the device are actuated to the closed state by the fuel strand F, the left pair of arms jaws will also close. The addition of F will reset the open state. (from F.C. Simmel, B. Yurke) [9]

Deoxyribozymes: Deoxyribozymes are DNA molecules that have the capability for catalytic activities. These catalytic entities have very high selectivity and product turnover rates (1010-fold over background). They are single turnover enzymes in contrast to many natural enzymes, which have multiple turnover capabilities. Although relatively little is known about the actual structure and mechanisms of this entity, some perceived catalytic reactions are: DNA phosphorylation [90], DNA adenylation (capping) [91], DNA deglycosylation [92], porphyrin metalation [93], thymine dimer photoreversion [94], and DNA cleavage [95]. The DNA ligase deoxyribozyme can join two DNA substrates via reaction of a 5- hydroxyl group having an activated 3phosphorimidazolide. To initiate activity, it would require either Zn2+ or Cu2+. [100]

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There are several inherent challenges that will have to be addressed prior to the application of these entities toward practical utility in joining DNA substrates. One issue is that activated termini are not readily obtained [96] and as well, their sequence prerequisite can significantly minimise the substrates that may be employed. [97] Catalytic enhancements can be devised however, by adding functional groups via chemical alteration. [98] Functional groups such as metal chelators (pyridine, or imidazole) can be integrated using mutant DNA polymerases. [100]

When aiming to initiate a reaction on a substrate that is not a nucleic acid, the substrate might be coupled to a nucleic acid having the sole purpose of binding with the DNA enzyme, via regular Watson-Crick base pairing. This combination may include a linked section that could be cleaved, enabling removal of the nucleic acid after catalysis. Other methods for binding the DNA to the substrate without use of base pairs might be through identification of particular sequences that simultaneously bind to substrates and undergo catalysis, or by creating DNA aptamers (DNA molecules that bind targets) that might be utilised as the basis of binding energy. [99, 100]

Speculatively, it may be possible to create modular DNA catalysts by methodically combining DNA aptamer and enzyme domains, in which the aptamer would bind the to substrate and the enzyme would catalyse and produce the desired end product. [100]

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DNA- Three State Device: A variation of the scissors mechanism is one in which the device has two mechanically strong states and an intermediate state. In its rest state, the extensions of single strands join to form a somewhat circular geometry. Subsequent removal of fuel strands F1 and F2 reconfigures the device into a stretched, or alternately, a closed state. By applying appropriate removal strands this device can be cycled through its three conformations. Verification of all three states was confirmed by the FRET technique. [9]

The most mechanically robust positions for this device are its closed and stretched states when the salt concentration is correct. Force transduction to other potential components might be translated by the operation between these two states. [9]

DNA Crossover Junction-Based Device: This device employs branch migration via strand displacement to cycle between conformational states. It consists of a more intricate double crossover molecule made of a number of four-way junctions that are linked together. The animated section of the mechanism [34] is made up of two helices of DNA held together by multiple crossovers. Rotation of 180 of the lower portion of the device may be used to create a molecular switch. Because of the rigidity of the crossover molecules, these devices are quite durable and would be capable of transducing their produced force efficiently.

The fuel strands for this device are functionalised with biotin, which will readily bind with streptavidin (the bacterial analogue of biotin and commonly used molecular linker system).

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[113,114] The non-covalent adhesion between streptavidin and biotin can tolerate a force of 5 pN for 1 min, but will break within 1 ms if 200 pN are applied. [115] This affinity would indicate that an active process for sustained waste removal is possible. Waste products can be purged from the system using magnetic beads having streptavidin-coats. [35]

DNA G-Quartet Device: The base guanine can form bonds with all other bases as well as itself. Specifically, when monovalent cations are present at areas rich in guanine sequences, the formation of G-quartets can occur. The in-plane alignment of four guanine bases is supported by hydrogen bonds, and potassium ions facilitate their assembly. [20, 36] DNA non-coding telomeres harbour stable Gquartets. [9]

This device is made of a single strand of DNA and presents a tetraplex construction. The 17 base sequence is designed such that two stacked G-quartets are formed bound by two upper loops and one lower loop. To activate the device, an external complement strand is added or removed. This hybridization changes the conformation from a tetraplex to a duplex. [9]

DNA Motors- Free-Running and Clocked: The mechanisms mentioned above are basic molecular motors that transfer the free energy of fuel and removal strand hybridization into mechanical action. Free-running motors are activated by internal switching mechanisms, whereas a clocked motor will cycle through its states having the requirement of alternating fuel supplies to step through its activation sequence. [112] Clocked motors are practical when external components must have co-ordination with their

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motions. An analogous example of a clocked motor in biology is the regulatory protein which is altered by GTP hydrolysis. [37]

Simultaneous application of fuel and removal strands to the devices discussed above will not translate to efficient state-cycling functionality. This is due to direct hybridization of these strands before any contact with the motor is achieved. Bio-motors, for the most part, are free running (they will cycle endlessly on a single fuel), as when an ATP or GTP molecule is supplied, they will move through their complete set of conformational states. The fuel molecules do not deteriorate because they are metastable, and will only give up their stored energy once they are bound with the motor molecule.

Molecular motors made of DNA have been devised that engage accessory strands to prevent fuel and removal strands from direct binding. [38,39] This is accomplished by protecting the fuel strand- F1, with an auxiliary strand section- P, which binds with and forms an intermediate structure having two duplex arms and a small loop of single-strand. Observations of the resulting kinetics have found that this preemptive fuel strand binding slows down hybridization with the removal strand- F2 by a factor of 100, when correlated with an unprotected fuel strand. [9]

A catalytic strand section- C is now introduced, and is designed to bind more readily with the F1/P complex than with F2. When this section hybridizes with F1/P via branch migration, the loop is opened giving the complex a greater affinity for F2. Next F2 attaches to the fuel strand triplet (F1/P/C) and displaces both P and C, giving waste products F1/F2 and P. After one

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cycle, the C strand is recovered. Strand C can be regarded as a very basic molecular machine. It cycles between a resting single-stranded configuration and a stretched duplex state when combined with the F1/P/C complex. This simple nucleic acid molecular motor can also be viewed as a new species of deoxyribozyme. [39,40]

Conceivably, structures could be made involving multiple motors, each being activated by a different F1 and F2 strand. These might enable the construction of geometries that incorporate a sophisticated series of actions. Organisation of motions would be governed by the procession of F1 and F1 strands provided. In other words, the co-ordination of movements would be programmable. [9]

Although the mechanisms comprised of nucleic acids (as described above) have so far been employed to handle only dye molecules in these investigations, they represent a potentially viable, controllable, and quite versatile set of tools for the manipulation of nanometric scaled objects. They illustrate a step in the direction of a vision for the possible parallel integration of many such devices working together, performing specific tasks to build subsequent elemental units, sub-components, and components to produce nanscopically precise and commercially useful macroscale products. Computer models and simulation capabilities will be important factors, and time saving tools toward the design and verification of DNA-based devices preceding their actual construction. [54]

Precise Manipulation of Proteins using DNA: The exploitation of DNA hybridization can lead to the generation of self-assembled ordered

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arrays of proteins. [184,185] The premise behind the DNA Guided Assembly of Proteins (DGAP) is that DNA may be used as an assembly jig in solution, and that covalently bound and precisely oriented nano-assembled proteins might be fabricated. [186] In DGAP, a number of DNA sequences are adhered to specific locations on the surfaces of target proteins.

Building blocks comprised of proteins (e.g. biomolecular motors, proteinaceous fibers, enzymes, antibodies) can be compelled to assemble in desired configurations via complementary sequence matching. These assembled entities may then be further stabilised using covalent inter-protein linkages. This protein positioning technique might also be employed for the assembly of entities other than proteins. These may be functionalised at numerous locations using explicit DNA sequences. [134]

DNA Manufacturing Complex- Infrastructure Elements: A solid infrastructure of appropriate scaffolding and/or dedicated substrates might facilitate the possibility of nucleic acid molecular manufacturing when employing position cycling mechanisms like the ones discussed above. When planning to assemble (cumulatively) nanometrically precise macroscale end products via a programmable DNA manufacturing complex, it seems evident that success might be more likely when this complex is bonded and somewhat constrained (possibly at multiple points) to a stable framework, to minimise the omnipresent effects of thermal noise. [112]

This strategy may allow for the more precise positioning of components, rather than if they were floating haphazardly and constantly being jostled within an aqueous medium. Though for

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smaller and simpler selfassembling components and sub-components the aqueous mode may have its benefits.

Current microfabrication techniques have allowed the interfacing of organic species to inorganic substrates. These are employed in the DNA-chip manufacturing industry where DNA microassays are applied to plastic or glass wafers. Photolithographic processes developed for making computer chips are used to tether many strands of DNA to these surfaces. The utilisation of photosensitive materials and thin chemically doped layers assist in the process. [42] An important challenge to address, which once solved, will open up a whole new range of DNA manufacturing possibilities, is the transition from 2D to 3D fabrication processes. [43]

DNA scaffold structures that can be amplified may play an essential part in the creation of increasingly complex nanometric products. One might speculate that autocatalytic polynucleotide systems may be coupled with intricate DNA-based 3D structured building blocks (e.g. switches, motors, actuators) to produce quite elaborate self-replicating molecular mechanisms. [134,180,197-203]

Non-covalent 3D nano-entities have been created utilising the self-assembly of covalent junctions within trisoligonucleotides, (synthetic 3-arm junctions in which the 3-ends of three oligonucleotides are linked). Specific objects can then be replicated by employing an electrophoretic process called eSPREAD, (surface-promoted replication and exponential amplification of DNA analogs, also called cloning and amplification technology, CAT). This procedure can be used for the cloning of nanomachines on the electrophoresis chip substrate.

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[178-180]

Subsequent to the adherence of rigid starting members to a substrate, (trihelical DNA and a novel 4-stranded form of DNA (the G wire'') have been developed. [173-177] These entities may serve as an extra stiff construction struts giving a relatively solid starting foundation, prefabricated elements (self-assembled structural components) might now be added to fulfil a design plan. This may include the build-up of an oriented support structure prior to the installation of the actual fabrication mechanisms themselves.

Some basic structural elements comprised of DNA might have the geometry of cubes with corners made of triple-armed junctions, two-dimensional crystals, and six-armed junctions, which can be bound together to construct a scaffolding, and octahedrons. [43] Investigations into the specific rigidity of these constructs may elucidate how they might be reinforced (if required) should they be too floppy in their basic form. A four-way junction would also be a useful interconnect to hold other entities together.

These entities can be held together in a robust manner by utilising DNAs sticky ends (singlestrands that extend beyond the main duplex structure). These will bind directly to a mirrorimage strand. Some tools that can assist in constructing a framework for a DNA fabrication complex are restriction enzymes to snip DNA at specific locations, and ligases, which can initiate covalent bonding between duplexes. DNA molecules can also be decorated by various molecules, fullerenes, dendrimers, and functional proteins by adhering to available atoms on the exterior of the phosphate backbone. [43,185,187,190]

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The use of polymerases may assist in the elucidation of novel, single-stranded DNA molecular devices that might be used to controllably induce relatively complex mechanical behaviours from DNA loops configured by an octahedron scaffold. This procedure (directed molecular evolution) [195] may be prompted to evolve into forms of molecular assemblers and disassemblers analogous to chaperones, or polymerase clamp loaders. [134] Polymerases are unique enzyme catalysts that use DNA as a substrate/template in the chemical reaction to guide the structural configuration of the product, which is also DNA. The clamp loader acts as a molecular facilitator, deriving energy from ATP binding and hydrolysis to interface and load a sliding clamp (closed protein ring) onto primed DNA, forming a topological link between the two. [193,194]

Dr. Eric Winfree, from the California Institute of Technology has devised molecular tiles. The edges of minute blocks of DNA can be programmed to bind together in patterns. [53] So far, only simple patterns have been made this way; therefore the goal being set is to design patterns having far more complexity. Potential real-world applications are estimated at ~5 to 10 years. [52] Perhaps a programmed arrangement of tiles could form the basis of a working substrate onto which appropriate DNA assembling devices would be mounted.

One scenario for a DNA fabrication complex may be to have the scaffolding and substrates mounted to a surface and immersed in an appropriate liquid environment (e.g. in shallow sectioned trays). This might allow for the free movement of all the required individual construction elements without them clumping together or eroding in some way. Substrates

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might be designed in such a way that they contain large surface areas to facilitate efficient production. A elementary DNA decamer (10-unit monomer chain or polymer) has been fabricated that can form an extended and staggered quadruplex array, attaining lengths of ~1000nm. [174-177] Extensive DNA arrays having various connectivities and irregular interstices have been designed as well. [178].

As discussed earlier, smaller components (made from metals, carbon etc.) could be prefabricated beforehand, perhaps within thousands of small solution-filled cavities in a preassembly tray. (Seeman, et al has fabricated a tiny section of nylon on the backbone of a nucleic acid). [43,44] These parts could have uniquely-designed guider-strands attached to them that might be used to direct the component to its required position and lock-in to a complement docking-strand on the substrate.

A further verification of proper placement could be accomplished by the implementation of a DNA barcode. DNA barcodes have been devised by bonding double-crossover (DX) units, representing 0, and double-crossover and junction (DX+J) units representing 1 in deliberately designed configurations, and lashed together in parallel constructs. [43] These barcodes may perhaps be read and registered via a laser-scanning set-up, and double-checked against the original design plan.

Once all of the components (inorganic structures) in this step are in position, and have been verified, a nuclease could be added to the mix resulting in the dissolution of the original DNA infrastructure. This step would leave behind the desired product (e.g. nanoelectronics grid,

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photonic element, quantum computing element, or nanometrically specified materials).

This process could be repeated as another DNA assembly seed substrate layer is added. Conversely, these layers might conceivably be stacked one on the other in place, until the designed functional macroscale object emerges. At this point of completion, the DNA dissolution solvent could be added.

Another productive step might be to construct custom substrates ahead of time, and then freezing them for storage until required. These units might be frozen for years and still be viable. DNA is readily re-natured, and its structure is very much a function of the conditions of the solution. [1] DNA can be concentrated via precipitation using ethanol or isopropanol. The overdrying of DNA will make re-solution more problematic and can cause denaturation. [103]

The characterisation of subtle electron flows involved in the process of enzymatic catalyzation of is an application that might utilise DNA-based scaffolding (DX and DX+J linkages) in that this structure may be used to immobilise reactive enzymes for use in electron transfer schemes. [1] These enzymes might catalyse certain molecules (e.g. glucose) and transfer electron flow through to nanowires acting as probes for the testing of nanoelectronic components.

Discussion: Diamondoid and sapphire materials have been deemed the materials of choice for advanced nanotechnology (e.g envisaged nanorobots). [89] Whether a DNA-based fabrication complex could construct such materials seems uncertain. There are problems with this idea, which

25

involve differences in scale and energy. DNA is on the order of 20-50 scale, and diamondoid entities are on the 1.5 scale. Ultimately, DNA may be able to have control of these small distances; however, the problem of energy is more pronounced.

Most DNA transformations involve low free energies (e.g. hydrogen bonds are a few kcal/mol). Carbon-carbon bonds in hydrocarbons are about 80 kcal/mol, and are likely to be similar in diamond. Therefore, it is improbable that the small forces generated by DNA could be used to manipulate carbon atoms to form a diamond lattice. [1] In addition, input molecular fragments might react with DNA in adverse ways. [89] DNA as a substance is very pliable, and is very efficient at building other soft molecules. However, it is unlikely that it will have the capacity to directly fabricate diamondoid entities. [72]

Perhaps fullerenes could be catalysed by DNA-positioned enzymes? Large (200-atom) graphite sheets, as well as buckybowls, have been built underwater. [116] A growing graphene/fullerene edge might be shaped by positioning an enzyme to deprotect it in specific spots. [112] Accelerating a slow deposition process can also be used to build engineered structures. [89] In addition, the potential fabrication of intricate silica structures by a DNA orchestrated manufacturing process would be a tantalising area for investigation. [118] (note: silica-based diatoms with elegant geometries are constructed at low energies). [112,117]

DNA damage in biological systems occurs on a constant basis. Extensive repair mechanisms have evolved however, to uphold its functional integrity. A derivative of DNA may be more effective for use in manufacturing processes. [1] Other issues to investigate are the creation of

26

controlled feedstock presentation into the complex, and proper disposal of waste products from the system. Many useful products might be built using DNA, both by employing it as a structural material and for use as a programmable jig to assist in assembling (aligning, joining, etc.) non-DNA components, be they organic or inorganic. [72]

Replication of DNA Structural Elements- Octahedron: Explorations into the capability for self-assembly and limited self-replication are important considerations when aiming to fabricate products destined for macro-world applications. DNA may have great difficulty in fabricating any rigid substance atom by atom and may be better suited to assembling rigid entities already constructed by chemical or other means. It may also be far more adept at self-assembly than positional assembly, and will be limited to feedstock inputs that can tolerate an aqueous environment. [72] Many millions if not billions of identical units working together with equal numbers of other dedicated mechanisms will be required for building up (from molecular scale levels) useful products. Cloning methodologies honed from the molecular biology discipline might lead the way in this area.

An octahedron the size of a cellular ribosome, and designed to self-assemble from a single strand of DNA has been developed by a group of researchers at the Scripps Research Institute. The diameter of the structure is ~22nm, and it can be easily cloned, amplified, evolved, and adapted for a myriad of applications by virtue of its single-stranded starting conformation. Heating, (as well as sequential cooling steps) are also required in the self-assembly process.

27

Some sections are designed to fold back on themselves, giving stability to the overall structure. It has six vertices, twelve edges, and eight triangular faces. The twelve edges (struts) possess particular sequences that are addressable and enable the possibility of constructing more complex higher-order structures, and encapsulation of other molecular species. These structural units may also provide the ordered scaffolding required for nanoelectronics and photonics. [4546] The strands of DNA can be amplified exponentially via polymerases (although this method is rather error prone) or grown on bacteria and then purified. [112]

Once assembled, this tetrahedral geometry will strongly resist deformation, even though there are no inherent covalent bonds between the different parts of the chain. The entire structure can be further stiffened by the addition of smaller DNA loops. [112] The internal cavity can hold a ~14nm diameter sphere, and the triangular opening has clearance for an ~8nm sphere. These structures use molecular recognition to translate the information encoded in DNA sequences into 3D positional data. [46]

DNA Adhesion to, and Dissociation from Nano-Entities and Substrates: The true power of a DNA-based molecular manufacturing system may lie in its potential capacity to facilitate the repeatable manipulation and positioning of a myriad of inorganic nanoentities to form (in the end) usefully functional (nano and/or macroscale) products with virtually infallible nanometric precision.

In DNA hybridization, an exquisite organising mechanism is manifest that might configure a wide range of specifically designed nano-components to be in close enough proximity that they

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then may be interfaced and connected, (either mechanically, chemically, nanoelectronically, or with thermal, magnetic, or photonic assistance). Experimental remote control of DNA hybridization has been demonstrated via the inductive coupling of a GHz radio frequency magnetic field to a 1.4nm gold nanocrystal covalently linked to a DNA duplex. [172]

The self-assembly of DNA-based nanotubes have been fabricated, as well as several DNAbased dendrimers. [181-183] There have also been a number of investigations into the use of natural and artificial nucleic acids in combination with inorganic entities. [153-171] Quantum dots may be attached to DNA via an intermediate organic layer, and metallic nanoparticles can be fused as well to produce nanowires. [1]

DNA Binding to Nanotubes: DNA has been tethered to the tips of nanotubes. [224] Nanotubes are oxidised by treatment with acids to introduce carboxyl groups to the tips. [225] This procedure also adds carboxyl groups to any defects in the sidewalls of the nanotube. [226] DNA molecules are then attached to the carboxyl groups resident on the nanotube with functional linkers. [227-231] This arrangement might have advantages when aiming to precisely orient nanotubes into nanoscale devices.

Covering nanotube surfaces with DNA gives a negative surface charge which will produce a stable nanotube dispersion within the aqueous environment necessary for DNA directed assembly. In contrast to this, if only the nanotube tips are covered the addition of a surfactant is required to avoid nanotube massing via hydrophobic interactions between their exposed surfaces. Substantial DNA coating coverage of the nanotube sidewalls also allows flexibility as

29

per their length requirements when positioning them into an assembly (e.g. bridging the gap between two electrodes possessing complement oligonucleotides). A solid-state reaction using azide-photochemistry adheres DNA to nanotube tips and sidewalls. Subsequent to this is the synthesis of a DNA oligonucleotides from reactive groups on each photoadduct. [232]

Thiolated DNA: Gold nanoparticles have an affinity for thiolated DNA strands (thiols are the sulfur analogues of alcohols). [204-207] When preparing the DNA sequences there is a requirement for thiol modification in one terminal so as to immobilise DNA strands on the gold nano-particles. Subsequent to this preparation, the gold particles can be self-assembled due to the inherent gold-thiol affinity. This self-assembly process may also be applied to RNA and PNA (peptide nucleic acid). Additional fabrication options may present themselves with the potential for decorating a DNA duplex with proteins having exposed imidazole rings of histidines. They also have an affinity for various transition metal ions such as Mn2+, Fe3+, Co2+, Cu2+, and Zn2+. [208]

A trithiol-capped oligodeoxyribonucleotide has been synthesised that can be used to stabilise gold nanoparticles with diameters of ~30nm. DNA/nanoparticle combinations have been exploited in the construction of both 2D and 3D entities due to their distinctive optical, electrical, and catalytic characteristics. [209-215] Among the many strategies for the synthesis of DNA-nanoparticle hybrids are the direct adsorption of alkylthiol- or disulfide-terminated oligonucleotides on metal nanoparticle surfaces, [209] oligonucleotide covalent binding to a

30

pre-activated nanoparticle surfaces, [216] and adsorption of biotinylated oligonucleotides on particle surfaces coated with avidin. [70,217,218]

DNA Adhesion to Dendrimers: Dendrimers are synthesised incrementally, layer by layer with each layer redefining the precise dimension of the molecule. Each molecule has a nominal dimension of a few nanometers, though some have been fabricated up to 30 nm. The outer layer of the dendrimer molecule can be made to form a dense layered array of molecular groups that act attachment sites for the tethering of other useful molecules, such as DNA, in the peripheral branches [134,233-235]

DNA Velcro: Gold nanoparticles have been combined and then dissociated through the use of single strands of DNA. Attachment of nanoparticles to DNA has been demonstrated, but until recently a means for nanoparticle separation has not been shown. The capacity for strand separation would allow for the editing of already fabricated structures (e.g. part switching, base structure modification or expansion, repair or enhancement capabilities).

Each gold nanoparticle was ~15nm in diameter and bound by sulphur to the centre of the DNA strand. The particles were joined by adding a third DNA strand that hybridized with half of each gold-bound strand and attached to both, thereby forming a link. This link strand had one end longer than the other with the longer end hanging loose. To separate the nanoparticles a fourth strand section was added which would bind to the hanging end and peel it away as would the pulling apart of Velcro and separating the particles.

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Chad Mirkin (a pioneer of the use of DNA in nanoscale construction) states that this capability is a step towards creating a structure where you could have triggerable changes.[219]

DNA Adhesion to Surfaces: Surfaces can be modified with varied DNA composites such as DNA-carbon nanotubes [220] or oligocationic polymerDNA layer-by-layer deposited films. [221,222] In microarray fabrication, DNA can be adhered to phosphate and borosilicate glass. The glass is first cleaned with both an acid and base etch and then covered with a layer of 3-aminopropyltrieethoxysilane. An indicator of good DNA adhesion to the silicate glass is that features are well defined and distributed. [236]

Surface specific proteins can be used as linkers to bind DNA onto molecular templates. In biomimetics, combinatorial biology techniques can be utilised to elucidate entities with strong adhesion characteristics. [237,238] A larger and random library of peptides with the same number of amino acids, but having varied sequences, are used to find specific sequences that strongly bind to a desired inorganic surface. [237-242]

Potential Computing Requirements for a DNA Manufacturing Complex: It may be assumed that a nucleic acid manufacturing complex will require some computational assistance for certain functions. The reality may prove to be a co-operative scenario by which DNA-based computers might work in conjunction with molecular recognition/self-assembly processes in order to successfully fabricate products. However, since current DNA-based

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computing employs diffusing molecules rather than spatially organised structures, there would seem to be a mismatch between it and the sort of sensing and control useful in manufacturing. [89]

The requirements for computational power might include such applications as assisting in the organisation of component placement (e.g. disseminating DNA bar code/identifier data on site), and then to issue actuation directions to receiving mechanisms anchored to the substrate. Verification protocols to ensure correct component placement may be carried out by DNA-based computers, as well as by the administration of stringent testing regimes. DNA-computational devices can take several forms. One example emanating from the manipulation of DNA-topology has resulted in the fabrication of Borromean rings which are made of three or more interlocking rings. When one of the rings is removed, the remaining linkages collapse. Proofs for logical statements could be represented by the integrity of linkages. [47-50] Magnetic nanoparticles self-assembled by the nucleic acid scheme, have potential for crafting nanoscale memory devices. [55]

Dr. Leonard Adleman, at the University of Southern California, assembled the first DNA computer, and demonstrated the power it possessed in solving complex problems. [51] Computational speed estimates were put at ~1.014 operations per second, or 100 Terflops (100 trillion floating-point operations per second) compared to the worlds fastest super-computer at 35.8 Teraflops. A jumble of 1,018 strands of DNA could clip along at 10,000 times the speed of its fastest silicon counterpart. [52] To engage in any meaningful computation however, DNA computers (being special purpose) require algorithms. [112] In the human body, transfer of information for the production of molecular entities is rapid and for the most part, very efficient. 33

A molecule of haemoglobin (made of 500 amino acids) is produced 500 trillion times/sec. in the human body. This translates to (15 x 1018) read operations by ribosomes per minute. [56]

The difference in calculating modes is that silicon chips must test every possibility separately until encountering the correct answer, whereas DNA will test all possibilities simultaneously. For DNA, the required storage area for a bit is 1nm3. One of several drawbacks inherent to DNA computation is that the rate of answer data output is very slow. There are accuracy issues as well, due to pair mismatches, and it relies heavily on the precision of enzymatic action involved. These difficulties will escalate exponentially as calculations necessary to solve a problem become more complex. Also, the cost of versatility can translate to instability, and a built-in redundancy that accommodates fault-tolerance might culminate in an enormous slowdown in DNA reactivity, and hence construction processes. [77]

Although the general consensus is that DNA computers will never match their silicon-based analogues, the true power lies in their potential for computing in vivo, and to establish control at the level of molecules and cells. [52]

An interesting aside as relates to the storage of DNA encoded information is elucidated by researchers at the Pacific Northwest National Laboratory. Naked DNA is destroyed easily, however, work has been conducted toward the long time storage and protection of DNA when inserted inside extremophile bacterium, Deinococcus radiodurans. This bacterium (considered by some to be essentially immortal, though all viable bacteria are potentially immortal and all can be 34

destroyed) [89] can survive UV radiation, desiccation, partial vacuum, high temperatures, and massive doses of ionising radiation. Its DNA repair capabilities, even when severely damaged, seem to enable an uncanny ability for reconstitution. [78, 79]

One millilitre of water can contain 1 billion of these bacteria. The storage capacity via this method may increase dramatically by storing different sequences in separate bacteria. Information can be stored as specific sequences sandwiched between two marker sequences. The entire strand is then inserted into a cloning vector (a circular DNA molecule that self-replicates inside a bacterium) and is put into the extremophile bacteria for very long-term storage. To read this information the DNA is run through a polymerase chain reaction, that can locate the marker sequences and replicate the strands whose sequences can then be analysed and read. [79]

Fractal Sierpinski Triangle Self-Assembly using DNA: A cellular automaton, employing the two dimensional self-assembly of DNA tiles has recently been developed. The binary function XOR (exclusive or), is computed via its update rule and subsequently constructs a fractal pattern of Sierpinski triangles as it grows. This was accomplished through the translation of abstract tiles into DNA tiles based on double crossover entities. The nucleated growth of DNA tiles into algorithmic crystals was guided by long single stranded DNA molecules. The error rates in the ensuing pattern were at ~1% - 10%. [128]

This demonstrates that engineered DNA self-assembly can be considered a Turing- universal biomolecular system having the capacity for the application of any desired algorithm for computation or assembly tasks, as well as enabling computer science-derived insights to be 35

applied to self-assembly. The successful adaptation of algorithms to molecular construction might include and link the essential concepts of energy and entropy toward the elucidation of how physical processes evolve order. [128]

Several types of DNA tiles have been designed and verified to grow into micron scale 2D periodic crystals. [119-121,123] The interfaces between the tiles are programmable via sequence-specific hybridization and single stranded templates may serve as inputs to the self-assembly process. [121,124-126] A new form of aperiodic crystal (algorithmic crystal) is manifest by Sierpinski tiling. Every molecular (Wang) tile is a DNA-double crossover molecule having four sticky ends (single strand overhangs comprised of five bases) operating as binding domains that are programmable. [127,128]

The initial output for the algorithmic self-assembly procedure must be provided by an appropriate nucleating structure. The assembly of a row of input tiles is guided by a long single oligonucleotide which serves as a scaffold. Only four DNA strands are required to assemble the input tiles. The bottoms of these tiles is provided by the nucleating strand, and an extra capping strand is used to construct a duplex between tiles on the nucleating strand. [128]

All four features required for Turing-universal computation by crystallisation are satisfied by the self-assembly of DNA Sierpinski triangles. 1) Formation of extended crystals. 2) Programmable interactions between DNA tiles determined by sticky end sequences. 3) Selective associations of tiles enforced by co-operative binding of more than one sticky end. 36

4) Controlled nucleation of growth initiated by template containing input information. [128]

Computation by DNA self-assembly, when applied to molecular construction might enable the control of direction and extent of workpiece growth, allowing for the creation of precise geometries. [129] Several challenges, however, need to be overcome before this system can be deemed viable. There currently exist several types of errors, including dislocated sections of the lattice, untemplated crystals, tile mismatches, and errors in the guidance of nucleation. The careful control of assembly conditions gives rise to the possibility of error reduction. Another path, perhaps more reliable, might be the creation of fault tolerant tile sets that perform similar logic. [130-133]

DNA- Electronic Elements: In one instance, a nucleic acid-based molecular manufacturing complex might be applied to fabricating nanoscopically pure materials for integration into other products. This would involve the exploitation of DNA hybrization principals to guide and join molecular elements together. However, many fabrication applications might be geared toward the formulating of nanoelectronics entities. Nanoscopic electronic interconnectivity will be an essential ingredient for making these devices. The potential capacity for designing these unseeable connections is an attractive feature of DNA-based component/entity synthesis. [55]

When it ever becomes possible to construct nanoelectronic components in this way, one can imagine that these mechanisms (and the molecular manufacturing infrastructures employed for making them) will be poised for pervasive integration into the majority of future 37

commercial/industrial products. This would constitute a further step in the evolution of our increasingly wired world. Devices such as transistors, complex circuits, biosensors, and molecular computers/quantum bits might be built by exploiting DNAs fine-tuned recognition properties. [55,67,68]

DNA Nanowires: Templates made of DNA have been used as deposition guides for producing nanowires made of palladium and silver. [59,60] Wire width of 2nm may be realised by placing gold quantum dots along a DNA template. The ends of the DNA nanowire can be configured with sequences that can attach to complementary sequences tethered to connection pads by hybridization. The two ends will automatically seek each other and bind together correctly in solution. This method can be adapted to 3D wiring as well. Gold quantum dots will also catalyse subsequent metal deposition building up a continuous metal nanowire [61]. Complex metallic structures can be self-assembled by attaching gold particles to single strands of DNA. [70] DNA is a good prospect as a nanowire template due to its flexibility, narrow diameter, and end

targeting. Length can be controlled, as a DNA synthesizer would produce the DNA or enzymes following a template. The insulating options include surface oxidation, forming a glass coating via reaction with silanes, or coating with alkane thiols. The average spacing of gold clusters adhered to a double stranded DNA segment was ~2nm. Interestingly, this is about the same distance for the tunnelling of electrons between metal particles. Several ways of binding gold clusters to DNA are covalent attachment, photoreaction, intercalation, or binding of positively-charged gold particles to 38

negatively charged DNA. Silver and other metals have also been nested into DNA [61].

An alternate way of engineering DNA molecules to demonstrate metallic conduction involves replacing the imino proton of each base pair with a metal ion. Both metallic-like conduction and semiconducting properties with a bandgap of several hundred meV at room temperature have

been shown. Molecular devices based totally on DNA may be enabled by this engineered variance of conductance. [62-64] Another factor to consider in the mix is that there are still conflicting experimental results as to whether DNA is conducting, semi-conducting, or insulating.

DNA Conductivity: Charge transfers in DNA can cover distances of up to hundreds of angstroms; however, the transfer rate is highly influenced by the actual sequence of the bases. Its electronic structure can also be changed by attached ions. Each phosphate group can be engaged as a tunnel junction. Another consideration here is that when DNA is deposited onto a surface, there is an induced structural compression that also alters its electronic properties. [66]

Application of thin coatings of metals can produce DNA nanowires, but due to its complete coverage it loses its ability to selectively bind with other molecules. When in a high pH (8.5) environment, DNA will adsorb nickel, zinc, and cobalt into the central axis of its helix. This species has been called M-DNA (formed from B-DNA) as it can conduct but also retain its capability to bind with other molecules, and presumably hybridise with other DNA strands. In a nucleic acid- based manufacturing scheme, these might be used to self-assemble and form the ultra39

fine electronic interconnects required for nanoelectronic circuitry.

Quantum effects may help to characterise electron transfer in M-DNA as single electrons are involved. Electrons may hop from one base pair to the next, as has been supposed for B-DNA; however, the electron-flow rate measured in M-DNA is orders of magnitude larger than that of B-DNA. [83,84] A three-way junction can be formed from three strands of DNA and different functional groups can be attached on each of three arms. [81] If, for instance, a thiol functional group is attached then it can be used to bind to a gold electrode. [84]

Work has been done in the functionalisation of nanotubes with DNA. Open-ended single walled nanotubes having terminal carboxylic acid groups react with amino-terminated DNA strands. This specific reaction will covalently bind the nanotube to the DNA strand. This hybrid structure can now be assembled onto surfaces or into structured networks, directed by DNAs self-assembly principals. [71,188,189] To simplify the creation of functional nanoelectronic circuitry patterns, molecular lithography processes might be used to apply DNA onto appropriate surfaces. [69] By exploiting the self-assembly properties of DNA, carbon nanotubes might be assembled onto substrates or configured into structural networks. Nanotubes may be sorted into metallic and semiconductive species by DNA self-assembly processes. [191,192]

When engaged in the fabrication of nanoelectronic circuits, precise capabilities for DNA scission may be required at some points in the assembly process. DNA-fullerene hybrids can cleave DNA specifically at guanine residues when exposed to light. [85] Other tools for DNA cleavage are water-soluble fullerene derivatives that are efficient in the production of singlet oxygen species. 40

[86-88]

DNA can also be written directly onto a surface by means of dip-pen nanolithography. This technique is quite versatile in that patterns can be applied using various oligonucleotides, and these, subsequently can be utilised to build up more complex structures. [73] Modifications of semi-conductor surfaces by DNA create the opportunity for the fabrication of nanometric electronic devices on these surfaces. [74,75]

Nanocrystalline diamond (NCD) thin films, grown on silicon substrates, can be covalently modified with DNA oligonucleotides, giving a stable and highly selective platform for custom surface hybridization processes. This may be accomplished by chemically modifying clean, Hterminated NCD surfaces creating a homogeneous coating of amine groups that can provide sites for DNA attachment. NCD may serve as a good interface between biological entities and inorganic circuitry. [76]

Conclusion: There are several criteria that may facilitate and define the emergence and practical application of a nucleic acid-based manufacturing complex. A distinction should be made as to what calibre of precision the products fabricated by this manufacturing capability might possess. Would they be nanometrically precise, or atomically precise? Dr. Drexler points out that as a matter of terminology, "molecular manufacturing" is defined in terms of productive molecular machine systems building complex structures with atomic precision. [89]

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The following preliminary thoughts reflect only this authors (F. Boehm) present perception, which offers but one of many potential organisational strategies, involving many additional unexplored elements. One ideal worth striving for would be toward the development of a completely programmable, and totally automated system to keep the fabrication process free of any contaminants and debilitating effects of cumulative waste products. A cascading sequence of events would most likely entail numerous co-ordinated functions to occur at very specific times. A perceived composition might be:

1. Initially, the self-assembly of smaller modular entities in massively parallel arrays comprised of sub-component fusing wells. These wells would be fed by exquisitely directed flows of prepared highly pure feedstock concentrations of the required elemental materials (perhaps extrapolating fluidic array designs and technologies). Nanoparticles comprised of specific elements would be bonded to single-stranded DNA .

Nanoparticles might be drawn together in certain orientation, contingent on single-strand hybridization modes. A potential technique for positioning atoms or clusters of atoms in this way may be discovered in the future. There may be a requirement for many sub-components to be built up from metallic constituents as parts of nanoelectronic interconnects, transistors, capacitors, etc. Affinity studies between metallic and non-metallic nanoparticles and DNA will require investigation. Capabilities for directing localised electronic charges may play an important role toward enhancing and assisting the bonding of these infinitesimally minute entities in conjunction with DNA. 42

DNA bar codes, or single strand identifiers might also be added at this stage for part identification and tracking, as well as perhaps several encoded single strand guiding handles. The fusing wells might contain an exit valve for purging the contents when this assembly is complete. Conversely, they could be removed by arrays of end-of-arm robotic mounted pipettes.

2. Sub-components would now be transported to an intermediate assembly chamber, and then conceivably to many subsequent intermediate assembly chambers (dependent on the complexity of the desired end-product) to draw these sub-components together for precise mounting on a substrate. This might be accomplished by incorporating dedicated guide strands residing in the aqueous mixture contained within the chamber. For example, assuming a guide strand sequence of 20 bases, 10 of these bases would hybridize with their complements attached to the sub-component, and the remaining 10 would hybridize with their complements tethered to the substrate at a specific location.

Subsequent single strands dangling from the sub-component could find use as fine-positioning tools as they would bond with their complements also located in precise locations on the substrate thereby ensuring correct orientation of the part. Final placement verification might occur by introducing very short single DNA strands combined with addressable gold nanoshells (varying wall thickness), or tuned quantum dots (dimensional variations) that would hybridize with identification strands, tagging the parts.

Once in place, the patterns formed by nanoshells or quantum dots might be read by external 43

scanners/computers, and digitally superimposed on preconfigured patterns conveying correct positions for all entities. This process may be repeated along and up the assembly sequence, involving assembly of larger components.

Corrective mechanisms employed in cases of misplaced entities would be an area to explore. It may turn out to be easier to discard faulty assemblages, rather than institute correction protocols in these cases. If all identifiers and verifiers are stringently checked prior to assembly, there should be very low off-pattern assembly incidences.

3. Verified substrates, studded with finished components would now be transferred to a final assembly tray. These substrates might still be microns to millimeters in size, so again they might be moved by fluidic means, by pipettes, (or if larger) by grasp-force sensitive robotic placement fingers and arms. The tray would house a well-organised and stratified infrastructure composed of a stable physical framework, immersed and securely anchored to the bottom surface, containing again, a finely regulated (chemically, thermally, controlled turbulence etc.) aqueous environment.

The framework might be comprised of basement scaffolding, interspersed at various levels and orientations (dictated by the design of desired end product) with supportive substrates that have been prefabricated and functionalised prior to installation into the assembly mainframe. These substrates may be stockpiled and stored (by freezing etc.), selected by their inherent actuation and assembly capabilities, mounted to the framework (via hybrization) by pylons, and re-activated by the chemical mix of the buffer solution. 44

The final phase of assembly would proceed in the same manner as described above, giving the nanoscopically precise final product. The completed product would now be transferred by robotic means to final testing and packaging stations.

Conclusion: One may summarise ones thoughts on the potential feasibility and application of nucleic acidbased manufacturing capabilities by perhaps focussing on some of DNAs outstanding attributes. Its molecular recognition talents, displayed by the meticulousness with which it can perform its hybridization feats, may be best put to use in organising, positioning, and verifying placement and orientation of microscopic components (perhaps atomically fabricated by another means) into larger entities. It may take shape as an integral facet of a hybrid manufacturing complex, having a pervasive presence within it.

Its characteristic and powerful feature of self-assembly, and its potential for flexible programming of this self-assembly, may be indispensable in any directed nanofabrication system. As mentioned above, it might play many other important roles in molecular manufacturing processes. Uses such as structural scaffolding, enzyme immobilisation grids, functionalised substrates having integrated actuators and other placement and positioning tools, information storage and conveyance devices (DNA computers), tagging, tracking, and verification might be possible. These are most likely just a small sampling of many more possible capabilities that are yet to be discovered.

In any case, an exciting and intriguing path lies ahead for molecular/nanometric manufacturing 45

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