A Proposed Path from Current Biotechnology to a Replicating Assembler

[Copyright(C) 1996 by James B. Lewis, Bruce Smith, and Markus Krummenacker; Molecubotics; 11/96]



A General Molecular Manufacturing Technology

Drexler (1981, 1986, 1992) has argued that it is possible to develop a technology capable of inexpensively fabricating large and complex structures to atomic precision, meaning with each atom placed to contribute to the designed function of the product. This proposed technology has been named molecular manufacturing, or molecular nanotechnology, or simply nanotechnology. Theoretical studies (Drexler, 1992) show that such manufactured products will have properties and functions many orders of magnitude beyond the properties and functions of products manufactured using current technology, and will in addition be inexpensive.

Assemblers, Replicators and Mechanosynthesis

Molecular manufacturing will require developing a class of programmable machines capable of covalently joining atoms or molecular fragments into any of a large number of possible bonding arrangements. Thus such machines, which have been termed assemblers, will be able to fabricate almost any device whose construction can be specified in atomic detail.

Assemblers that have been designed to be able to make exact copies of themselves have been termed replicators.

A key capability of assemblers will be the ability to do mechanosynthesis. Drexler (1992) has defined mechanosynthesis as "Chemical synthesis controlled by mechanical systems operating with atomic-scale precision, enabling direct positional selection of reaction sites ..."

The assemblers envisioned by Drexler for use in molecular manufacturing would be very strong, stiff structures that would work in a vacuum, be able to handle very reactive chemical moieties, such as free radicals, and be able to position such species to a precision of better than 0.1 nm. It is difficult to see how such complex devices could be manufactured directly except by already existing, more primitive assemblers.


It is expected (Drexler, 1992) that the ultimate assemblers will thus be developed through several generations of devices, with very crude and limited tools being used to make better tools, eventually culminating in the production of the desired general purpose replicating assemblers. Early generations of assemblers, relatively limited in their capabilities, have been termed proto-assemblers.

The obvious bottleneck to the development of nanotechnology is the identification of  the first generation of replicating, programmable assemblers that can be fabricated using current laboratory technology, or incremental developments of current technology, which will not require for their fabrication already existing replicating assemblers. To be useful, such proto-assemblers must be able, with appropriate programming, to fabricate second generation assemblers with enhanced synthetic capabilities.

It has been suggested (Drexler, 1992) that early generations of assemblers will deal not with very reactive single atoms or small groups of atoms in a vacuum, but rather with larger molecular building blocks (MBBs) in solution. Drexler considered two approaches: (1) Brownian assembly of fairly large MBBs, each of which is a polymer designed to fold into a specific shape, and (2) mechanosynthetic assembly of small MBBs guided by a scanning force microscope.

Building molecular devices (nanodevices) by Brownian assembly of engineered proteins or other polymers, designed to fold and form matching complementary surfaces with which they bind noncovalently to each other in specific arrangements, presents very formidable design challenges, which seem unlikely to be solved within the next few years at current rates of progress.

As for the second approach, initial analysis (Krummenacker, 1994) reveals that the choice of appropriate MBBs and methods of linking them together in precise three-dimensional networks is a very difficult problem in organic chemistry.

Therefore, there seems to not be an obvious way to make even proto-assemblers starting with current technology.

Protein Molecular Machines as the Earliest Proto-Assemblers

Drexler has suggested (Drexler, 1981, 1992) that the initial, crudest steps toward molecular manufacturing capability could be taken using protein-like molecular machines, and that development pathways exist which lead from them to more general molecular manufacturing technologies, for example the use of small synthetic organic groups, attached to protein-based machines, as tools for positional control of reactions.

Drexler discussed the engineering of protein-like molecules having complementary surfaces for self-assembly, and proposed that designing proteins to fold in a predictable fashion was much easier than predicting the folding pattern of natural proteins. He suggested methods for increasing the stability of the desired conformation of designed proteins. This approach can almost certainly eventually be made to work, but it entails a formidable challenge in protein design, as stated earlier.

DNA-Guided Assembly of Proteins (DGAP) for the Fabrication of Nanodevices

A novel approach invented by Bruce Smith uses DNA-protein conjugates produced by biotechnology as MBBs. However, rather than designing variations of proteins or protein-like polymers to fold into desired tertiary and quaternary structures to be used as artificial complementary surfaces for binding, this invention utilizes the well understood complementarity of DNA sequences to assemble proteins into specific configurations. After being positioned relative to one another using DNA complementarity, MBBs would be joined by forming covalent bonds, using solution chemistry of the kind which is familiar and routine in organic chemistry.

Drexler pointed in this direction in general terms in 1992 ("Nanosystems" p.446, after discussing the work of Ned Seeman (1991)):

"The ability of complementary segments of DNA to bind to one another provides a powerful, readily controllable mechanism for guiding the Brownian assembly of molecules. Hybrid structures of DNA and other macromolecules may prove particularly attractive."

Protein-DNA conjugates, with different DNA sequences attached at specific points on the protein surface, thus become the MBBs to be assembled by Brownian motion to build nanodevices. One of the advances in Smith's proposal beyond what Drexler suggested in 1992 is that "loose" binding of complementary DNA strands on different protein molecules confers specificity on subsequent rigid, covalent binding between protein MBBs by holding the MBBs accurately enough that each covalent binding site is accessible only to its intended partner. The resulting structures will be strong enough to use as components of molecular machines able to actively position similar MBBs, and to perform some forms of positionally-controlled chemical synthesis. A second advance is how to achieve a defined spatial arrangement of several different DNA sequences on the surface of the protein, even though it is not feasible to use a different attachment chemistry at each attachment site on the protein.

The modifications required of the proteins for permanent bonding are thus much simpler than if the proteins had to be redesigned to have complementary surfaces. An additional advantage of this approach is that the library of potential MBBs includes the vast collection of proteins produced by biological evolution (many of which have known structures) as well as synthetic variants thereof. Thus a wide variety of MBBs with specific functions (structural, ligand-binding, and catalytic) can be included in these first generation nanodevices.

The DGAP Invention:

Motivation for DGAP

The hardest problem in getting from today's technology to primitive molecular machines is devising any means to connect a reasonable collection of protein-sized molecules into specific arrangements. We can already make (or harvest) a wide variety of natural molecules (with known structures; perhaps slightly modified) to serve as machine parts resembling "blocks, ropes, springs, and semi-sticky surfaces" (as well as surfaces with catalytic activity), which if arbitrarily joinable would be adequate to make a wide variety of mechanical devices, including molecular computers and robots. It is likely that such structures, combined with organic molecules like those that can already be synthesized separately, could perform a wide variety of synthetic organic reactions with selectivity due to positional control (Drexler, 1981). Even though the first generation of molecular mechanical devices would not be able to perform diamondoid mechanosynthesis directly, it would lead to immense improvements in our capabilities in the molecular realm, and would be on a direct path to full-fledged nanotechnology.

One well-known scheme to join proteins together to form complex structures (Drexler 1981, 1986, 1992) is to learn to engineer folding polymers with many different specified surface structures, and to use this ability to make "bricks" which self-assemble in solution into a specific arrangement due to complementary surfaces. The protein design issues to implement this scheme are very challenging; there has been steady progress, but the level of success required for this application still appears to be several years away. In contrast, the DGAP method requires much less innovation to develop from already-demonstrated technologies.

The DGAP invention is a method for assembling almost-natural molecular building blocks (MBBs) into specific, almost-arbitrary arrangements, potentially with hundreds of distinct block locations in each assembly. Blocks at different locations in one assembly are allowed to be the same, which means that a small library of distinct block types could suffice for many designs. The assemblies of blocks could be joined recursively into larger structures.

MBBs could be joined in a fairly rigid fashion. Protein-based MBBs could be held together in a compact fashion with covalent tethers at several locations around the joined surfaces. Since the overall connectivity in a network of MBBs could be essentially arbitrary (thus 3-D and polycyclic), larger structures could be reasonably rigid as well. They are likely to be able to be made rigid enough for positional control of chemical reactions, though not strong enough, at least in the first generation, to apply the significant forces needed for some uses of mechanosynthesis. (See Drexler 1992, pp.462-463, for a rigidity and strength analysis of a similar system.) They will certainly be rigid enough for making "robot arms" which could move other similar building blocks into contact with the correct partners, and thus build similar devices more directly than by unassisted self-assembly.

Besides rigid joints, rotating or tethered joints between MBBs are also possible, either via different choices of chemistry for covalent bonding, or by the use of building blocks which already incorporate such joints.

Since slightly modified natural proteins could be used as building blocks, the resulting devices could incorporate biomolecules of interest in particular applications, such as antibodies or enzymes.

The Basic Insight of DGAP

The basic idea, compared to the designed-complementary-surface approach, is to try to remove or reduce the need to design molecular building blocks with a variety of complementary interfaces by separating the two functions of complementarity: (1) guidance of self-assembly and (2) a mechanically strong connection of the MBBs.

Specifically, one could attach to each building block (at particular sites on its surface, here called "C sites" for "complementary") a few different single-stranded DNA molecules, and (at "P sites", for "permanent") a few non-specific permanent binding sites. (The P sites will probably be sites capable of forming covalent crosslinks, though other choices are conceivable; several choices of chemistry for covalent crosslinking are already in wide use in molecular biology. Also, it will sometimes be convenient for an assembly consisting of a DNA strand and one or more P sites to be added to each C site already on the protein, rather than for the protein to start out with separate P sites and C sites.)

During assembly of several MBBs into one structure, the P sites are prevented from reacting initially by maintaining a chemical environment preventing reaction until the DNAs are hybridized, after which each P site is only able to contact that P site with which it is meant to react. The chemical environment is then changed to permit reaction (e.g. by the addition of appropriate crosslinking reagents, active only at the P sites).

That is, DNA hybridization (joining of very-easy-to-design, very-specific complementary "surfaces") guides self-assembly, allowing non-specific connection sites designed solely for their chemical/mechanical properties to be uniquely paired.

The problem of making a MBB by attaching a different DNA sequence to each of several specific sites on a protein is the only part of the DGAP invention without a direct precedent in current molecular biology. We have developed several possible methods of solving it (the best of which we are keeping under nondisclosure), and this is the main focus of our present research; fully developing one of these methods and putting it into practice will require some research and development in the laboratory. The kinds of equipment and procedures required for performing the R&D on any of our proposed DNA-attachment methods are standard in molecular biology.

For making large structures out of networks of linked MBBs, the average MBB must be directly linked to more than two neighbors, preferably at least 3 or 4 if the structures should be rigid. The number of C sites and P sites on each MBB needed to accomplish this depends on the specific design of an assembly of MBBs; for ease of designing assemblies without this connectivity being a limiting factor, we should be able to add different DNA sequences to at least 6 different sites on one protein (taking advantage of the possibility of connecting the C site to the middle of the DNA strand, so there would be 12 different DNA sequences leaving the MBB). The methods we are investigating should be able to accomplish this; if they can't, as few as 4 or even 3 separate C sites per protein would still be useful for many applications, with more design effort required for some of them.

Major Issues not addressed in this document

Substantial issues affecting the feasibility of the DGAP invention and affecting business plans based upon the DGAP invention have not been addressed in this document. Additional information is available from Bruce Smith, although some of this information will be governed by a non-disclosure agreement. These issues include the following.


Drexler, K.E., 1981,
"Molecular Engineering: An approach to the development of general capabilities for molecular manipulation"
Proc. Nat. Acad. Sci., 79: 5275-5278
Drexler, K.E., 1986,
"Engines of Creation: The Coming Era of Nanotechnology"
New York: Anchor Press/Doubleday.
Drexler, K.E., 1992,
"Nanosystems: Molecular Machinery, Manufacturing, and Computation"
New York: Wiley.
Krummenacker, M., 1994,
"Steps Towards Molecular Manufacturing"
Chem. Design Autom. News, 9: pp.1,29-39
Seeman, N. C., 1991,
"Construction of Three-dimensional Stick Figures from Branched DNA"
DNA Cell Bio. 10:475-286.