Creating A Medical Nanobot - Molecular Computing Using In Situ Cell Surface Identification

the concept of individual cell identification in vitro and in vivo, using specific DNA-antibody complexes. The cell surface membrane, a lipid bilayer with numerous specific proteins embedded on, in, and through it, has been extremely useful for the identification, quantification, and collection of specific cell types for both patient care and research. Determining in real time and in situ with viable cells the exact identification of the cells and having this information immediately reported using bioinformatics, would be very advantageous. The work of Rudchenko, Stojanovic and colleagues at Columbia University provides a beginning for such a system, using whole blood (Rudchenko M, et al., Nature Nanotechnology, 1023;8:580-586).

DNA-specific antibody complexes attach to the cell surface. Through a cascading process of movement on the fluid cell surface, these interacting complexes determine whether a cell is or is not the specific cell type being sought. Not only can this technique can be applied to identifying the cellular population of choice for collection and further testing, such as a cancer or organ stem cell, but alsothe process can be scaled, so that the identification of "unknown" cell types may be collected. Background information and the method are reviewed by Schaus T E and Yin P: Molecular Computing: In SituComputation of Cell Identity, Nature Nanotechnology, 2013;8:546-548. See Figure 1 below.

Figure 1 (Schaus & Yin): A three-input AND gate (A AND B AND C) using a programmed cascade of reactions on a cell surface. Three probe molecules (to antigens A, B and C), each of which are composed of an antibody (grey) conjugated to one strand of a synthetic DNA duplex (shown as a series of colored domains), are added to a solution and bind to their target antigens on the surface of a cell (brown). An initiator molecule (Ini) is then added (left panel), which binds to the complementary strand of probe A by means of the toehold sequence (purple), and removes it, exposing a new signal sequence (red). The newly active probe A then interacts with probe B in a similar fashion, and probe B subsequently activates probe C (middle panels). The exposed strand of activated probe C represents the unique output signal of the cascade, which is possible only when antigens A, B and C are all present. In the example shown, probe C binds, separates and de-quenches a fluorophore strand from a soluble reporter duplex (R), leaving the cascade in its fluorescent (yellow star), completed state (right panel).

The details of a specific example from human whole blood are in Figure 1 below: the automated and computed identification of T-lymphocytes with the phenotypes of CD45+/CD20+ and CD45+/CD20-.


Figure 1 (Rudchenko, et al.): Design considerations for automata operating on cell surfaces. a, Scheme of automata operating on a B cell with a C45⁺CD20⁺ phenotype (target) and on an example of a non-targeted cell with a CD45⁺CD20^- phenotype (for example, a T cell). Oligonucleotide components (colored horizontal lines) attached to antibodies (Y-shaped structures) are brought together on some cells and not others (for example, αCD45-1·2 and αCD20-3·4 are together only on B cells), leading to a cascade of oligonucleotide transfers driven by an increase in complementarity. The transfers result in a unique single-stranded oligonucleotide 4 being displayed only on targeted cells. b, Scheme of a typical strand displacement reaction used in the automata 0 + 1·2 + 3·4  0·1 + 2·3 + 4, controlled by a sequential exposure of toeholds (T₁ then T₃): single-stranded oligonucleotide 0 displaces oligonucleotide 2 from its complex with 1 via toehold interactions, that is, stronger complementarity and kinetic enablement due to the additional complementarity with exposed T₁. This generates an oligonucleotide stretch in strand 2 complementary to a toehold T₃ in strand 3 that can extend the reaction cascade by displacing oligonucleotide 4 from 3·4. This in turn generates the next oligonucleotide stretch complementary to toehold T₅, which can be used to extend the cascade to 5·6 (not shown), and so on (as indicated by double dotted arrows), or to label the cell with 5 carrying fluorescein. Without other components displaying T₅, the cascade stops at 4. c, An example of oligonucleotide sequences used in the automata.

One limitation of this cascading process is that it is limited by the least abundant probe present on the cell surface. A process using a probe that propagates its signal to multiple downstream partners would overcome this limitation. This propagation would lead to faster and higher signal generation, as well as creating a binary output typical of logic circuits.

The number of novel nanotechnology processes that can be utilized in both medical and non-medical settings is starting to move up the exponential curve. I sincerely hope that the promise and delivery of improved patient care and safety through the use of simple and complex medical nanobots will outweigh the potential safety risks involved in their development and use.

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