Sergei Rudchenko

Exercises in molecular computing.

Conspectus The successes of electronic digital logic have transformed every aspect of human life over the last half-century. The word “computer” now signifies a ubiquitous electronic device, rather than a human occupation. Yet evidently humans, large assemblies of molecules, can compute, and it has been a thrilling challenge to develop smaller, simpler, synthetic assemblies of molecules that can do useful computation. When we say that molecules compute, what we usually mean is that such molecules respond to certain inputs, for example, the presence or absence of other molecules, in a precisely defined but potentially complex fashion. The simplest way for a chemist to think about computing molecules is as sensors that can integrate the presence or absence of multiple analytes into a change in a single reporting property. Here we review several forms of molecular computing developed in our laboratories. When we began our work, combinatorial approaches to using DNA for computing were used to search for solutions to constraint satisfaction problems. We chose to work instead on logic circuits, building bottom-up from units based on catalytic nucleic acids, focusing on DNA secondary structures in the design of individual circuit elements, and reserving the combinatorial opportunities of DNA for the representation of multiple signals propagating in a large circuit. Such circuit design directly corresponds to the intuition about sensors transforming the detection of analytes into reporting properties. While this approach was unusual at the time, it has been adopted since by other groups working on biomolecular computing with different nucleic acid chemistries. We created logic gates by modularly combining deoxyribozymes (DNA-based enzymes cleaving or combining other oligonucleotides), in the role of reporting elements, with stem–loops as input detection elements. For instance, a deoxyribozyme that normally exhibits an oligonucleotide substrate recognition region is modified such that a stem–loop closes onto the substrate recognition region, making it unavailable for the substrate and thus rendering the deoxyribozyme inactive. But a conformational change can then be induced by an input oligonucleotide, complementary to the loop, to open the stem, allow the substrate to bind, and allow its cleavage to proceed, which is eventually reported via fluorescence. In this Account, several designs of this form are reviewed, along with their application in the construction of large circuits that exhibited complex logical and temporal relationships between the inputs and the outputs. Intelligent (in the sense of being capable of nontrivial information processing) theranostic (therapy + diagnostic) applications have always been the ultimate motivation for developing computing (i.e., decision-making) circuits, and we review our experiments with logic-gate elements bound to cell surfaces that evaluate the proximal presence of multiple markers on lymphocytes.

Autonomous molecular cascades for evaluation of cell surfaces

Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs1–4. Previously studied nucleic acid-based-automata include game-playing molecular devices (MAYA automata3,5) and finite-state automata for analysis of nucleic acids6 with the latter inspiring circuits for the analysis of RNA species inside cells7,8. Here, we describe automata based on strand-displacement9,10 cascades directed by antibodies that can analyze cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells.

Comparative Structural and Functional Features of the Human Fibrinogen αC Domain and the Isolated αC Fragment CHARACTERIZATION USING MONOCLONAL ANTIBODIES TO DEFINED COOH-TERMINAL Aα CHAIN REGIONS

The αC domain of fibrinogen (Aα-(220-610)) plays a central role in maintaining hemostasis by serving as a substrate for factor XIIIa and plasmin. Monoclonal antibodies that recognize eight distinct epitopes within the COOH-terminal two-thirds of the Aα chain were employed as structural probes to: 1) isolate the human αC domain, 2) compare the topography of the eight epitopes within the αC domain of intact fibrinogen and in purified αC fragments, and 3) explore the degree to which the αC domains role as a factor XIIIa substrate in intact fibrinogen is preserved within the structure of isolated αC fragments. Five antibodies were raised against small, synthetic peptide immunogens (Aα-(220-230), Aα-(425-442), Aα-(487-498), and Aα-(603-610)), and three were generated against larger cyanogen bromide (A)α chain derivatives with each epitope subsequently localized to discrete Aα chain sequences (Aα-(259-276), Aα-(529-539), and Aα-(563-578)). Human αC preparations were isolated from mild plasmin digests of fibrinogen by successive chromatography on concanavalin A-Sepharose, anti-Aα-(425-442)-Sepharose, and Superdex-75 fast protein liquid chromatography. Immunochemical characterization indicated that the NH2-terminal residue of αC fragments was either Aα-220 or Aα-231 and that, although the extreme COOH-terminal region, Aα-(603-610), was absent, all molecules were intact at least through Aα-(563-578). Solution phase competitive assays indicated that the release of the αC domain from intact fibrinogen was associated with several conformational changes, e.g. in the vicinity of Aα-(220-230), Aα-(259-276), Aα-(487-498), and Aα-(529-539), but that the relative accessibility of other localized structures remained unchanged, e.g. Aα-(425-442) and Aα-(563-578). Immunoblotting analysis of αC cross-linking in vitro revealed that isolated αC fragments could serve as a substrate for factor XIIIa. Immunoblotting studies of the Aα chain proteolysis that occurs during thrombolytic therapy indicated that αC fragments, similar in size and epitope content to those isolated from purified fibrinogen, were released in vivo early during fibrinolytic system activation. The collective findings provide new information about the fine structure of the fibrinogen αC domain and its functional implications and also draw attention to the as yet unexplored role of αC fragments in the pathophysiology of thrombosis and hemostasis.

Positive and Negative Regulation of c-Myc Transcription

The c-Myc proto-oncoprotein is well-known to play important roles in determining the growth and development of normal cells [1,2]. c-Myc is required for cells to exit G0 and to enter cycle and is induced as an immediate early gene in response to most mitogenic stimuli. In contrast to the requirement for c-myc expression in proliferating cells, c-myc expression is shut down in differentiating cells. In fact, addition of exogenous c-Myc blocks terminal differentiation of several hematopoietic cell lines [3–11] and of myogenic cells [12,13] while inhibitors of c-Myc expression accelerate terminal differentiation of promonocytic HL60 cells [14–16], M1 leukemic myeloid cells [17], F9 teratocarcinoma cells [18] and human esophagael cancer cells [19]. Finally, c-Myc plays a role in programmed cell death. Elevated levels of c-Myc can cause apoptosis in certain cells when other factors necessary for their proliferation are absent [20, 21]. However, decreased levels of c-Myc can also cause apoptosis in Ramos and WEHI 231 B cell lines upon treatment with anti-immunoglobulin [22,23].