Table of Contents

1. ForewordForeword
2. HistoryIntroduction & A Brief History of Molecular Computing
3. FoundationsFoundations
   1. PrimerBioBiology for Molecular Programmers
      • Central dogma & cell response to external stimuli
      • Compartmentalization & reactions?
      • Common DNA/RNA binding macromolecules (Box)
   2. PrimerThermoBasic Thermodynamics
      • Concept of entropy, enthalpy, Gibbs energy, Chemical Equilibrium
      • Thermodynamic energy landscapes, partition function
   3. PrimerODE_CRNDynamic Models ODEs and Chemical Reaction Networks
      • Stochastic Models
      • Deterministic Models
      • Box on rule-based systems / process algebras
   4. MethodMolProgExperimental Methods to Program Molecules
      • Synthesis of DNA
        • Phosphoramidite (chemical synthesis)
        • Ligation-based, Gibson assembly
        • Single-stranded DNA production, e.g. asymmetric PCR, bead pulldown, phagemid production, cell production, RCA, etc
        • In vivo assembly, e.g. with yeast
        • Upcoming methods: PER, TdT-based
        • Scaled production: bacteriophage/cell culture/IVT+RT
        • Modifications/redox/thiol/maleimide, NHS, succinimide, amine, enzymatic approaches, biotinylation, fluorization, dyes and quenchers etc.
        • BOX/TABLE: Discussion/comparison of yield, error rates, costs, challenges (e.g. repetitive sequences, modifications) for different ways of writing DNA
      • Purification (HPLC/agarose and polyacrylamide electrophoresis/centrifugation)
        • Agarose/PAGE purification
        • Chromatography (HPLC, LC, maybe capillary?, ion exchange, size exclusion for origami purification, affinity like via his-tags)
        • Centrifugation (filter, gradient, ultracentrifugation)
        • Precipitation/binding methods: magnetic beads, PEG precipitation
      • Experimental methods to produce and modify RNA
      • Experimental methods to produce and modify proteins
        • protein-DNA conjugation
   5. MethodRxnSetupExperimental Methods: Reaction Setup
   6. MethodMicroscExperimental Methods: Microscopy
      • Microscopy (AFM, TEM/cryoEM/SEM, fluorescence/superresolution - PAINT)
        • Contrast mechanisms and their drawbacks (stains being inconsistent, AFM always convolutes tip, etc)
      • Spectroscopy (fluorescence/CD/etc)
        • Table comparing different methodologies: labelled vs unlabelled, sensitivities, throughput?Background reporter signal?
        • Quenchers
        • Photobleaching (things that kill fluorophores permanently)
        • EXTINCTION COEFFICIENTS. basics of beer’s law. assumptions re: additivity of nucleotides. nearest neighbor model for absorption. denatured vs native.
      • Mass Spec
        • Detecting large amounts of small molecules in solution
        • Understand mass spectra (e.g. oligo synthesis)
   7. MethodAnalysisExperimental Methods: Analysis
      • Gel electrophoresis
        • Intercalating dyes vs fluorophores or radiolabeling
        • Separation quality as a function of gel % and DNA length?
        • Role of buffer conditions
        • Pulse field gradient electrophoresis + Other advanced methods (reference)
        • Capillary electrophoresis
        • Denaturing Gels
        • Stacking Gels
      • Sequencing
        • Sanger sequencing
        • NGS - Illumina, PacBio, DNAnanoball,
        • Nanopore
        • Microarrays

Structures

1. ChemistryFrom molecules to variables
   1. InfoMolsWhat are information bearing molecules?
      • nucleic acids (basic features and geometry)
      • proteins/peptides; Protein geometry (alpha helix, …)
      • Other (PNA, L-DNA, new nucleotides, polysaccharides, block-copolymers, other supramolecular programmable molecules)
      • Information in unordered mixtures of small molecules
   2. GlueThe interactions that glue molecules together
      • Chemical bonds and interactions (a primer in chemistry)
      • Intramolecular forces via covalent bonds: peptide bonds, ester bonds, delocalised aromatic bonds.
      • Intermolecular forces and non-covalent bonds: Van der Waals, hydrogen bond and hydrophobicity, stacking interactions, electrostatic interactions, Debye screening. (Table with relative bond strengths)
      • Entropic forces: depletion, steric, fractionation, crowding, polymers
   3. MolNDigital description of molecules.
      • Structure abstraction layers: Primary, Secondary, Tertiary structure
      • Sequence abstraction layers: domain level vs nucleotide level
      • Notations: Dot parens plus notation, DU+ notation, …
      • SMILES string for small molecules
      • Notions of “valid” conformations (nearest neighbor model)
      • Intuition behind coarse-grained representations and macrostates
   4. MolVDigital visualization of molecules
      • Intuition behind coarse-grained representations and macrostates
      • How can we represent molecules graphically
      • Intro into formal concepts for visualization.
2. BiomoleculesMolecules as construction material
   1. DNADNA properties
      • data sheet to find useful parameters.
      • Crossover motifs (anti-parallel and parallel)
      • Special motifs (I motif, G-quads, aptamers, triplex, Holliday junctions)
      • Non-Watson-Crick-Franklin base-pairing (wobble etc)
      • Differences between single and double stranded DNA (could be a box) [for example different persistence lengths, inter-basepair lengths].
      • Biophysical influence of buffer conditions on DNA structure
   2. RNARNA properties
      • data sheet to find useful parameters.
      • Special motifs (I motif, G-quads, kissing loops, aptamers, ribozymes)
      • Biophysical influence of buffer conditions on RNA structure and stability
      • Non-canonical base pairing
   3. DSDDynamic processes of nucleic acids
      • Hybridisation
      • Toehold-mediated strand displacement via branch migration
      • 4-way strand displacement
   4. ProteinProtein biochemistry
      • data sheet to find useful parameters.
      • Enzymatic activity and binding pockets, …
      • Special motifs of secondary structure (alpha helices, beta sheets), tertiary structure (fold families like b-barrels etc)
      • Quaternary structure: Multimeric protein complexes
      • Biophysical influence of buffer conditions on enzyme activity and protein structure
   5. XNANon-canonical polymers and interactions (LNA, PNA, XNA, etc)
      • Comparisons of interactions between canonical polymers (DNA/RNA, Protein/RNA binding)
      • Protein Non-canonical amino acids
      • data sheet to find useful parameters…
3. BiophysicsInterfacing biophysical and computational models for analysis and design.
   1. PPPolymer physics models for nucleic acids and proteins.
      • Static properties
      • Elastic models (FJC, WLC)
      • Transport phenomena (viscosity, diffusion)
   2. MDMolecular dynamics models.
      • Basics of simulation
      • Nucleic acid models
      • Protein models
   3. NNThe thermodynamic nearest neighbor model.
      • single DNA/RNA molecules
      • multi stranded nucleic acid systems
   4. NLKinNearest neighbor level stochastic simulations
      • Thermodynamic energy landscapes and their application (and limitations) for Gillespie-type kinetic simulations
   5. DLKinModels of hybridization, dissociation and branch migration.
      • The ad-hoc approach (based on experimental results)
      • Molecular Dynamics Models (e.g. oxDNA)
      • Secondary Structure Kinetics (with and without the nearest neighbor model, e.g. Dave Zhang 2009 and Niranjan 2013 CRN Models, Multistrand)
   6. SeqDesignDNA/RNA Sequence design.
      • Why does sequence design work? – properties of landscapes (Lenvinthal’s paradox?)
      • Sequence design as an artificial evolution to optimize a landscape.
      • Formulation of a thermodynamic objective function for multistable design.
      • Formulation of combinatorial WCF base-pairing objectives.
      • Incorporation of kinetic objectives into sequence design (possibly with reference to kinetic proofreading.)
   7. ProteinDesignSequence design of proteins to fold into a specific structure
      • Rational Protein engineering (David Baker, U Washington )
        • Box: theory/tools for rational design
      • Directed Evolution protein engineering (Frances Arnold)
        • Box: Theory of directed evolution
      • Alphafold2 box on learned structure prediction and the protein folding grand challenge
4. SelfAssemblyIntroduction into molecular self-assembly theory via natural examples.
   1. NatAssemblyMacSelf-assembly of macroscopic phases
      • Crystals
      • Gels and disordered phases
      • Liquid-liquid phase separation
      • Lipid membranes and bilayers
   2. NatAssemblyMicSelf-assembly of finite-sized structures
      • Assembly from distinct units (capsid, enzyme complexes, ribosome…)
      • Protein folding and cyclisation/looping (eg. hairpins)
   3. NatAssemblyNoneqBeating equilibrium in self-assembled systems
      • Molecular templating as a way to drive the formation of non-equilibrium structures (central dogma).
      • Molecular species such as ATP as a store of high free energy fuel.
      • Use of fuel to push assembled systems out of equilibrium (kinetic proofreading, chaperones).
      • Use of fuel to create dynamic non-equilibrium systems (motors, signal-processing architectures, things like microtubules which are a form of "dissipative self-assembly").
5. DNAstructuresProgrammed molecular self-assemblies (experiments)
   1. OrigamiDNA Origami
      • The concept
      • Design principles, cooperativity
        • Sequence design constraints
      • Design and simulation tools
      • “Wireframe origami” examples 1D / 2D / 3D structures
      • Production and purification
        • Custom scaffold design
        • Thermo/stoichiometry
   2. AssembledDNAScaffold-less DNA assemblies
      • ss Tiles,
      • dx Tiles
      • HCR
      • Simple polyhedra: Seeman cube, tetrahedron, Yamuna’s icosahedron, Mao’s Bucky ball and octahedron.
   3. MultiComponentPeriodic and Multi component assemblies
      • Shape complementary, base stacking
      • Self-limiting assemblies (rings)
      • Fractal assemblies
      • Lattices, ribbons, nanotubes, and crystals
      • Interlocked assemblies (e.g. origami rotaxanes) -> connection to mechanics
   4. DynamicDNADynamic rearrangements of structures
      1. DynDNAMotionDNA tweezers, walkers, and motors
         • DNA tweezers, DNA Walkers, burnt-bridge motor
      2. DynDNAMechNanomechanical devices
         • Mechanical constructs / active components/ machines / walkers (comparison with molecular motors/enzymes)
   5. ProgrammedRNARNA Structures
      • RNA nanoparticles / Tiles
      • Cotranscriptional RNA-Origami
      • Multi-stranded RNA tiling
   6. ProgrammedProteinSynthetic Protein Structure
      • Protein Complexes (e.g. nanocage self-assembly)
   7. LiquidDNAOther phases of DNA structures (Physical properties)
      • DNA hydrogels
      • DNA liquids
      • Coacervates
   8. SurfaceDNADNA-grafted structures on surfaces
      • Colloids
      • Polymer/DNA brushes (surface/colloid coatings)
      • Grafted nanoparticles

Circuits

1. ComputationIntroduction to Computation
   1. Computation_introIntro to computation / information processing
   2. conventionalCompConventional computation
   3. CompBackgroundBackground: boolean logic, turing machines, register machines, analog computation nondeterministic finite automata, cellular automata
   4. unconventionalCompExamples of unconventional computing
   5. naturalCompExamples of natural computing (e.g. neural networks, gene regulatory networks)
   6. molprogLangMolecular programming languages
   7. molprogCompilCompiling molecular programs
2. CRNProgramming molecular behaviors over time (CRNs)
   1. CRN_introIntroduction
   2. dCRNComputing with Deterministic CRNs
      • Theory examples: circuits, boolean circuits, oscillators, bistability, etc.
      • Well-mixed CRN’s as example of analog computing
      • Computing functions (e.g. \(y=kx\))
      • Approximate majority
      • Dynamic system: Oscillators, bistability
      • Biology example: (predator-prey / ecology models)
      • Computational power of deterministic CRNs
   3. CRNdistributionCompute with distributions
   4. sCRNComputing with Stochastic CRNs
      • Theory example and analysis: min/max and boolean logic programming with CRNs
      • Biology example: something simple
      • Computational Power of Stochastic CRNs
      • Time complexity of stochastic CRNs
3. NucleicAcidCircuitsNucleic acids as a universal substrate for molecular programming
   1. NucleicIntroIntroduction (reference Foundations for overlapping topics)
      • Reference to Structures section on background: nucleic acid hybridization and thermodynamics
      • Background: nucleic acid branch migration
      • Toehold-mediated strand displacement
      • Experiment examples: toehold exchange reaction
   2. StrandDisplacementCascadesStrand displacement cascades
      1. DNAforCRNsTheory & experiment: DNA as a universal substrate for CRNs
      2. EarlyBooleanDSDBoolean circuits: an early example
      3. DSDsequenceDesignBox: DSD sequence design strategies
      4. ApproxMajorityTwoDomainApproximate majority (two-domain design)
      5. AmplifyCatalyzeAmplification and catalysis
      6. ThresholdThresholding
      7. SignalRestoreBox: signal restoration comparison
      8. SeesawSeesaw circuits
      9. NucleicNeuralNetworksNeural networks
      10. OscillatorOscillators
   3. NucleicAdvancedAdvanced designs in DNA strand displacement cascades
      1. ToeholdActivationToehold activation
      2. 4wDSDTheory & Experiment: 4wDSD circuits
   4. NucleicLeaklessLeakless circuits
      • Why do circuits leak and why leaks are problematic
      • How to avoid leak. Examples of leakless circuits
4. EnzymeCircuitsProgramming behavior with diverse biomolecules
   1. EnzymeCircuitsIntroIntroduction
      • Background: enzymatic behaviors
   2. PolymeraseCircuitsDNA polymerase-based circuits
      • PEN circuits, predator prey
      • APR, PER circuits (whiplash PCR?)
      • Shah et al. work on logic gates
   3. TranscriptionalCircuitsTranscribed RNA-based circuits
      1. ToeholdSwitchesToehold switches
      2. ConditionalCrisprConditional crispr
      3. OritatamiOritatami
      4. GeneletsGenelets
   4. ProteinCircuitsProtein-based circuits
      • Protein-protein binding circuits
      • Phosphorylation based circuits
   5. GeneCircuitsGene circuits
      1. GeneLogicOscillatorsLogic gates and repressilator
      2. GeneNetworksTheory of synthetic gene regulatory networks (incl. Cell-level circuits)
         • References to good syn bio resources?
   6. SmMolCircuitsSmall molecule cascades
      • Autocatalytic reactions and applications
5. SpatiallyOrganizedCircuitsSpatially-Organized Circuits
   1. SpatialIntroIntroduction: advantages of spatial structures
   2. SpatialBackgroundBackground: compartmentalization in biology
   3. SurfaceCRNsSurface CRNs
      • Surface DNA circuits and DNA walkers
   4. DropletComputingDroplet-based computing / “synthetic compartmentalization”
   5. ReactionDiffusionReaction diffusion circuits
   6. MicrofluidicsBoxMicrofluidics Breakout Box
6. AdvancedAssemblyAdvanced topics in tile assembly (algorithmic self-assembly)
   1. TileAssemblyTile self-assembly
      • Tiling theory as a mathematical theory (Geometry, Wang tiles).
      • A-tam and K-tam.
      • Tiles and algorithmic self assembly, example algorithms
      • Selected models (3D, active tile-assembly, probabilistic…), the problems that led to their creation.
      • Error correction and proofreading strategies
   2. WangTileWang tile ATAM Turing machine implementation
   3. TileComplexityTile Complexity of shapes and patterns
   4. ActiveAssemblyNubots, amoebots, turning machines
7. CircuitsConclusionConclusion

Interfaces

1. IntIntroIntroduction to Interfaces & Applications
2. CompIntIntegrating Molecular Programming with Traditional Computers
   1. CompIntIntroIntroduction
      • Pros
      • Cons
   2. LgDataStorageLarge-Scale Molecular Data Storage (focusing on DNA)
      • History
      • Encoding
      • Synthesis
      • Storage
      • Recovery
      • Readout
      • Decoding
   3. SmDataStorageSmall-Scale Molecular Data Storage (focusing on small molecules)
      • History
      • Encoding
      • Synthesis
      • Storage
      • Recovery
      • Readout
      • Decoding
   4. CompIntFutureEmerging/future applications
      • Implementing algorithms
      • New bases and chemicals
3. ChemPhysIntChemical and Physical Interactions
   1. MaterialMaterial science
      1. DNATemplateDNA as a template for non-DNA molecules
         • Encoding template
         • Positioning template
         • Framing template
      2. SurfaceAssistSurface-assisted methods
         • DNA nanostructure placement on surfaces
         • DNA lithography
         • Large lattices
   2. ChemCtrlChemical control
   3. MagCtrlMagnetic control
      • Fundamentals of magnetic control at the nanoscale
      • Magnetic control of molecular systems
   4. OptCtrlOptical control
      • Primer on optical materials
      • DNA plasmonic and photonic circuits
      • Dynamic control of optical DNA devices
   5. ThemCtrlThermal control
      • Thermal control of duplex
      • Thermal control of stacking and shape complementarity
      • Thermo-responsive materials
      • Engineering thermodynamic landscape of DSD
   6. ElecCtrlElectrical control
      1. ElecCtrlFieldElectrical field interface
      2. ElecCtrlRedoxRedox interface
   7. ChemPhysAdvAdvanced topics and future directions
4. BioIntInteracting with Biology, Medical Diagnostics and Therapeutics
   1. BioEnvBiological environments
      1. CellFreeCell-free systems
      2. BioChallChallenges of working in a biological medium
   2. DiagDiagnostics
      1. DiagIntroIntroduction to molecular diagnostics and precision medicine
      2. BiomarkerDetectTargeting and binding biomarkers
         1. NucDetectNucleic acid detection
         2. AntibodyDetectAntibodies
         3. AptamerDetectAptamers
         4. AffimerDetectAffimers and other affinity reagents
      3. BioSenseSensors and readouts for biomarker quantitation
         1. ElectroSenseElectrochemical
         2. FluorColSenseFluorescence & colorimetry
         3. SizeExcluSenseSize exclusion
            • Other alternatives
   3. TherapyTherapy
      1. TherMolProgTherapeutic molecular programming
      2. NucAcidTherTherapeutic Nucleic Acids
         • Genomic interactions
         • Antisense oligonucleotides
         • siRNA, mRNA, miRNA mimics
         • Aptamers
         • Delivery of nucleic acids
         • Approved or under-trial DNA/RNA-based drugs
      3. DrugDelDrug Delivery
         • Benefits and applications of targeted drug delivery
         • Techniques for carrying and storing drugs using molecular programming
         • Mechanisms for target site detection and release
   4. CellIntCellular Interfaces
      • Introduction to artificial cells and cellular mimics
      • Synthetic organelles and cellular components
      • Cellular interactions
        • Manipulating signal pathways
        • Channel receptor mimics
      • Applications and research trajectory
   5. BurBioIntBurgeoning Biological Interfaces