In vitro DNA synthesizer
From Biohack
- Synth2008 Fwd: Hplusroadmap Synthetic Biology Use a laser-inducable writozyme to make a plasmid, which then inserts genes
- Methods:
- hack polymerase to be pausable and accept the next (programmed) nucleotide
- something on the surface of polymerase that prohibits it from doing its thing
- molecular engineering: making a rotating belt of tools for the polymerase molecule
- laser-induced partial denaturation of polymerase (?)
- beta clamp method
- find a very, very slow polymerase, or inhibit its ability to move beta clamp, etc.
- hack polymerase to be pausable and accept the next (programmed) nucleotide
Another few ideas --- we can incorporate the biosynthesis pathways for unnatural nucleotides and use these as smoke signals to the polymerases that use mostly template-independent synthesis (not attached to the template, but still able to receive an input template nucleotide, i.e. the type that I just mentioned); the hard part here is making a polymerase that is only able to allow unnatural nucleotides in the 'template strand' slot, so how would this work? You still need to run the same type of selections and therefore this runs into the exact same problems as selecting for polymerases that incorporate only one nucleotide.
Another method: In vitro transcriptional-translational phosphoramidite chemistry
Papers
Selective Destruction of Protein Function by Chromophore-Assisted Laser Inactivation
Selective destruction of protein function by chromophore-assisted laser inactivation.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=281775
D G Jay
Chromophore-assisted laser inactivation of protein function has been achieved. After a protein binds a specific ligand or antibody conjugated with malachite green (C.I. 42,000), it is selectively inactivated by laser irradiation at a wavelength of light absorbed by the dye but not significantly absorbed by cellular components. Ligand-bound proteins in solution and on the surfaces of cells can be denatured without other proteins in the same samples being affected. Chromophore-assisted laser inactivation can be used to study cell surface phenomena by inactivating the functions of single proteins on living cells, a molecular extension of cellular laser ablation. It has an advantage over genetics and the use of specific inhibitors in that the protein function of a single cell within the organism can be inactivated by focusing the laser beam.
Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals.
J C Liao, J Roider, and D G Jay
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=43429
Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no significant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process.
Genetically targeted chromophore-assisted light inactivation (awesome) (bingo)
http://www.ncbi.nlm.nih.gov/pubmed/14625562
Tour O, Meijer RM, Zacharias DA, Adams SR, Tsien RY.
http://150.176.130.202/research_programs/pdf-files/Tour_zacharias_tsien%20Nat%20Biotech%202003.pdf
Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and alpha1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by approximately 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or alpha1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.
So, it takes 30 seconds for ReAsH-based CALI illumination to work its magic. polA writes bases at 20 bp/sec. This needs to get significantly slower. What's the main mechanism that makes polA so much more slow? Surely this has been investigated -- crystallographic race tests of different polymerases with chemical considerations on speed / reaction time. How could it be slowed without inhibiting actual functionality? (think capacitor -- but this, then, requires more energy). Doesn't need to have a timer / clock onboard the polymerase protein (it wouldn't hurt, but that's rather hard to construct), so what then?
Does the protein fold back up after the laser illumination is removed?'
Molecular dynamics simulation of protein denaturation: solvation of the hydrophobic cores and …
Model system for investigating laser-induced subcellular microeffects
Gereon Huettmann, Jesper Serbin, Benno Radt, Bjoern I. Lange, and Reginald Birngruber
Background: Laser induced protein denaturation is of fundamental interest for understanding the mechanisms of laser tissue interaction. Conjugates of nanoabsorbers coupled to proteins are presented as a model system for investigating ultrafast protein denaturation. Irradiation of the conjugates using repetitive picosecond laser pulses, which are only absorbed by the nanoabsorbers, could result in effects with a spatial confinement of less than 100 nm.
Materials and Methods: Experiments were done with bovine intestinal alkaline phosphates (aP) coupled to 15 nm colloidal gold. This complex was irradiated at 527 nm wavelength and 35 ps pulse width with a varying number of pulses ranging form one up to 104. The radiant exposure per pulse was varied form 2 mJ/cm2 to 50 mJ/cm2. Denaturation was detected as a loss of protein function with the help of the fluorescence substrate 4MUP.
Results and discussion: Irradiation did result in a steady decrease of the aP activity with increasing radiant exposures and increasing number of pulses. A maximal inactivation of 80% was reached with 104 pulses and 50 mJ/cm2 per pulse. The temperature in the particles and the surrounding water was calculated using Mie's formulas for the absorption of the nanometer gold particles and ana analytical solution of the equations for heat diffusion. With 50 mJ/cm2, the particles are heated above the melting point of gold. Since the temperature calculations strongly depend on changes in the state of matter of the particles and water, a very sophisticated thermal model is necessary to calculate exact temperatures. It is difficult to identify one of the possible mechanisms, thermal denaturation, photochemical denaturation or formation of micro bubbles from the dependance of the inactivation on pulse energy and number of applied pulses. Therefore, experiments are needed to further elucidate the damage mechanisms. In conclusion, denaturing proteins irreversibly via nanoabsorbers using picosecond laser pulses is possible. The confinement of the heat to the nanoabsorbers when irradiating with picosecond pulses suggests that the denaturation of proteins could be possible with nanometer precision in cells with this approach. However, the mechanism of protein inactivation, which is part of present investigations, is crucial for the precision of such nanoeffects.
(meh) Photorepair of RNA polymerase arrest and apoptosis after ultraviolet irradiation in normal and XPB deficient rodent cells
http://www.nature.com/cdd/journal/v9/n10/abs/4401072a.html
V Chiganças, L F Z Batista1, G Brumatti, G P Amarante-Mendes, A Yasui and C F M Menck
Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.
