De novo Synthesis of Nucleic Acids of Specified Sequence
The chemical synthesis of nucleic acids is based on the use of phosphoramidites and protection chemistry.
- C5' hydroxyl / DMT (dimethoxy trityl)
- N bases / benzoyl or
- Phosphite group / cyano ethyl
The chemical synthesis proceeds from the 3' to 5' direction in an iterative process (detritylation / activation /
coupling / capping / and oxidation steps (see U. Penn. web site link for details).
At the end of the synthesis, the oligo is released and deblocked (NH4OH at 55o for 8 hours)
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Analysis of Nucleic Acids
1. Use of Radio Isotopes
How to Define Units for Radioactivity
- each isotope has a characteristic decay / characteristic decay particle, E, and half-life.
- for nucleic acid chemistry - 32P is the most common and useful isotope;
beta emitter / 1.71 MeV, half-life of 14.2 days
- Radioactivity is measured in Curies (Ci); 1 Ci = 3.7 x 1010 disintegrations / second
1 microCurie ( 1mCi = 2.22 x 106 disintegrations / minute
Define Specific Activity = disintegrations / unit mass = mCi / 1mmole
In practice, instruments measure counts per minute (CPM) which is related to DPM.
How to Measure Radioactivity
1. Geiger Counter: count ions produced to in a gas chamber by the passage of a decay particle
2. Scintillation Counter: Embed radioactive sample totally within chamber, usually in a solvent (toluene / p-Xylene / 1,4-Dioxane) mixed with a "fluor" (PPO / POPOP). The radioactive decay particle excites many solvent molecules as it looses energy, these in turn excite the fluors (POP / POPOP) used in scintillation counting which emit fluoresence at wavelengths that can be readily counted by surrounding photomultipliers. Reduce background from thermal noise by using "coincidence counters". Another advantage of liquid scintillation counters is that the number of photons produced is roughly proportional to the energy of the beta particle counted, so the counter can be "set" to detect different kinds of decay by setting "energy windows" on the detector.
3. Film: Overlay photographic film over radioactive sample, the intensity ("blackening") of the film gives a rough measure of the amount of radioactivity in the sample.
4. PhosphorImagers: Use a PhosphorImager Screen to take the place of film. Advantages are that the PI screen is more sensitive than film, can be exposed in less time, and is reusable. The phosphors on the screen absorb energy at one wave-length and emit it at another that can be read out by photomultipliers. Phosphorimager screens are composed of fine crystals of BaRBr:Eu+2. Radioactive sample excites the Eu+2 and mobile electrons become trapped in "storage" phosphors that can retain the "trapped" signal for up to a couple of hours. An instrument called the "Phosphorimager" scans the screen using a helium-neon laser that emits red light at 633 nm. The charged BaFBr- complexes absorb the red light, the trapped electrons are freed, Eu+3 is reduced back to Eu+2* and as Eu+2* returns to the ground state Eu+2 it releases energy as blue light in proportion to the initial radioactivity that is then collected by the Photomultiplier Tube (PMT). The scanned image and resulting PMT counts generate a "digital" image of the "film". The PI screen can be regenerated by exposure to strong light and reused almost indefinitely. (Molecular Dynamics is one maker of PhosphorImagers, find more details on the use of PI on their web site.).
How to Label Nucleic Acids
Nucleic acids are normally labeled using 32P by incorporating either alpha or gamma labeled nucleoside triphosphates.
Manipulation of DNA and RNA:
1. DNA Polymerase: Nick DNA with DNase, remove nucleotides by 5'-->3' exonuclease activity of E. coli DNA polymerase I. Replace excised nucleotides with alpha radiolabeled nucleotides by E. coli DNA polymerase I.
2. Use of Phosphatases and Kinases: Use gamma labeled nucleotide to label 5' phosphate.
II. Separation of Nucleic Acids - Gel Electrophoresis
Electrophoresis refers to moving charged particles due to an applied voltage. The force experienced by the particle is given by Coulomb's Law:
F = z e E (z = # charges; e = electronic charge; E = electric potential)
at steady state velocity, Force of acceleration due to potential = Force of friction = f(vel) where f is the frictional coefficient, so f(vel) = zeE. The electrophoretic mobility (m) is the ratio of the velocity (vel.) of the particle to the electrical potential (E).
(m) = vel/E = ze/f
Frictional coefficients can be calculated for a variety of sizes, but in practice biochemists rely on relative mobilities - that is the mobility relative to that of reference standards of known molecular weights. A plot of relative mobility vs. log(MW) is nearly linear for restriction fragments of DNA.
Gels can be made of a variety of materials (agarose, polyacrylamide, etc.) and cast in different ways (tube gels, vertical slab gels, horizontal slab gels, etc.) The porousity can be varied by varying the concentration (1% vs. 2% agarose) or % concentration and % cross-linking as in the case of polyacrylamide gels.
Gel electrophoresis uses the porousity of the gel to separate particles by size (actually their charge / size (mass) ratios). Since nucleic acids carry one negative charge per nucleotide and thus have nearly uniform charge/mass ratios, nucleic acids can be readily separated in the gel by size (length) if the shapes are uniform.
Gel Electrophoresis can be carried out under "Native" or "Denaturing" (SDS / Urea) conditions. During native gel electrophoresis samples retain native or near native conformations and charges, thus mobility is usually proportional for double-stranded DNA, but highly unpredictable for native proteins and single-stranded nucleic acids which can fold back on themselves to form a variety of secondary structures. However, nucleic acids run under denaturing conditions (7M urea) separate uniformly according to length. SDS is used to denature proteins and coat the protein with negative charge, mimicing the situation naturally occurring with nucleic acids. This generates nearly uniform charge to mass ratios for proteins and thus allows them to be separated by size (more on this later).
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Headlines for Exam I -
Review of Amino Acids - know all 20 a.a. structures by name (3-letter and 1-letter codes)
Protein Structure:
Peptide Bonds: Torsion Angles - Phi / Psi angles / Ramachandran Plot
1o, 2o, 3o, 4o Structures / helices / sheets / domains / representative folds
Molecular Interactions and Protein Folding
Review of Thermo: DG = DH - TDS / van't Hoff plots
Thermo of "folding" - DS for unfolding is always (+); DH = ??
DH term is dominated by many noncovalent interactions:
Review of Non-Covalent Interactions
Can Molecular Mechanics use Newtonian Physics to Predict Folded State?
Need to define Molecular Force Fields - find Emin using Molecular Dynamics
Define Bonding and Non-bonding Potentials
Non-bonding Potentials:
Electrostatic - role of "D", the dielectric
Dipole-dipole interactions - important in alpha helices (aligned dipoles)
van der Waals (dispersion forces) - weak but numerous
H-bonds - give specificity to interactions
Hydrophobic Interactions / Conformational Entropy
Can use the various potentials to find preferred conformational angles, dipole interactions, etc., but since this is a many variable problem, and solute-solute interactions are often balanced by solute-solvent interactions, this has not been very successful to date in predicting protein structure. However, these potentials are very useful to optimize a structure once a fairly good starting model is found (X-ray, NMR, etc.). To improve the "convergence" of such methods, molecular simulations are used to allow the molecule to sample greater conformational space. In the computer, the molecule can be "heated" to very high temperatures (3000o) to give it sufficient energy to overcome local minima and sample alternative conformational spaces. It is allowed to sample this larger conformational space for several nanoseconds (torsion angles twisting occurs in psec) in the computer simulation and then slowly "annealed" by lowering the temperature in steps while doing energy minimizations.
Folding Pathway -
Levinthal Hypothesis - How can proteins fold in a finite amount of time if they have to sample large regions of conformational space?
New Model - use of "molten globules" of pronounced, local secondary structure but of loose tertiary structure. As folding proceeds, steps lead to more compact tertiary structures that allow the expulsion of solvent and the tight packing of hydrophobic groups in the interior away from solvent. Molecular Chaperones help enable the folding process.
Predicting Protein Folding
1. Molecular - Dynamics Simulations
2. Monte-Carlo Simulations
3. Statistical Methods - use database of known structures
Chou / Fasman - 1974 - Propensity for a.a. residues to be in helix, sheet, or turns.
Many web-based programs that allow one to input an amino acid sequence and look at the predicted secondary structure averaged over many computational procedures.
Use of Molecular Transitions to Study the Energetics of Folding
Many biomolecules undergo sharp transitions (proteins: coil / alpha helix ; DNA: d.s. vs. s.s.). Often these structural transitions can be followed using CD spectroscopy. Such sharp transitions are usually the result of cooperative (all-or-none) transitions. The "zipper" model attempts to model this process by dividing the rates into a slow "nucleation" step followed by rapid "propagation" steps. Following these transitions as a function of Temperature allows one to plot logK vs. 1/T to obtain thermodynamic information (-DH/R) about folding from a van't Hoff plot.
Denaturation (Non-native state): There are many denatured states of macromolecules. Denaturation can occur with changes in pH, Temperature, adding detergents (SDS), water soluble organic agents (urea, guanidine HCl), changing ionic strength, reducing agents, etc.