HV blog

We have now many methods to construct atomic-level machines. Bionanotechnology is the easiest way to do that because there are already nanomachines that can work for us. We don't have to implement everything, atom by atom, by ourselves. To do this we can make those machines to construct molecules they already can make but with some little changes, or we can design nanomachines entirely and make those already implemented workers to make them for us. But this is much more difficult than introduce some changes. For example, if we know a protein that actually works it is easier to give it some new features than to construct a whole protein from scratch because it is still a expensive problem to predict the structure of that protein, and it is important for the operation of that protein.

Recombinant DNA technology is the main process to construct this proteins. It is about changing the DNA that stores the information to create the protein and then wait until ribosoms create it for us. We use two natural enzimes for this, restriction enzimes and DNA ligase. Using this we can cut and paste DNA to make our own sequence.Some of this enzimes are being sold in the market, so now it is easy and not so expensive to manipulate DNA. There is a variety of natural biomolecules for handling DNA, like:Restriction enzimes: isolated from bacteria. Used to cut DNA.DNA ligase: reconnects broken DNA strands.DNA polymerase creates a new DNA strand by using another strand as a template.
Once we have a new DNA we can duplicate it using: DNA cloning and polymerase chain reaction.
The DNA cloning create identical chains. To do this we can inject DNA in a virus and let it to inject the DNA in some bacteria. Then they will duplicate, so we will have a lot of identical cells.
The polymerase chain reaction is for coping a small sample of DNA. We use another bacteria for this. Forte it to duplicate. Using this method we can have as many DNA as we need.

Once we have all this DNA we can create the proteins byr forcing the cells to produce it. This is done using expression vectors. They have a highly active promoter sequence that forces the cell to create mRNA. It is taken from a virus. This mRNA will be later processed to create the proteins. The cells used are bacteria. But they have some limitations, animals and plants modify their proteins after create them, but bacteria don't. This can be a serious problem because the immune system can react against them because of those differences. Another problem is that proteins tend to aggregate when they reach high concentrations forming dense inclusion bodies.Proteins can be created as well without the help of living help using a test tube and enzimes that make them. But the synthesis of protein from the mRNA is still a challenge. Stracts of cells can be used then to form those proteins. But  because of the complexity of the system it is only used for small tasks and for research. This is a controlled method and has not the interferences of other enzimes in the bacteria, that's why it is good for research.In other cases we prefer to make some little changes to a natural protein. Then we can use the site-directed mutagenesis. This is to introduce some specific mutations in an existing DNA sequence to create already existing proteins but with some changes. This method has revolutionized molecular biology. It is useful to determine the function of specific aminoacids. It is also used to improve the stability  of proteins.

We can also combine two different proteins to get a new functionality with the older ones. We can specify the attach point for this task.

When we need to obtain some kind of detector we can use the immune system. Its main function is to detect, so we can use that feature for our interest. Combining the knowledge of the immune system with the modern methods of antibody production is possible to get a large number of high-affinity recognition molecules.

To be able of all this manipulations we need to understand how bionanomachines work. To do this we can use some methods like x-ray crystallography to get atomic structures. We obtain a 3D map of the molecules that help us to understand. The resolution of this map depends of the quality of the crystals used, this is a major in this technique.

Another method is the nuclear magnetic resonance. It is used to determine molecular structure in chemistry. It characterizes the local environment of atomic nuclei inside molecules. We can alter the atomic nuclei inside molecules using a radio frequency, then, when they relax, emit radio frequency that shows the structure of the molecule. But this method is not good for big molecules, so it can be only used with some nucleic acids or small proteins. However, a 2D map can be used for greater molecules.Electron microscopy is a method that can reveal molecular morphology. It uses electrons to look at the molecules. But it has some limitations. Due to imperfections in the magnetic optics and problems with the specimen preparation it is not possible to see individual atoms, only overall morphology. The good point about this method is that it can provide information about the structure of large molecules that are impossible to study with the other previous methods. But the information can be combined with the others to get a more accuracy study.The atomic force microscopy is a method used for getting the surface of a molecule. It is like touch the molecule and trace a topographic map from that data.  This method was developed to study samples in a water environment where other methods cannot be applied. Today the problem is solved by immersing the sample in solvent. Because the samples are inside a similar environment as the one inside cells it is really good because gives data of the nanomachine as it is in its real environment.The molecular modeling using computers has revolutionized the study of biomolecules. Computer graphics allow us to visualize the molecules in a familiar manner. The best visualizations captures the keys of the molecules and shows them in 3D models that are easy to understand.Some free software available for visualize molecules is:
RasMol
ProteinExplorer
Chime
(http://www.rcsb.org/pdb/software-list.html)

The computer modeling can be used to predict biomolecular structures and functions that depends on that structures. This can be for optimization of the structure of a molecule. For normal mode analysis getting the forces working in a molecule. The molecular dynamics, how the molecule works in a variant environment, and free energy perturbation, shifting the system smoothly to learn what does the molecule.

Another commercial and academic software is:
Insight (BioSym): commercial
Sybyl (Tripos): commercial
Amber (UCSF): academic

There is a problem with proteins (the protein folding problem), we need to predict the folded structure before starting with any other study. But this is hard because every amino acid has interactions with other neighbors, so we can't know easily the structure. Another problem is to estimate the stability of the trial during the experiment. Proteins have lots of bounds inside that stabilize them. But outside water they act different.This two problems make hard to predict protein folding. Currently the best predictions are from homology modeling. Taking a similar protein and estimate the structure by similarities is easier than any other method.Simulations of biomolecular interaction are for knowing how molecules interacts with other molecules. The most successful methods combine two capabilities, a fast algorithm to search how the molecules can fit together and an energetic model that predicts the energy of the interaction. Current methods are:AutoDock (Scripps Research Institute): a genetic algorithm to evaluate energies.Dock (UCSF): a geometric matching algorithm to search in databases.Using this new functionalities new computer-assisted molecular dessign is possible. They allow us to create improved molecules. In practical application it is used for design mutations to increase stability and shifting in proteins. Many of these techiniques are used in the drug industry.