Chemical synthesis of DNA is a crucial technology for molecular biology; however, the current methods limit the speed with which new genetic modifications can be introduced in living cells.
In the last 15 years an enormous progress has been made in the field of protein engineering. Soon it might become feasible to design new enzymatic systems, performing functions not found in nature.
A method capable of generating long, fully experimenter-specified DNA molecules directly in the cell nucleus (or cytoplasm of prokaryotic cells), immediately ready to be incorporated into the genome and transcribed into RNA, would greatly expand the options for the genetic engineer, and accelerate progress in medicine. The outline of a possible protein-engineering solution to this challenge follows.
Natural DNA synthesis proceeds from a single-stranded DNA template under the action of a DNA polymerase. If a molecule could be formed to act as a template, the DNA polymerase would be capable of synthesizing the complementary strand. The challenge is to design a molecule, which could be easily synthesized by the cell itself and which could be controlled by an outside source of information to encode the desired sequence. A possible solution would be to design carrier proteins for each of the four nucleotides and then assembling them in the order specified by a sequence of light pulses. The proposed system would consist of four nucleotide carriers, with light-sensitive dissociable domains (with incorporated retinal molecules), a modified DNA synthetase, as well as additional molecules needed for initiation of synthesis and DNA processing.. A brief description of the synthesis cycle is given in the legend to fig. 1, showing the concept of nucleotide carriers combining to produce a DNA template. The total number of proteins that would have to be designed de novo or substantially re-engineered would be 11. A description of the caveats and specific design problems will be included in the poster presentation.
Figure 1. The cytosine carrier (gray) approaches the growing template stack with an adenine carrier (yellow). DS- dissociable subunits. RD- recognition domain. MD- main domain. The cytosine DS was removed from the adenine carrier by a light pulse specific for the unit's light sensitive retinal molecule (not shown). Interaction between the RD on the cytosine carrier and the void left by the DS allows binding of the carrier to the template stack. The initiator protein is in blue.
While such a project might appear daunting, a proof-of-principle system with only two types of carriers, in a cell-free medium might need only 6 engineered protein elements. Once in place, the system would allow extremely fast advanced genetic engineering with very simple equipment - a set of four laser diodes controlled by a computer and a small vessel with culture medium. With the breathtaking pace of development, the project described here could become feasible, perhaps in as little as 10 to 15 years.