DNA-recorded synthesis

DNA-Recorded Synthesis Assisted Libraries Encoded Self-assembly chemical library... 

In this chapter, we will first discuss the basic principles of DNA-templated organic synthesis, as it is the mechanistic foundations for DNA-encoded libraries then we will discuss the progressive development of DNA-templated libraries and the evolution into a novel drug discovery tool. We will also discuss the DNA-recorded library, which also encodes library molecules with DNA but is conceptually different. These discussions will naturally involve specific drug discovery programs in which these libraries were applied and finally, we will discuss the outlook of DNA-eneoded libraries in the future of drug discovery. 

DNA-recorded library is another approach to encode small molecule libraries on DNA templates. Instead of preparing a template pool in which every library compound has already been encoded, in a DNA-recorded library initially all templates share an identical sequence, or even are simply one template. Usually, the chemical reactions on each template are encoded spatially such as the split-pool-split method i.e. the initial template pool is separated into several batches for discrete reactions with various building blocks. Before all the sub-pools are combined, an enzymatic tag is added to the template to record the synthesis. For every step, an additional tag is added. As a matter of fact, DNA-recorded library, similar to Harbury s DNA routing strategy discussed above, does not use DNA to template the reactants into proximity and to increase the effective molarity. 

Figure 8.22 Neri s DNA-recorded library synthesis scheme. The encoding is done by enzymatic extension. Reprinted, with permission, from Buller et al 2008 Elsevier.

In this approach, DNAs are tagged on small molecules and serve as barcodes to record both the structural information of the small molecules and the library information. DNA-recorded libraries, which consist of large numbers of encoded small molecules, have been generated in time- and cost-effective manner. Repeated cycles via a well-established procedure, known as spUt-and-pool synthesis strategy in combinatorial chemistry, ensure production of huge compound libraries with diversity. 

Increased DNA synthesis due to repair can be measured by recording tritium-labelled thymidine incorporation. 

It is apparent from this that each m-RNA molecule can be used to read out aprotein several times over (just asa tape can be played many times on a tape recorder). How long the m-RNA lasts varies from minutes to days, depending on the cell and protein concerned. Some types of template, at least, must be capable of making several hundred protein molecules before needing renewal, as experiments demonstrating protein synthesis in the absence of nuclear DNA reveal. 

In the experiment shown in Figure 9.17, the SV40-infected cells were treated with cisplatin. After 24 h, [ H]thymidine was added and, after 24 more hours, the cells were harvested, and SV40 DNA was isolated the amount of DNA synthesis was recorded by comparing incorporated radiolabel with results from control experiments where no platinum was present. The data show that, when 25 platinum was present, SV40 DNA replication was reduced to about 5 percent of control. Quantitation reveals that, at 2 platinum atoms bound per thousand nucleotides (drug-per-nucleotide, or (DIN)h, — 0.002), synthesis is only 10 percent that of control. 

Pyrosequencing is based on sequencing by synthesis. The assay takes advantage of the natural release of pyrophosphate whenever a nucleotide is incorporated onto an open 3 DNA strand. The released p5u-ophosphate is used in a sulfurylase reaction releasing ATP. The released ATP can be used by luciferase in the conversion of luciferin to oxyluciferin. The reaction results in the emission of light, which is collected by a CCD camera and recorded in the form of peaks, known as pyrograms (Fig. 1). When a nucleotide is not incorporated... 

RNA turns over in the cell, whereas DNA is the nearly permanent record of inherited information. The consequences of most errors in RNA synthesis will be short-lived, perhaps a protein synthesized with a mistake in its sequence for a short time during the life of the cell, but then the RNA in question may be degraded and the error will disappear. By contrast, an uncorrected error in DNA replication will be passed to the next generation. 

Figure 2.4. Effect of novobiocin on (a) growth, as recorded by changes in optical density, (b) DNA synthesis, (c) RNA synthesis, and (d) protein synthesis in E.coli in nutrient broth at 37°C. Novobiocin concentrations (ng/ml) 0, 0—0 20, — 100, — 500, A—A. (From Morris and Russell [72], by courtesy of Microbios.)...

Enzyme microreactors have also received wide application not only in the analysis but also in the synthesis of DNA. A nanoscale platform for real-time single-molecule sequencing by recording the DNA synthesis process by an immobilized DNA polymerase has been developed and has been covered by US patent application number 7,329,492 [62]. The process relies on fluorescence resonance energy transfer (FRET) between the fluorescence receptor moiety on the dNTPs and the fluorescent dye attached to the donor DNA that emits an enhanced signal upon nucleotide incorporation. Immobilization of polymerase enables increased sensitivity detection as the DNA strand extends imattached. 

The year 1953 was important not only because of the Staudinger Nobel Prize, but also because it was the year that the structure of the macromolecule DNA was reported by James Watson and Francis Crick [5], which is the subject of another article in this volume by Ned Seeman [6]. Yet another important milestone was recorded in 1953, namely the first pubhshed use of a computer to carry out a simulation of liquid, albeit a liquid composed of argon atoms [7]. This seminal work, which was carried out on the famous MANIAC machine at Los Alamos National Laboratory, by Nick Metropolis and collaborators, was never honored with a Nobel Prize, but nonetheless had an enormous impact across the whole breadth of the physical and life sciences. Indeed, it is inconceivable today that one would attempt a research program dealing with either natural or synthetic macromolecules without the aid of computer simulation as a complement to their design, synthesis, and characterization [8]. 

When horse lymphocytes were stimulated with mitogenic agents [39] significant heat production, that preceded DNA synthesis, was recorded after 2 days of incubation, reaching a maximum at 4-5 days. A similar response was observed in human lymphocytes [40] reaching a maximum of heat production... 

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