What was the primary use of oligonucleotides at the time?
Beaucage: As I mentioned earlier, the kind of work Marv Caruthers was doing— that is, studying protein-DNA interactions by chemically modifying protein binding sites with synthetic DNA oligonucleotides—was motivational at the time. More importantly, the emerging field of
oligonucleotide-based site-specific mutagenesis, which typically consists of modifying DNA sequences at predetermined locations in a gene of interest in order to alter the expression, structure, and function of proteins, required the use of accurately engineering synthetic
oligonucleotides. This work was pioneered by the late Michael Smith of the University of British Columbia in Vancouver, who shared the 1993 Nobel Prize in Chemistry for his contribution leading to modern genetic engineering. There was also a growing need for synthetic oligonucleoties as primers for DNA sequencing purposes and as hybridization probes for diagnostic applications.
Were there any competing technologies for making
oligonucleotides?
High-Impact Papers by Serge L. Beaucage,
Published Since 1990
(Ranked by average citations per year)
| Rank |
Paper |
Total
Citations |
Average
cites
per
year |
| 1 |
D.M. Klinman, et al., "CPG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin-6, interleukin-12, and interferon gamma," Proc. Natl. Acad. Sci., USA, 93(7):2879-2883, 1996. |
355
|
71
|
| 2 |
S.L. Beaucage, R.P. Iyer, "Advances in the synthesis of oligonucleotides by the phosphoramidite approach," Tetrahedron, 48(12):2223-2311, 1992. |
247
|
27
|
| 3 |
S.L. Beaucage, R.P. Iyer, "The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications," Tetrahedron, 49(12):6123-94, 1993. |
192 |
24 |
| 4 |
S.L. Beaucage, R.P. Iyer, "The functionalization of oligonucleotides via phosphoramidite derivative," Tetrahedron, 49(10):1925-63, 1993. |
149
|
19
|
| 5 |
R.P. Iyer, et al., "3H-1, 2-benzodithiole-3-one 1, 1-dioxide as an improved sufurizing reagent in the solid-phase synthesis of oligodeoxyribonulcleoside phosphorothioates," J. Amer. Chem. Soc., 112(3):1253-4, 1990. |
182
|
17
|
| 6 |
R.P. Iyer, et al., "The automated synthesis of a sulfur-containing oligodeoxyribonucleotides using 3H-1, 2-benzodithiol-3-one 1, 1-dioxide as a sulfur-transfer reagent," J. Org. Chem., 55(15):4693-9, 1990. |
166
|
17
|
|
|
Beaucage: When I proposed the phosphoramidite approach to oligonucleotide phosphoramidite synthesis in the early 1980s, the phosphotriester method and the phosphite coupling procedure for oligonucleotides synthesis were still popular. However, refinements of the phosphoramidite chemistry made it by 1984 what it is today: the method of choice for synthesizing
oligonucleotides. In 1986, the hydrogen-phosphonate chemistry was rediscovered essentially at the same time by a Swedish group and an American group. Even though this chemistry offers some advantages over the phosphoramidite method, the hydrogen phosphonate approach to oligonucleotide synthesis has not nearly been used as heavily as the phosphoramidite chemistry. In my opinion, most of the oligonucleotides being currently synthesized worldwide originate from the phosphoramidite approach.
How has the demand for synthetic oligonucleotides changed over the years?
Beaucage: From the late 1970s through the mid 1980s the biotech industry was growing at a fast pace. There was then a pressing need for constructing protein-encoding genes from synthetic
oligonucleotides, and expressing these gene constructs in bacterial or eukaryotic microorganisms to produce biologically important and, in some cases, life-saving proteins. With the advent of the polymerase chain reaction by the mid-to-late 1980s, the demand for synthetic oligonucleotides in gene constructs decreased because minute amounts of any gene of interest could be amplified by PCR to enable cloning and the ensuing large-scale production of proteins encoded by such genes. However, the availability of synthetic oligonucleotides primers was urgently needed to fuel those PCR amplification experiments and to construct engineered gene controls regions for optimal expression of
PCR-amplified genes. In this regard, one cannot help wondering about the fate of the Nobel Prize-winning PCR technology and DNA-sequencing techniques without readily available oligonucleotide primers. It would have definitely taken considerably longer to benefit from the sequencing of the human genome without the facile access to synthetic
oligonucleotides. The phosphoramidite approach to oligonucleotide synthesis has undoubtedly been instrumental in the colossal impact that these award-winning technologies have had on biomedical sciences.
By the late 1980s and most of the 1990s, intense scrutiny was becoming focused on the use of modified oligonucleotides as potential antisense drugs to inhibit the expression of genes responsible for a plethora of human diseases. The search for therapeutic oligonucleotides in the treatment of various types of cancer and infectious diseases is still ongoing. By the late 1990s, the emergence of "molecular beacons" and densely packed oligonucleotide microarrays as diagnostic tools had created a heavy demand for synthetic
oligonucleotides. A molecular beacon is a relatively short DNA oligonucleotide designed to form a hairpin loop. The loop serves as a fishing pole for any complementary single-stranded nucleic acid targets. A fluorophore and a fluorescence quencher are each attached to one of the two termini of the molecular beacon. When the fluorescence quencher is near the fluorophore across the double-stranded stem of the hairpin loop structure, no fluorescence can be detected upon irradiation. However, when the loop finds, let's say, a complementary RNA target, the disturbance created by hybrid formation causes the hairpin stem structure to fall apart. As the distance between the fluorophore and fluorescence quencher increases, the fluorophore begins emitting fluorescence when irradiated. The beacon technology is used in PCR experiments as a means to measure, in real time, the concentration of any
amplicons. It's definitely a fantastic tool for the detection of single-stranded nucleic acids. Another powerful diagnostic tool is oligonucleotide
micoarrays. One way to generate these microarrays entails the covalent attachment of synthetic oligonucleotides on, for example, a flat glass surface, in arrays of tightly packed rows or columns. An oligonucleotide array can carry several thousands, or even millions, of synthetic
oligonucleotides. Each of these has a predetermined sequence that can be used to monitor the simultaneous expression of an equal number of genes in, let's say, a cancer cell treated with different drugs, to evaluate the role of these drugs on the production of specific proteins. Alternatively, oligonucleotide microarrays can be used to screen genetic defects in the human genome for diagnostic purposes, or to rapidly identify life-threatening pathogens.
What do you think is in the future for synthetic
oligonucleotides?
Beaucage: I think that given the diagnostic power of oligonucleotide
microarrays, these are here to stay. I foresee the synthesis of oligonucleotides directly on arrayable surfaces as being the predominant method for the production of oligonucleotide
microarrays. My laboratory is certainly committed to modifying the phosphoramidite chemistry in order to make it more amenable to parallel oligonucleotide syntheses on "chips."
Aside from the DNA chip technology, the use of modified oligonucleotides as investigational tools for controlling gene expression, and determining structure and function of RNA and proteins, is so essential to biomedical sciences that it's also going to stay with us for a while. It's also likely that synthetic oligonucleotides conjugated to DNA and/or RNA modifying entities will widen the pharmaceutical spectrum of therapeutic oligonucleotides in specific areas—such as gene therapy, to name one.
I am very pleased that my work on the development of the phosphoramidite method for oligonucleotide synthesis has been beneficial to the life sciences over the years. It is still one of my dearest objectives to push the frontiers of knowledge further in the discovery of simpler and faster methods for producing synthetic
oligonucleotides, and defining new applications for these exceptionally useful biopolymers.
 continued from
Science
Watch®, September/October 2001, Vol. 12, No. 5
Citing URL: http://www.sciencewatch.com/sept-oct2001/sw_sept-oct2001_page4.htm |
|
.
