Polynucleotide synthesis using a processing enzyme

Details Polynucleotide synthesis using a processing enzyme Polynucleotide synthesis using a processing enzyme

2002-01-02

2003-09-23

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007600, C435S091100, C536S024320, C536S024300

Reexamination Certificate

active

06623929

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method for polynucleotide synthesis.
BACKGROUND OF THE INVENTION
At present the demand for synthetic polynucleotides is large, due in most part to the need for oligonucleotides of known sequence to be used as primers within the Polymerase Chain Reaction (PCR) or within polynucleotide sequencing strategies. More recently, demand has increased even further with the advent of polynucleotide hybridisation arrays. These arrays have, attached to a solid support (or chip), either oligonucleotide probes that hybridise with the sample to be tested, or sample to which labelled oligonucleotide probes can hybridise (Lysov, Dovi. Akad. Nauk SSSR (1988) 303:1508-1511; Bains et al, J. Thero. Biol., 135:303-307; Dramanac et al, Genomics, 4:114-128). This hybridisation pattern is then used to reconstruct the target polynucleotide sequence. This technique has been further facilitated by the utilisation of light-generated oligonucleotide arrays (Fador et al, Proc. Natl. Acad. Sci. USA (1994) 91:5022-5026).
All current techniques are restricted in the length of synthetic polynucleotide that can be produced and the accompanying problem of low yields. They also employ a significant number of manipulations and hence take a significant period of time to execute.
There is therefore a need for an improved method for the synthesis of polynucleotides which significantly increases the maximum length of the polynucleotide synthesised and increases the rate at which such a polynucleotide is synthesised. Such a process would preferably be carried out by an automated process, reducing the complexity and cost associated with existing methods.
SUMMARY OF THE INVENTION
The present invention is based on the realisation that electromagnetic radiation can be used to generate conformational changes within a polynucleotide processive enzyme, such that by controlling the radiation applied to such an enzyme, the sequence of the polynucleotide strand produced can be pre-determined. This enables the production of “synthetic” polynucleotides in real-time by manipulating the normal in vivo polynucleotide assembly process.
According to the present invention, a method for synthesising a polynucleotide comprises the steps of:
(i) reacting a polynucleotide processive enzyme with a nucleotide substrate under appropriate conditions; and
(ii) exposing the enzyme to a controlled environment (including radiation) so as to affect the three-dimensional conformation of the enzyme and hence determine/affect the sequence of the polynucleotide produced.
DESCRIPTION OF THE INVENTION
If radiation is used to control the conformation of the processive enzyme, then it may be applied to a sample using a number of techniques. These include evanescent wave spectroscopy techniques, in particular surface plasmon resonance (SPR) spectroscopy.
The application of radiation to the processive enzyme via the application of laser technology (Light Amplification by Stimulated Emission of Radiation) is particularly applicable to the present invention due to the monochromatic and controllable nature of the radiation produced by such devices.
The control of the conformational structure of processive enzymes can be accomplished by controlling the environment in which they act It has been shown that variations in such conditions as pH and sait content/concentration of the reaction medium can have an effect on the three-dimensional structure and hence on the activity of such enzyme systems (Wong et al, Biochemistry (1991) 30:526-537).
The addition of the specified nucleotide, and hence the synthesis reaction, may be accomplished by directly creating the ability of the processive enzyme to undergo a conformational change that IS specific for the addition of a particular nucleotide, depending on the form of radiation delivered. This could be achieved by engineering (via state-of-the art genetic manipulation techniques) a processive molecule (or molecule associated with it) such that it contained a chemical/moiety/peptide group or groups that enable the molecule to convert or transduce radiation into a conformational change. These chemical/moiety group or groups may be so positioned so as to select for the nucleotide to be added to the growing polynucleotide chain. The method may therefore proceed on a “real-time” basis, to achieve a high rate of polynucleotide synthesis.
The present method for the synthesis of a polynucleotide, as indicated above, involves the control of the environment in which a polynucleotide processive enzyme is placed, and hence of the three-dimensional conformation of said enzyme. This three-dimensional conformation in turn selects if and/or which substrate nucleotide is added to the growing polynucleotide strand.
The term “polynucleotide” is used herein as to be interpreted broadly, and includes DNA and RNA, including modified DNA and RNA, as well as other hybridising nucleic acid-like molecules, e.g. peptide nucleic add (PNA).
The term “polynucleotide processive/polymerisation enzyme” is used herein as to be interpreted broadly, and pertains to ubiquitous proteins that can attach one nucleotide to another in order to create a polynucleotide. Such a group will, of course, include all polymerases, both DNA- and RNA-dependant and also such enzyme groups as terminal deoxynucleotidyl transferases (Kato et al, J. Biol. Chem., (1967) 242:2780; & Frohman et al, Proc. Natl. Acad. Sci. USA, (1988) 85:8998).
Using a polynucleotide processive enzyme in order to control the synthesis of a polynucleotide offers several advantages for the success of this method. Firstly, the problem of reaction yield in solid phase synthesis is avoided due to the highly efficient catalytic nature of organic molecules. Secondly, speed of synthesis and polynucleotide strand length are several orders of magnitude greater than those currently available, again due to the requirements of the enzyme systems in their native environments.
Another important aspect of the invention is the realisation that, although a large number of polynucleotide processive enzymes require an existing polynucleotide template to initiate polynucleotide synthesis in their native environment/form, this is not always the case. As the effectiveness of the nucleotide (Crick-Watson) base pairing and hence of complementary strand construction is ultimately dependent on the three-dimensional conformation (and resulting kinetic parameters) of the processive enzyme, this system can be disrupted and utilised in order to externally control the sequence of nucleotides polymerised. In the specific case of the utilisation of polymerases for the present invention, therefore, the “synthetic” polynucleotide strand produced may not (and in most instances will not) be a complementary copy of the template polynucleotide strand. Disruptions to polymerase function via active site mutation are known in the art (Freemont et al, Proteins (1986) 1:66-73) but, critically, they are not conformationally/spatially modulated. Such disruption/mutation could take the form, as in the present invention, of a reduction in the natural fidelity of the polymerase such that it does not discriminate against dideoxynucleotides. This would allow the mutated polymerase to insert any nucleotide in solution into the growing polynucleotide chain independently of the nucleotide sequence of the polynucleotide template. The nature of such binding site modifications that are fixed upon molecular cloning (i.e. not capable of external real-time conformational modulation) are known in the art (Ollis et al, Nature (1985) 313:762-766 & Freemont et al, Proteins (1986) 1:66-73) and are directed at the polymerase active site. For example, it has been shown that Phe
762
of
E. Coli
polymerase I is one of the amino acids that directly interact with the substrate nucleotide (Joyce et al, Ann. Rev. Biochem. (1994) 63:777-822 & Astake et al, J. Niol. Chem. (1995)270:1945-54). Converting this amino acid to a Try results in a mutant DNA polymerase that does not discriminate against dideoxynucleotides. See U.S.

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