ABSTRACT
In this study we develop a sequence-specific precipitation separation system of oligonucleotide (ODN) using a conjugate between poly(N-isopropylacrylamide) (PNIPAM) and ODN. PNIPAM is known as a thermoresponsive polymer and dehydrates to precipitate above its phase transition temperature in an aqueous milieu. The principal advantage of this separation system using the conjugate is that the hybridization reaction between the conjugate and oligonucleotide is conducted in homogeneous solution. The conjugate was prepared by copolymerization between N-isopropylacrylamide and a vinyl-derivatized (dT)(8). The obtained conjugate efficiently precipitated (dA)(8) from solution when the solution contained more than 1.5 M NaCl. The conjugate containing 3 nmol of (dT)(8) residue was able to precipitate 1.4 nmol of (dA)(8), suggesting that the (dT)(8) residue of the conjugate formed a triple helix with (dA)(8). From an equimolar mixture of (dA)(8) and its one point mutant, the conjugate selectively precipitated (dA)(8): the highest selectivity was obtained for the isolation of (dA)(8) from the mixture consisting of (dA)(4)dT(dA)(3) and (dA)(8). When the conjugate was applied for the precipitation of five oligo(dA)s having different chain lengths, the longer oligo(dA)s tended to be precipitated by the conjugate more efficiently than the shorter ones. The conjugate could be used repeatedly for precipitation of (dA)(8) without showing any loss in precipitation efficiency.
Subject(s)
Acrylic Resins/metabolism , Oligonucleotides/genetics , Oligonucleotides/metabolism , Acrylic Resins/chemical synthesis , Acrylic Resins/chemistry , Base Pair Mismatch/genetics , Base Sequence , Molecular Structure , Molecular Weight , Nucleic Acid Denaturation , Nucleic Acid Hybridization , Oligonucleotides/chemistry , Oligonucleotides/isolation & purification , Poly A/chemistry , Poly A/metabolism , Poly T/chemistry , Poly T/metabolism , Sodium Chloride/pharmacology , Solubility , TemperatureABSTRACT
The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants. We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli. We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids. One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3, 4,3',4'-tetradehydrolycopene. This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products. One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E. coli. This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry.
Subject(s)
Carotenoids/biosynthesis , Genetic Engineering/methods , Carotenoids/analogs & derivatives , Carotenoids/metabolism , Chromatography, High Pressure Liquid , Cloning, Molecular , Escherichia coli/metabolism , Gene Library , Intramolecular Lyases/genetics , Intramolecular Lyases/metabolism , Models, Chemical , Oxidoreductases/genetics , Oxidoreductases/metabolism , Plasmids/genetics , Transformation, GeneticABSTRACT
The conjugate between oligo(dT)16 and thermo-responsive polymer, poly(N-isopropylacrylamide), was prepared for isolation of poly(A)+ RNA from total RNA. The hybridization reaction between the conjugate and poly(A) (average length: 320 base) was equilibrated in 10 min, and all the poly(A) (16 nmol base for 24 nmol base of conjugate) was precipitated when raising the solution temperature to 35 degrees C. The precipitate was dissolved in water, and poly(A) was dissociated from the conjugate by heating to 65 degrees C. This separation system was successfully applied to the isolation of poly(A)+ RNA from total RNA.
Subject(s)
Acrylic Resins , Oligodeoxyribonucleotides , Poly A/isolation & purification , RNA, Messenger/isolation & purification , Chromatography, Affinity , Hot TemperatureABSTRACT
Poly(N-isopropylacrylamide) having a terminal psoralen group was synthesized and covalently bound to plasmid pBR 322 DNA through a photochemical reaction. The resulting conjugate exhibited temperature-responsive precipitation due to the aggregation of poly(N-isopropylacrylamide) chains when heated above 31 degreesC. This system was used for the one-pot separation of restriction endonuclease EcoRI.
Subject(s)
Acrylic Resins/chemistry , DNA-Binding Proteins/isolation & purification , DNA/chemistry , Chromatography, Affinity , Deoxyribonuclease EcoRI/isolation & purification , Electrophoresis, Polyacrylamide Gel , Furocoumarins/chemistry , Indicators and Reagents , Photochemistry , SolubilityABSTRACT
We have synthesized a vinyl monomer having a psoralen moiety, which can form a photoadduct with double-helical DNA. The monomer 1 was proved to have an ability to crosslink DNA double strands through a photochemical reaction when irradiated by UV light. The resulting DNA having vinyl groups was copolymerized with a comonomer such as acrylamide and N-isopropylacrylamide to give rise to a DNA-vinyl polymer conjugate. A conjugation based on covalent bondings was verified by using gel electrophoresis; the conjugation efficiency was found to be dependent upon concentration of the monomer which had been used in the antecedent photochemical reaction. This monomer will be a useful tool when anchoring double-helical DNA on polymeric materials for separating and sensing DNA-binding substances.
