Method for synthesizing single-stranded stem-loop DNAs, the products and uses therefor

FIELD OF THE INVENTION
The invention concerns the field of recombinant DNA. More particularly, the invention relates to a method of synthesis of new and useful single-stranded DNA which have a stem-loop configuration (ss-slDNA). The invention relates to an in vitro and in vivo method of synthesis. Further, the invention relates to the replicating vehicle which produce these ss-slDNAs. Moreover, the invention relates these novel structures and discloses uses for these structures. There is described a method for amplifying ss-slDNAs with or without genes encoding a target protein.
BACKGROUND
Duplication of part of the genome is known to occur via an RNA intermediate which is reverse-transcribed into complementary DNA (cDNA) by reverse transcriptase. For a review, see Weiner et al., Ann. Rev. Biochem., 55, 631 (1986). The consequential reverse flow of genetic information is considered to have played a major role in the evolutionary diversification of eukaryotic genomes. A similar mechanism may very well have been responsible for genomic evolution in procaryotes in the light of the recent discoveries of bacterial transcriptases. See Inouye and Inouye, TIBS, 16, 18 (1991a) and Inouye and Inouye, Ann. Rev. Microbiol., 45, 163 (1991b). Gene duplication through cDNA synthesis by reverse transcriptase is believed to have played an important role in diversification of genomes during evolution.
The invention arose in connection with basic research related to genome evolution. The synthesis of a unique ss-slDNA during plasmid DNA replication was demonstrated. It may be speculated that slDNA production may be widely prevalent during both procaryotic as well as eucaryotic chromosomal DNA replication. The chromosomal genetic elements followed by IR structures may always be subject to duplication into slDNA at a frequency depending on the stability of the IR structure and the property of the polymerase(s).
It has been shown that there are many inverted repeat (IR) structures (approximately 1,000 copies in E. coli), known as REPs for repetitive extragenic palindromic sequences or PUs for palindromic units. Higgins et al., Nature, 298, 760 (1982), Gilson et al., EMBO. J., 3, 1417 (1984) and Gilson et al., Nucl. Acids Res., 19, 1375 (1991). These structures appear to be associated with specific cellular components including DNA polymerase I, and may be playing a significant role in chromosomal organization. Gilson et al., Nucl. Acids Res., 18, 3941 (1990) and Gilson et al., EMBO. J., 3, 1417 (1984). It should also be noted that approximately 6% of the human genome are occupied with elements called Alu whose transcriptional products have been shown to contain substantial secondary structures. Simmett et al., J. Biol. Chem., 266, 8675 (1991).
Since slDNA synthesis does not require RNA intermediates nor reverse transcriptase activity in contrast to that of cDNA synthesis, slDNA may be more frequently produced than cDNA. Thus, slDNAs might have played a major role similar to cDNA in the genomic evolution of both procaryotes and eucaryotes by duplicating genetic elements which then were dispersed or rearranged within the genome.
SUMMARY OF THE INVENTION
In accordance with the present invention, a fundamental finding has been made. It has been found that portions of the genome can be directly duplicated from the genome. This gene duplication it has been found, requires neither an RNA intermediate nor reverse transcriptase and occurs during DNA replication.
Briefly described, the invention provides a method (or process) for synthesizing a novel and useful single-stranded DNA (ssDNA) molecule. The method involves using a DNA inverted repeat (IR) and necessary components to start and synthesize a single-stranded structure. The invention also provides a system for synthesizing such ssDNA from and with the necessary components including a DNA inverted repeat. The invention further provides competent replicating vehicles which include all the necessary elements to synthesize the novel ssDNA molecules. The invention also provides novel and useful ssDNA molecules which form a unique structure. The structure has a stem constituted of duplexed DNA of complementary bases; the stem terminates at one of its ends by two termini, respectively a 3' and 5' terminus, and at the other end by the ssDNA forming a loop.
The invention contemplates carrying out the method and the systems in vitro or in vivo.
Various uses of the new molecules are described, including a method for providing random mutations in genes with the aim of generating proteins with improved or novel biological properties. Another interesting contemplated use is to integrate the ssDNA molecules of the invention into DNA to generate a triplex DNA of increased stability. Another use is the application of the polymerase chain reaction (PCR) using a single primer.
The invention and its several embodiments are described in greater detail hereinafter.
By the method of the invention there is produced a ssDNA molecule whose structure comprises a stem portion of duplexed DNA of annealed complementary bases within the ssDNA, which stem forms, at one of its ends, the 5' and 3' termini of the ssDNA molecule and, at the other end, a loop, which is constituted of a single-strand of non-annealed bases which joins the two single-strands of the stem. In a specific embodiment, strand ending in the 3' terminus is longer than the other strand. The slDNAs may include DNA segments capable of encoding a protein, more particularly any gene(s). The gene will be located between the loop and the termini. The gene may be a gene carrying a mutation(s).
A mechanism postulated for the synthesis of slDNAs is illustrated in FIGS. 5A and B. The synthesis is believed to involve, in summary, the following course: DNA is initiated at the origin of replication (OR), replication of a first strand (or "the" or "a" strand) then proceeds using one of the strands of the double-stranded DNA as template (by the same mechanism as chromosomal DNA replication (see Tomizawa et al., Proc. Natl. Acad. Sci. USA, 74, 1865 (1977)), proceeding through the IR structure resulting in the replication of the entire plasmid genome when a plasmid is used. However (as is described in greater detail hereinafter), part of the first strand forms a loop structure as the first strand synthesis is disrupted or terminated within the IR (FIG. 5, from steps 2 to 3). The loop forms a short duplexed region which functions as priming site for continuing DNA synthesis. DNA chain elongation resumes from the newly formed 3' end, now forming the second strand (or "the other" strand) utilizing the nascent first strand as a template. An alternative (or concurrent) postulated synthesis course is described hereinafter. Thus, the direction of DNA synthesis is reversed by template switching to duplicate the DNA fragment (FIG. 5, from steps 4 to 5). The newly synthesized slDNA molecule dissociates itsef from the parent template strands which undergo another round of replication to form another slDNA. The slDNA is isolated, if necessary, purified.
In another embodiment of the invention, there is provided a DNA self-replicating vehicle, e.g., a plasmid into which there has been inserted a DNA fragment which contains an inverted repeat (IR) structure, the DNA fragment which will serve as template for synthesis of the ssDNA and a suitable priming site, e.g., an origin of replication (OR), such as the E. coli origin of replication (ColE1). The IR is situated downstream of the OR. The self-replicating vehicle is replicated in a suitable host, e.g., an E. coli. The host will contain at least one DNA polymerase to synthesize the ssDNA from the template. In this specific embodiment, it is presumed that there are two polymerases, a first DNA polymerase contributing to the elongation of a portion of the ssDNA from the OR to the IR, the first strand, and a second DNA polymerase which synthesizes the balance or second strand of the ssDNA strand. As the synthesis of the first strand terminates, complementary bases anneal to form a single-stranded non-annealed loop. The synthesis of the second strand then takes place. A new DNA structure is synthesized which is constituted of a duplexed DNA stem and a single-stranded loop structure at the opposite end. For this new DNA structure the term "stem-loop" or "slDNA" has been coined.
Another specific embodiment provides a replicating vehicle in which a promoter, specifically the lac promoter-operator has been deleted. An slDNA was nonetheless produced, supporting the synthesis model proposed, as described further herein.
The plasmid may be constructed to contain any selected DNA sequence capable of encoding a protein between the priming site and the IR in which event the slDNA synthesized will contain the DNA sequence or a mutation thereof.
The slDNAs of the invention need not be synthesized by a self-replication vehicle, but may be synthesized in an appropriate in vitro system constituted of any segment of DNA, linear or not, which contains any IR and the necessary elements to synthesize the ssDNA. Such system is described hereinafter.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A Illustrates a plasmid of the invention, pUCK106.
FIG. 1B Shows the DNA sequence of 215-bp (SEQ ID NO:6) inserted at the XbaI site of pUCK19.
FIGS. 2A, 2B and 2C show an ethidium bromide staining of polyacrylamide gel of the production of an slDNA from pUCK106 and its characteristics.
FIGS. 3A, 3B and 3C show an autoradiograph of a dried polyacrylamide gel of dimer formation of the slDNA from pUCK106.
FIGS. 4A, 4B, 4C, 4D and 4E illustrate determination of the DNA sequence of the slDNA from pUCK106.
FIG. 4A shows an autoradiograph of a dried polyacrylamide gel of the determination of the DNA sequence of the 5' end region of the slDNA (SEQ ID NO.2).
FIG. 4B shows an autoradiograph of a dried polyacrylamide gel of the 5' end sequencing of the loop region of the slDNA (SEQ ID NO.3).
FIG. 4C shows an autoradiograph of a dried polyacrylamide gel of the 3' end sequencing of the loop region of the slDNA (SEQ ID NO.3).
FIG. 4D shows an autoradiograph of a dried polyacrylamide gel of the DNA sequence of the 3' end region of the slDNA (SEQ ID NO.4).
FIG. 4E shows the structure of the slDNA from pUCK106.
FIGS. 5A and 5B illustrates two possible models of slDNA synthesis.
FIG. 6, lanes 1 and 3 show the HaeIII digest of pBR322 as size markers. Lane 2 shows the slDNA from the preparation from pUC7.
FIG. 7 shows the gene for .beta.-galactosidase in a transformed vector.

DEPOSIT OF GENETIC MATERIAL
Plasmid pUCK106 has been deposited on Aug. 28, 1991 in accordance with the Budapest Treaty with the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Md. 20852 under Accession No. 68679.
Plasmid pUCK106.DELTA.lac.sup.PO has been deposited on Aug. 28, 1991 in accordance with the Budapest Treaty with the ATCC under Accession No. 68680.
EXAMPLES
The following examples are offered by way of illustration and are not intended to limit the invention in any manner. In these examples, all percentages are by weight if for solids and by volume for liquids, and all temperatures are in degrees Celsius unless otherwise noted.
For convenience and clarity, the Examples refer to and provide also a detailed description of the Figures.
Example 1
FIG. 1A illustrates pUCK19 (circular map), a specific plasmid made as shown below. The open bar in the circular map represents the kanamycin-resistant gene (from Tn 5). The straight open bar shown at the upper right hand side represents a 215-bp DNA fragment (SEQ ID NO:1) inserted at the XbaI site, which contains 35-bp inverted repeat (IR) sequences. Solid arrows show the IR structure. The solid circle design is the origin of replication (Ori). The longer open arrow indicates the direction of DNA replication from the OR. The smaller open arrow shows the position of the lac promoter-operator (lac.sup.PO).
FIG. 1B shows the DNA sequence of 215-bp inserted at the XbaI site of pUCK19. This plasmid is designated herein as pUCK106. The open arrows indicate the IR sequences. The HindIII (AAGCTT) site shows the center of the IR.
Mismatched positions in the IR are shown by two open spaces in the arrows with mismatched bases C and T inserted in the spaces.
The DNA fragment discussed above (which contains the IR) has the following sequence (SEQ ID NO.1): ##STR1##
Example 2
This example illustrates the production of an slDNA from pUCK106 and its characteristics.
(A) E. coli CL83 was transformed with either pUCK19, pUCK106 and with pUCK106.DELTA.lac.sup.PO and a plasmid DNA fraction was prepared. A DNA preparation (after ribonuclease A treatment) was applied to a 5% acrylamide gel for electrophoresis. The gel was stained with ethidium bromide. Lane 1 shows the HaeIII digest of pBR322 as size markers. The numbers shown are base pairs; lane 2, the DNA preparation from cells harboring pUCK19; lane 3, pUCK106; and lane 4, pUCK106.DELTA.lac.sup.PO.
pUCK19 is a kanamycin resistant variant of pUC19.
(B) The slDNA from pUCK106 was purified by polyacrylamide gel electrophoresis followed by various restriction enzyme digestion. The digests were analyzed by polyacrylamide (5%) gel electrophoresis, and the gel was stained by ethidium bromide. With reference to FIG. 2, lane 1, the HaeIII digest of pBR322 as size markers; lane 2, slDNA without digestion; lane 3, slDNA digested with XbaI; lane 4, with HindlII; and lane 5, with PvuII.
(C) Heat-denaturation of the slDNA from pUCK106. The purified slDNA (as described above) was solubilized in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The slDNA solution was incubated in a boiling water bath for 3 minutes and quickly chilled in an ice bath. Samples were analyzed as described in A. With reference to FIG. 2, lane 1, the HaeIII digest of pBR322 as size markers; lane 2, slDNA without heat treatment; and lane 3, slDNA heat-denatured followed by quick cooling.
Example 3
This example illustrates dimer formation of the slDNA from pUCK106.
(A) The purified slDNA from pUCK106 as described in FIG. 2 was solubilized in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM MgCl.sub.2. The slDNA solution was incubated in a boiling water bath for 3 minutes and then gradually cooled. The renatured slDNA was digested with XbaI and the DNA fragments thus generated were labeled at their 5' ends with [.gamma.-.sup.32 P]ATP and T4 polynucleotide kinase. These products were applied to a 5% polyacrylamide gel. After electrophoresis, the gel was dried and subjected to autoradiography.
With reference to FIG. 3, lane 1, the HaeIII digest of pBR322 as size markers; lane 2, the EcoRI and HindIII digest of .lambda.DNA as size markers. Numbers shown are in base pairs; lane 3, the slDNA from pUCK106 without treatment; lane 4, the XbaI digest of the untreated slDNA; lane 5, the slDNA after heat-denaturation followed by gradual cooling; and lane 6, the XbaI digest of the slDNA from lane 5. Bands are marked from "a" to "e" at the right hand side.
(B) Characterization of fragment "d" in FIG. 3A. Fragment "d" purified from the gel; lane 3, the HindIII digest of the purified fragment "d"; and lane 4, the purified fragment "d" was heat-denatured and quickly chilled as described with respect to FIG. 2.
(C) Schematic representation of bands "a" to "e" shown in A and B. X and H represent XbaI and HindIII sites, respectively. These are two lother HindIII sites in fragment "a" very close to the XbaI sites (within fragment "c"). These HindIII sites are not shown.
Example 4
This example illustrates determination of the DNA sequence of the slDNA from pUCK106.
(A) Determination of the DNA sequence of the 5' end region of the slDNA. 0.2 .mu.g of the isolated and purified slDNA was used for sequencing by the chain termination method. Primer "a" (.sup.5' GGTTATCCACAGAATCAG.sup.3') (SEQ ID NO.5) which corresponds to the sequence 96-bp downstream from the origin (see FIG. 4B) and SEQ ID NO.3 was used as primer.
(B) 5' end sequencing of the loop region of the slDNA. 0.5 .mu.g of the slDNA was digested with SacII and the DNA fragments thus generated were labeled at the 5' end with [.gamma.-.sup.32 P]ATP and T4 polynucleotide kinase. The DNA fragment migrated at approximately 40-bp was isolated and sequenced by the Maxam-Gilbert method.
(C) 3' end sequencing of the loop region of the slDNA. The SacII digest slDNA was labeled at the 3' end with [.gamma.-.sup.32 P]dideoxyATP using terminal deoxynucleotidyl transferase. The DNA fragment containing the loop region was isolated and sequenced by the Maxam-Gilbert method.
(D) DNA sequence of the 3' end region of the slDNA. The slDNA was digested with AflIII (see FIG. 4E). The 5' ends were labeled with [.gamma.-.sup.32 P]ATP and T4 polynucleotide kinase. The labeled products were separated by a sequencing gel. The single-stranded DNA which migrated at 76 bases was isolated and sequenced by Maxam-Gilbert method. By reference to FIG. 4D the numbers represent the residue numbers from the origin of pUCK19.
(E) Structure of the slDNA from pUCK106. The slDNA consists of a single-stranded DNA of 1137 to 1139 bases. The 5' end of the slDNA appears to be heterogeneous; some start from +1 while other start from -1, +2 and +3. The +1 position corresponds to the origin of ColE1 DNA replication. Thus, various slDNAs have different length 5' ending strands. At the 3' end a sequence of 16 bases is extended beyond the +1 position of the 5' end. The loop is considered to be formed with the 4 base sequence (AGCT) corresponding to the sequence at the center of the IR structure, where a HindIII site (AAGCTT) is designed to be placed. The base pair corresponding to the mismatch in the IR structure in pUCK106 was converted from C.cndot.T (in pUCK106) to C.cndot.G (in the slDNA) is shown between the SacII and PstI sites. The position of primer "a" used for DNA sequencing in FIG. 4A is shown by an arrow.
Separation and purification of the slDNAs are performed according to standard techniques as by following the procedures described in Molecular Cloning, A Laboratory Manual, Sambrook et al., 2d Ed. (Sections 1.121-1.40) ("Sambrook").
Example 5
This example illustrates two possible models of slDNA synthesis. The double-stranded DNA around the origin of the ColE1 DNA replication is shown on the top. The shaded circle represents the DNA replication complex which initiates DNA replication from the origin. The open arrows on the DNA strand indicate the position of the 35-bp inverted repeat (IR) structure (see FIG. 1B) and SEQ ID NO.1 in the DNA sequence. The mismatched base pair (C.cndot.T) in the IR structure is also indicated within the arrows.
At step 1, the DNA replication fork proceeds from the origin (+1 position) to the position indicated by the shaded circle. The newly synthesized first strand is shown extending from the origin (a solid circle) to the replication fork. The DNA replication complex reaches immediately before the mismatched T residue in the IR structure that is shown by solid arrows. At step 2, the 3' end of the nascent strand detaches from the DNA replication complex and a secondary structure is formed by the IR structure. At step 3, DNA synthesis reinitiates from the 3' end of the stem-loop structure utilizing either the nascent strand (model A) or the upper parental strand (model B) as template. At step 4, DNA synthesis proceeds beyond the origin by 16 bases.
In model A, the primer RNA which remains attached at the 5' end of the DNA may be used as the primer RNA. Subsequently, the RNA may be removed resulting in the formation of slDNA. In model B, DNA synthesis terminates at the terH site by a similar mechanism known for the termination of the second strand DNA synthesis.
It is conceivable that both models A and B can explain the synthesis, and the synthesis may proceed by both routes concurrently, at least for part of the time. Thus, an appropriate template will be used for the second strand other than the strand which was the template for the first strand.
Example 6
Construction of pUCK106.DELTA.lac.sup.PO
When the 199-bp PvuII-HincII fragment containing the lac promoter-operator was deleted from pUCK106 (see FIG. 1A), the resulting pUCK106.DELTA.lac.sup.PO produced a new slDNA which migrated faster than the slDNA from pUCK106 as shown at position (b) in lane 4, FIG. 2A. The size of this new slDNA was 360-bp in length, which is shorter than the pUCK106 slDNA by a length nearly identical to the size of the deletion in pUCK106.DELTA.lac.sup.PO.
In FIG. 2A, lane 3 shows the slDNA from the DNA preparation from cells harboring pUCK106.DELTA.lac.sup.PO.
This experiment supports the model for slDNA synthesis proposed above and also indicates that the lac promoter-operator is not essential for slDNA synthesis. This notion was further supported by the fact that the addition of isopropyl-.beta.-D-thiogalactopyranoside, an inducer of lac, did not affect the production of the slDNA from pUCK106. However, the reason for the reduction of slDNA synthesis from pUCK106.DELTA.lac.sup.PO is not known at present.
Example 7
The synthesis of slDNA was not dependent upon the primary sequence of the IR structure used for pUCK106. Interestingly, the pUC7 vector by itself, which has an IR structure at the polylinker site is also able to produce an slDNA corresponding to the DNA fragment from the origin to the center of the polylinker site.
The isolated and purified slDNA produced from pUC7 was 252-bp in length. A plasmid fraction was prepared and treated as in Example 2 and applied (and stained) to acrylamide gel for electrophoresis.
In FIG. 6, lanes 1 and 3 show the HaeIII digest of pBR322 as size markers. Lane 2 shows the slDNA from the preparation from pUC7.
The slDNA of pUC7 was amplified by PCR (which is described in further detail herein) and lane 4 (at arrow) shows the slDNA.
Example 8
Confirmation of the slDNA Structure
The slDNA structure and mechanism described above (illustrated in FIGS. 5A and 5B) was confirmed as follows.
The DNA fragment that was synthetically constructed was provided intentionally with mismatched bases CT for CG. See FIG. 1B in the open spaces of the IR. After synthesis of the slDNA (with the second strand snapped-back over the first strand) the mismatch has been repaired. FIG. 4E, now CG appears. If the structure were not a snap-back structure, the polymerase would have read right through the IR, would have read the T and inserted an A; the new strand would still have contained the mismatch. To replace the mismatched T, that IR portion necessarily had to snap-back onto the first strand, thus allowing the polymerase to use the first synthesized strand as template to synthesize the second strand and as it synthesizes it, insert the complementary base G in place of the mismatched T. This unequivocally establishes the synthesis mechanism and structure of the slDNAs of the invention.
DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION
The inventors have made the fundamental discovery that part of the genome is directly duplicated from the genome. The mechanism discovered requires neither an RNA intermediate nor reverse transcriptase (RT) as is well known. See Weiner et al., Ann. Rev. Biochem., 55, 631 (1986); Kornberg, in DNA Replication (W. H. Freeman and Company, San Francisco, Calif., 1980), pp. 101-166. New and useful DNA structures named stem-loop DNAs (slDNAs) have been discovered.
By way of introduction, the invention provides several embodiments. One embodiment is a method (or process) for synthesizing a novel and useful ssDNA molecule (or structure). Another embodiment is an in vivo and in vitro system for synthesizing such molecule which includes a DNA fragment which contains a suitable priming site, an inverted repeat (IR) and other necessary components for synthesizing the slDNAs.
An in vivo system for synthesizing such molecules uses a competent self-replicating vehicle and other components of the system. Another aspect of this embodiment is the self-replicating vehicle which contains an inverted repeat, a DNA to serve as template for the replication of a first strand of the slDNA, a suitable priming site for the template to start DNA synthesis in the opposite orientation and when needed, the second strand of the parent DNA and other components further described.
Another embodiment is the novel ss-slDNA molecules.
The invention provides a method for synthesizing a novel single-stranded DNA (ssDNA) molecule. The molecule comprises a stem-loop structure (slDNA) which stem is constituted of duplexed DNA of annealed complementary bases within the ssDNA, which stem forms, at one end, the 5' and 3' termini of the ssDNA molecule and at the other end, a single-stranded loop of DNA joining the opposite ends of the duplexed DNA. The method is carried out in a system which contains the conventional components of DNA synthesis, and the following components:
(a) a template DNA containing a suitable priming site and an inverted repeat (IR) downstream of said priming site,
(b) a primer for the template to allow the start of DNA polymerization, and
(c) a DNA polymerase to replicate the slDNA from the template.
The method comprises the steps of:
(1) priming the DNA template to allow the start of DNA polymerization,
(2) synthesizing the ssDNA from the primer using one of the double-strand of the DNA as template thereby forming one strand, continuing the DNA synthesis of the strand into the IR sequence and allowing the synthesis to cease within the IR sequence,
(3) allowing complementary bases within the newly synthesized strand at the IR sequence to anneal forming, at one end, a loop of a non-duplexed region and a duplexed region, the duplexed region functioning as a priming site for continued DNA synthesis,
(4) resuming DNA synthesis using as template the newly synthesized strand and/or the other strand of the DNA,
(5) forming the slDNA, and
(6) separating and isolating the slDNA.
The method is carried out in a system which contains all the necessary conventional components for DNA synthesis. These components may be present inherently as when the method is carried out in vivo; they will normally be introduced into the system when the method is carried out in vitro.
The method calls for the presence of a DNA which has a suitable priming site and an inverted repeat downstream of the priming site. The DNA serves as a template for directing the DNA replication. The primer can be any oligonucleotide (whether occurring naturally or produced synthetically) which is capable of acting to initiate synthesis of a primer extension product which is complementary to a nucleic acid strand. The method of initiation of DNA synthesis is of course well known. See Watson, Molecular Biology of the Gene, 3rd Ed., W. A. Benjamin, Inc.; DNA Synthesis: Present and Future, Molineux and Kohiyama, Eds., (1977) Part V, "G4 and ST-1 DNA Synthesis In Vitro" by Wickner; Part VII, "DNA Synthesis in Permeable Cell Systems from Saccharomyces cerevisiae" by Oertel and Goulian. Different polymerases may contribute to the synthesis of the second strand, as opposed to that contributing to the synthesis of the first strand. The primer may be a DNA or RNA primer. Synthesis is induced in the presence of nucleotides and an agent of polymerization, such as DNA polymerase at a suitable temperature and pH.
The method of the invention uses an agent for polymerization such as an enzyme, which catalyzes the synthesis of the strand along the template. Suitable enzymes for this purpose include, for example, any DNA polymerase like E. coli DNA polymerase I, or III, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, reverse transcriptase (RT), vital polymerases, including heat-stable enzymes. Any polymerase that will recognize its site of initiation for DNA replication is appropriate. Generally, when the process is carried out in vivo, these genetic elements will be present; if not or if performed in vitro, they will be added to the system.
Any polymerase that will recognize the site of initiation may cause or contribute to the polymerization. While the inventors do not wish to be bound to any particular theory at this time, it is not to be excluded that the polymerase that contributed to the synthesis of the first DNA strand also contributes to synthesize the other or second DNA strand. The method thus provides continuing the synthesis of the second strand until it terminates at terminus 3' beyond the 5' terminus of the first formed strand. Thus, there is formed the duplexed stem of the slDNA.
Information on DNA polymerases is available. For instance, see Kornberg, in DNA Replication (W. H. Freeman and Company, San Francisco, Calif., 1980), pp. 101-166, Chapter 4, DNA Polymerase I of E. coli, Chapter 5, Other Procaryotic Polymerases, Chapter 6, Eucaryotic DNA Polymerases and Chapter 7.
Other polymerases, such as those that are useful in second-strand synthesis in cDNA may be considered.
In a specific illustration described above, it is postulated that the polymerases are two: DNA polymerase III and polymerase I.
When it is desired to replicate a desired or target nucleic acid sequence which is capable of encoding a protein, e.g., a gene as part of the synthesized slDNAs, the sequence will be positioned upstream of the IR. For example, when the replication takes place in a replication vehicle like a vector, e.g., pUCK106, which has an origin of replication (OR), the target nucleic acid sequence will be positioned in between the IR and the OR.
The method of the invention uses a double-stranded DNA fragment which has, as described, a priming site and also an inverted repeat (IR), i.e., two copies of an identical sequence present in the reverse orientation. The IR may be present as a sequence or "palindrome".
As will be described hereinafter, certain enzymes will be preferred over others, such as RT when it is desired to favor introducing random point mutations in a nucleic acid sequence.
The synthesis of the first strand proceeds in a contiguous manner along the dsDNA template through the IR resulting in the replication of the entire plasmid genome. At a certain frequency, the synthesis of the DNA strand is ceased within the IR, forming a short region of sequence which is complementary to itself, forms a loop and at the IR sequence anneals to itself. This double-stranded region within the newly synthesized strand is recognized as the priming site for the second or other DNA strand. Synthesis of the second strand starts using the first formed strand and/or the other parent strand of the DNA as template. Thus, template switching is believed to occur.
As the second strand is synthesized by the polymerase incorporating the nucleotides, the stem is formed of the annealed complementary bases resulting in its duplexed structure with internal complementarity.
The synthesis of the second strand proceeds all the way past the first nucleotide of the first synthesized strand through the RNA primer of this first strand which eventually degrades, thus providing a 3' overhang. In one specific illustration, the DNA synthesis is believed to terminate at the terH site by a mechanism similar as described in Dasgupta et al., Cell, 51, 1113 (1987).
By appropriate manipulations the length of the 3' end overhang can be controlled, e.g., lengthened, as by moving the terH site downstream from the priming site.
Further, instead of a stem with a 3' end overhang, it is considered feasible to block the synthesis of the second strand before the end of the first strand by placing an appropriate termination site, e.g., terH upstream of the priming site. Thus, it is contemplated that either strand can be longer by a predetermined length with respect to the other.
As the synthesis of the second strand ceases, the template and the formed slDNA separate.
The method of the invention may be repeated in cycles as often as is desired. If any of the necessary components are becoming depleted, they are resupplied as it becomes necessary.
When the method is carried out in vivo there is provided a suitable competent replicating vehicle which carries the necessary template DNA fragment having a priming site and an IR downstream of the priming site. The DNA fragment carrying the IR will normally be inserted into a restriction site unique in a polylinker sequence. A plasmid has been used in which the polylinker has an IR (and symmetrical restriction sites). When not inherently present, the DNA fragment will be provided with a primer to the DNA sequence; the polymerase may be indigenous to the vehicle or not. The vehicle will contain all other necessary elements for replication and forming the slDNAs.
From the foregoing it will be understood that any self-replicating vector which contains a DNA fragment to serve as template, an IR sequence, the necessary elements to prime and continue the replication of the strand that will form the slDNA, is suitable to synthesize the slDNAs.
Another major embodiment of the invention provides the new ss-slDNAs. These structures have already been described hereinabove. Additional description is provided hereinafter.
The new structure which has been named "stem-loop" or "slDNA" is single-stranded. An illustration of an slDNA is shown in FIG. 4E (from pUCK106) and FIG. 6 (from pUC7).
Typical slDNAs contain a duplexed double-stranded stem of annealed complementary bases and a loop of a single-strand of nucleotides connecting the two strands of the stem. The single-strandedness of the loop is another interesting feature of the slDNAs of the invention. slDNAs can include loop of a very short sequence of nucleotides or considerably longer ones. The slDNAs may contain a nucleotide sequence just long enough to form the loop and base pairing forming a short duplexed double-strand. The minimum size should allow for base pairing enough to provide for priming site for the start of synthesis of the second strand. The minimum size of the loop may be limited by the strains on the bases that would prevent their pairing into a stable structure. The maximum size is influenced by the use intended for the slDNAs. The loop illustrated in FIG. 4B is constituted of four bases; loops of 10, 20 or more bases can be conceived. The single-strandedness of the loop of the slDNAs is a feature which may be quite useful in the utilities proposed for the slDNAs.
Yet another interesting structure contemplated for the slDNAs is the double slDNAs. In this structure, the free ends of the single-strands of two slDNAs are ligated. The structure is expected to be extremely stable. On exposure to conditions which normally denature DNAs, these ssDNAs are likely to "snap-back" to their original structure. Joining of the strands will be carried out by conventional procedures with DNA and/or RNA ligases. Such structures can also carry selected genes for encoding proteins and thereby thus provide interesting new practical possibilities.
It is believed that the stability, an important property of the slDNAs, tends to increase with longer duplexed tails; thus, such structures are favored when this is a property which is to be emphasized. It should be noted that all slDNAs generated from a single replicating vehicle are not necessarily identical in size. In the illustration shown above, slDNA from pUCK106, the 5' end of the first strand appears to be heterogeneous, some strands starting from the +1 position (which corresponds to the origin of ColE1 replication) (see Tomizawa et al., Proc. Natl. Acad. Sci. USA, 74, 1865 (1977)), while other strands start from -1, +2 and +3. Thus, the DNAs may be considered as family of analogous slDNAs.
It may be noted that the presence of one or more mismatch in the DNA fragment beyond the IRs does not adversely affect the synthesis nor the structure of the slDNAs. This is illustrated by the mismatch T for G, in this case 25 nucleotides away from the center of the palindrome (see FIG. 1B and SEQ ID NO:1). This mismatch was repaired in the synthesis of the slDNA.
Several utilities for the slDNAs of the invention are proposed herein which take advantage of the ssDNA overhang of one end of the tail over the other. It will therefore be appreciated that this is an important feature of the new structures of the invention.
The synthesis of the slDNAs in the illustrated plasmid is descriptive of a best mode of the invention. However, any vector which contains the IR and other components described herein is suitable for synthesizing the slDNAs.
IR is a structure frequently occurring in procaryotes and in eucaryotes. Any such IR may be used in the invention. IR sequences may also be prepared synthetically. An illustration is the IR synthesized and shown in FIG. 1B.
Sequences of IR or palindrome sequences have been obtained from E. coli (Gilson et al., Nucl. Acids. Res., 18, 3941 (1990)); Gilson et al., Nucl. Acids Res., 19, 1375 (1991) reported palindromic sequences from E. coli and Salmonella enteritica (a palindromic unit sequence 40 nucleotides long). Chalker et al., Gene, 71, (1):201-5 (1988) reports the propagation of a 571-bp palindrome in E. coli; inverted repeats are reported by Lewis et al., J. Mol. Biol. (England), 215, (1):73-84 (1990) in Bacillus subtilis (a 26-base pair repeat), and Saurin, Comput. Appl. Biosci., 3, (2):121-7 (1987) discusses the use of a new computer program to search systematically for repetitive palindromic structures in E. coli. The following U.S. patents disclose palindromic sequences: U.S. Pat. Nos. 4,975,376; 4,863,858; 4,840,901; 4,746,609; 4,719,179; 4,693,980 and 4,693,979.
Palindromes have been defined to include inverted repetitious sequences in which almost the same (not necessarily the same) sequences run in opposite direction. Though some are short (3-10 bases in one direction), others are much longer, comprising hundreds of base pairs. Watson, Molecular Biology of the Gene, 3rd Ed., pps. 224-225.
The IR in the DNA fragment can vary considerably in size. Without intending to be limited slDNAs with inverted repeats of 10 to 30 or longer nucleotides may be considered. Inverted repeats have been reported to contain more than 300-bp. Current Protocols., Section 1.4.10.
The slDNAs can be the synthesis product of procaryotic or eucaryotic host expression (e.g., bacterial, yeast and mammalian cells).
Examples of appropriate vectors such as a plasmid and a host cell transformed thereby are well known to one skilled in the art. Among appropriate hosts for plasmid carrying the necessary components are procaryotes and eucaryotes. Procaryotes include such microorganisms as those of the genus Escherichia, in particular E. coli; of the genus Bacillus, in particular, B. subtilis.
Plasmids capable of transforming E. coli include for example, the pUC and the ColE1 type plasmids. See U.S. Pat. No. 4,910,141. Plasmids capable of transforming E. coli include for example, ColE1 type plasmids in general. Other appropriate plasmids for transforming E. coli include: pSC101, pSF2124, pMB8, pMB9, pACYC184, pACYC177, pCK1, R6K, pBR312, pBR313, pML2, pML21, ColE1AP, RSF1010, pVH51, and pVH153.
Plasmids capable of transforming B. subtilis include: pC194, pC221, pC223, pUB112, pT127, pE194, pUB110, pSA0501, pSA2100, pTP4, pTP5 and their derivatives. Plasmids capable of transforming both B. subtilis and E. coli are described in J. Bacteriol., 145, 422-428 (1982); Proc. Natl. Acad. Sci. USA, 75, 1433-1436 (1978) and Principles of Gene Manipulation 2nd Ed., Carr et al. Eds., University of Ca. Press, Berkeley, 1981, p. 48.
Of special interest for carrying out the synthesis of the slDNAs in eucaryotes are plasmids capable of transforming S. cerevisiae: pMP78, YEp13, pBTI1, pLC544, YEp2, YRp17, pRB8 (YIp30), pBTI7, pBTI9, pBTI10, pAC1, pSLe1, pJDB219, pDB248 and YRp7. Also to be considered are YIp5, pUC-URA3, pUC-LEU2 and pUC-HIS3. See page 285 and pages 373-378 in Methods in Enzymology, Vol. 194, "Guide to Yeast Genetics and Molecular Biology", edited by Guthrie and Fink (1991), Academic Press, Inc. Other yeast vectors are described at pages 100-104 in Experimental Manipulation of Gene Expression, edited by Masayori Inouye, Academic Press, Inc. (1983).
Further, of particular interest are shuttle vectors which can be used to transform E. coli as well as yeast like S. cerevisiae. Such vectors include the following: pKB42 and pYC1. Other examples are listed in the section on "Cosmid Vectors for Low and Higher Eucaryotes" in A Practical Guide to Molecular Cloning, 2nd Edition by Bernard Perbal (1988), Wiley and Sons. Other suitable vectors are described in Vol. 2, Sections 13.4.1, 13.4.2 (1989), Current Protocols. Other suitable vehicles include such popular multicopy vectors like YEp24 (Botstein et al., Gene, 8, 17 (1979)) and pJDB207 (Beggs, Genetic Engineering (Ed. Williamson), Vol. 2, p. 175, Academic Press (1982)). Others that may be selected include plasmids of the classes YIp, like YEp51 and YEp52.
Examples of commercially available eucaryotic vectors for carrying out the present invention are pSVL and pKSV-10 in for example, COS, CHO and HeLa cells. Other examples are listed in A Practical Guide to Molecular Cloning.
Culturing and fermentation of the transformed hosts is carried out by standard and conventional methods known in the art. See for example, Methods in Enzymology, Vol. 185, Gene Expression Technology (Goeddel, editor) 1990 (in particular, Growth of Cell Lines); for yeasts, see Methods in Enzymology, Vol. 194, Guide to Yeast Genetics & Molecular Biology; growth conditions for E. coli are described in Current Protocols in Molecular Biology, Vol. 1, at pages 1.1.1, 1.1.2, 1.1.3, 1.1.4 and 1.3.1 and Molecular Cloning: A Laboratory Manual, 2nd Edition at page 1.21 and purification of plasmid DNA is described at page 1.23, culturing growth conditions suitable for mammalian cells are described in Current Protocols, Vols. 1 and 2 at pages 9.0.4-9.0.6, 9.1.1, 9.1.3, 9.2.5, 9.4.3, 11.5.2, 11.6.2 and 11.7.3.
In summary, when the necessary components for initiating and the synthesis of the single first strand are supplied in a replicating vehicle, namely, a DNA template fragment with an initiation site, and an IR (and the polymerase(s)), a slDNA is expected to be formed.
When the synthesis of the slDNAs of the invention is performed in vitro, the synthesis will be performed in a medium which includes conventional components for the synthesis of nucleotide strands using the DNA fragment as template. Generally, the synthesis will take place in a buffered aqueous solution, preferably at a pH of 7-9. Preferably, a molar excess of the oligonucleotides over the DNA template strand. The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are also a component of the synthesis mixture. Heating and then cooling of the solution is performed. The polymerase is then added and the synthesis proceeds. These components are called "conventional components" for the purpose of the invention described herein.
Oligonucleotide synthesis may be carried out by a number of methods including those disclosed in U.S. Pat. No. 4,415,734, and in Matteuci et al., J. Am. Chem. Soc., 103 (11):3185-3191 (1981), Adams et al., J. Am. Chem. Soc., 105 (3):661-663 (1983), and Bemcage et al., Tetrahedron Letters, 22 (20):1859-1867 (1981).
The methods of the present invention make use of techniques or genetic engineering and molecular cloning. General techniques of genetic engineering and molecular cloning are included in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, 1990, A Practical Guide to Molecular Cloning, 2nd Ed., Bernard Perbal (1988), Methods in Enzymology, Volume 68, Recombinant DNA (Wu, editor), Academic Press, New York, 1979, Methods in Enzymology, Volume 185, Gene Expression Technology (Goeddel, editor) 1990, Current Protocols in Molecular Biology, Vols. 1, 2 and 3.
The slDNAs of the invention have several interesting utilities. The method of the invention may be used to provide random mutations in a selected gene. Such a system is illustrated in FIG. 7 which shows the gene for .beta.-galactosidase in a transformed vector.
The method comprises synthesizing an slDNA containing a gene of interest located in the stem, isolating the slDNA, cutting out the gene from the slDNA, cloning it into an appropriate replicating vehicle and expressing the protein encoded by the gene. The proteins can be tested for the desired activity. By standard procedures the colonies can be screened to identify those with mutated genes.
An illustration for generating and identifying mutations proceeds as follows. Into pUCK19 (See Example 1) which harbors the lacZ gene, there is ligated the 215-bp DNA fragment containing the 35-bp IR (described in Example 1) between the DNA fragment and the OR (as shown in Example 1). The plasmid is transformed into competent E. coli CL83 which is grown under standard conditions. Thereafter, the lacZ gene is isolated and inserted into pUCK19 (without the IR-containing fragment). pUCK19 is digested with KasI and EcoRI and the isolating lacZ gene is transformed into E. coli CL83. The frequency of mutation is scored by the known in vivo test using .beta.-gal, a colorless substrate, which is hydrolyzed to give a dark blue product. When the colonies are colorless, this indicates that no .beta.-galactosidase has been produced.
Other genes of lac gene family, e.g., lacY or lacA, or other suitable genes can be used for this screening test. The gene encoding the protein (or polypeptide) of interest may also be used to determine the frequency of mutations and selection of appropriate gene encoding the derived protein (or polypeptide).
This screening procedure permits selection of other polymerases that are more likely to introduce or increase the rate of mutation in the target gene. Thus, a selected gene such as the lacZ gene can be used as the screening gene. Reverse transcriptase known to have less replication fidelity, appears to be an enzyme of choice for the purpose of introducing mutations in a target gene encoding a desired protein.
The desired target protein(s) is then expressed by a competent selected transformed host carrying the mutated gene and selected for its desired biological properties.
The frequency of mutation is believed to be influenceable by selecting appropriate enzymes that are known to have less fidelity in replication. Thus, when target DNA fragments or genes are amplified, the system depends on the degree of replication fidelity or the infidelity, that is, the frequency of error made by the DNA polymerases in replicating the inserted DNA fragment or gene. Thus for each replication error, random mutations are introduced into the genes. The higher the degree of infidelity of the DNA polymerase, the greater the number of mutated genes, and vice versa.
In one above-illustrated embodiment of the invention in which it is believed the PolIII and PolI were active, it is expected that the former has less fidelity, since PolI is known to have a self-correcting function. Thus, the replicating fidelity of the system can be regulated by appropriate selection of the DNA polymerases. Generally, it is believed that random mutations are more likely to be introduced by the polymerase synthesizing the second strand. An interesting candidate would be RT.
Genes carrying the desired mutation(s) are useful in chromosomal cross-over. By this method the genes of interest or the slDNA carrying the mutated gene of interest can be made to integrate and exchange genetic information from one similar molecule to another. The mutated gene locates within the genome a sequence that is similar to the vector sequence and the homologous gene is replicated by the mutated gene.
In this manner there can be generated new strains of microorganisms (or hosts) that contain the mutated gene and will express a desired protein. The slDNA carrying the mutated gene can be made in vitro or in vivo.
The polymerase chain reaction (PCR) is a rapid procedure for in vitro enzymatic amplification of a specific segment of DNA. The standard PCR method requires a segment of double-stranded DNA to be amplified, and always two-single stranded oligonucleotide primers flanking the segment, a DNA polymerase, appropriate deoxyribonucleoside triphosphate (dNTPs), a buffer, and salts. See Current Protocols, Section 15.
The ss-slDNA of the invention can be amplified with a single primer. This feature considerably simplifies the amplification, helps to overcome the problem of one primer finding its proper initiation site, renders it more economical and helps overcome problems associated with the traditional PCR method.
The amplification of the slDNA comprises denaturing the slDNA to form a single-stranded DNA (from 3' to 5' ends). The reaction follows the usual sequence: priming from 3' end, polymerase reaction, denaturing and annealing. It is carried out for 25 cycles leading to million-fold amplification of slDNA. The slDNA can be carrying a gene for encoding a target protein.
A recent report in Science, 252, 1643-1650 (Jun. 21, 1991) entitled "Recent Advances in the Polymerase Chain Reaction" by Erlich et al. discusses problems associated with primers and improvements that are being proposed to the PCR method.
Accordingiy, a method for amplification of the ss-slDNA structures of the invention by a method using one primer is of great interest. The slDNA could be carrying a gene of interest, such as a mutated gene having improved biological properties.
The in vivo production of the slDNAs of the invention can be so manipulated to provide a desired sequence. The slDNAs so produced may then be used as antisense DNA.
A fascinating utility that is being considered is the role that slDNAs of the invention can play on the formation of triple helix DNA, or triplex DNA, and the resulting new triplex slDNA structures. A recent report in Science, 252, 1374-1375 (Jun. 27, 1991), "Triplex DNA Finally Comes of Age", highlights the timeliness of the present invention. Triplex DNA can be formed by binding a third strand to specific recognized sites on chromosomal DNA. Synthetic strands of sizes preferably containing the full complement of bases (such as 11-15 and higher), are discussed. The slDNAs of the invention with long 3' (or 5') ends (and the loop of non-duplexed bases) would appear to be excellent candidates. The resulting triplex DNA is expected to have increased stability and usefulness. New therapies based on the triple helix formation, including in AIDS therapy and selective gene inhibition and others are proposed in the Report.
It can be seen that the present invention is providing a significant contribution to arts and science.
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__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 5(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 215 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CTAGAGATATGTTCATAAACACGCATGTAGGCAGATAGATCTTTGGTTGTGAATCGCAAC60CAGTGGCCTTATGGCAGGAGCCGCGGATCACCTACCATCCCTAATGACCTGCAGGCATGC120AAGCTTGCATGCCTGCAGGTCATTAGGTACGGCAGGTGTGCTCGAGGCGAAGGAGTGCCT180GCATGCGTTTCTCCTTGGCTTTTTTCCTCTGGGAT215(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:AGGCCGCGTTGCTGGCGTTT20(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 60 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: both(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:ACCATCCCTAATGACCTGCAGGCATGCAAGCTTGCATGCCTGCAGGTCATTAGGGATGGT60(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 34 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:CCGGCGCAACGACCGCAAAAAGGTATCCGAGGCG34(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GGTTATCCACAGAATCAG18__________________________________________________________________________

Number	Name	Date
4357421	Emtage et al.	Nov 1982
4965188	Mullis	Oct 1990
5162209	Scheele	Nov 1992
5215899	Dattagupta	Jun 1993

Method for synthesizing single-stranded stem-loop DNAs, the products and uses therefor

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Entry
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