5′-thio phosphate directed ligation of...

Details 5′-thio phosphate directed ligation of... 5′-thio phosphate directed ligation of...

2001-07-20

2003-10-21

Riley, Jezia (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C536S023100

Reexamination Certificate

active

06635425

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the field of ligation of nucleic acids, using phosphorothioate derivatives as a means of ligating single stranded oligonucleotides, and where such oligonucleotides contain a point mutation, as a means of detecting single nucleotide polymorphisms.
BACKGROUND OF THE INVENTION
The ability to detect single base differences in DNA is of great importance in molecular genetics. Specific identification of point mutations is playing an increasingly important role in diagnosis of hereditary disease and in identification of mutations associated with drug resistance. Because of the high fidelity of ligation, enzymatic ligation methods have proven useful in a number of novel gene detection techniques.
Polynucleotide ligases are ubiquitous cell proteins that are required for a number of important cellular processes, including replication, repair and recombination of DNA. One of the best-characterized enzymes that joins DNA ends is T4 DNA ligase, first isolated some three-decades ago (for reviews see 1-3). This and related proteins catalyze ATP-dependent phosphodiester bond formation between the 5′-phosphate and 3′-hydroxyl groups of adjacent DNA strands (4,5). Duplex DNA molecules with either cohesive ends or blunt ends can serve as ligation substrates (6,7). T4 DNA ligase can also repair nicked DNA duplexes efficiently and thus the enzyme is often used for joining two DNA segments that are hybridized adjacent to each other on a complementary strand.
The use of circular DNAs (which can be formed by ligation, internal or external, of single stranded oligonucleotides) in such methods of amplification as rolling circle amplification technology (RCAT™) promises to greatly improve the performance of gene-based diagnostic testing and to facilitate the detection of a wide variety of infectious agents, cancerous cells, and genetic variations (also called polymorphisms) (71). Since the discovery of circular DNA to serve as a template for DNA polymerases (72), there has been increasing demand for the synthesis of circular oligonucleotides. Although there have been reports of successful enzymatic ligation to produce circular DNA, the yields of circles (less than 100 nucleotides or 100 nt) have been modest. Non-enzymatic ligation strategies have been somewhat more successful in the synthesis of small circular DNAs (less then 50 nt) on solid supports. Several approaches using non-enzymatic methods have recently been described. However, synthesis of circular DNA (larger than 50 to 100 nt or nucleotides) by non-enzymatic methods is a slow, entropically disfavored process and remains a synthetic challenge.
Kool et al have reported a method of circle synthesis (See Kool, E. T. et. al, Nature Biotechnol (2001)19, pp. 148-152; Kool, E. T. et. al.,
Nucleic Acids Res,
(1995)I. 23 (17), pp. 3547-355; Kool, E. T. et.al (1999) 27(0.3), pp 875-871. However, Kool's published report uses a chemical ligation approach wherein the ligation reaction produces a DNA containing sulfur in the bridging junction as 5′-S-thioester linkage at the site of ligation. In this approach, two oligonucleotides bound at adjacent sites on a complementary strand undergo autoligation by displacement of a 5′-iodide with 3′-phosphorothioate group (see, in general, the cited references herein). In addition, Kool et. al has reported a reagent free autoligation approach where 5′-S-thioester bonds formation between 3′-phosphorothioate and a 5′-iodide, which acts as a leaving group.
Other published methods report a chemical ligation approach for the synthesis of single stranded circular DNA wherein the modified circular DNA comprises a single 5′-S-thioester linkage with sulfur located in the 5′-bridging region. Other strategies have used sulfur atoms to replace specific non-bridging phosphate oxygens in RNA. Eckstein, F,
Angew. Chem. Intl. Ed. Engl.
22, pages 423 (1993) have synthesized phosphorothioate-linked polyribonucleotides as early as 1967 using DNA-dependent RNA polymerase from
E.coli
. Several other polymerases proved useful in the synthesis of the phosphorothioate linked ribo and deoxy oligonucleotides.
When a single base pair mismatch exists at either side of the ligation junction, the efficiency of the enzyme in ligating the two oligonucleotides decreases markedly. This high sequence selectivity has resulted in the development of novel sequence detection methods using this enzyme. These approaches include the ligase detection reaction (LDR) (8-10) and the ligase amplification reaction (LAR) (11). T4 DNA ligase displays selectivity against single base mismatches on the order of 2 to 6 fold in yield and conditions such as the presence of spermidine, high salt and low enzyme concentration have been reported to improve the fidelity of ligation to as high as 40 to 60-fold (9, 10). The thermostable DNA ligase from
Thermus thermophilus
(Tth DNA ligase) has been reported to have higher fidelity (mismatch discrimination of 450 to 1500-fold in rate) and has been measured for the wild-type enzyme with optimized mismatch location at the 5′-side of junction (12,13), making its use preferable in some sequence detection methods including the ligase chain reaction (14-16).
Since the discovery of ligases there have also been developed a number of non-enzymatic approaches to joining the ends of two DNA strands (17-29). These chemical ligations have been achieved via oxidative coupling of terminal 3′-phosphorothioates and displacement of 5′-bromide from a monoacetylamino bromide (19), 5′-tosylate (17) and a 5′-iodide (30). The resulting DNA contains the modified 5′-S-thioester linkage in the bridging position. Strategically placed sulfur atoms in the backbone of nucleic acids have found widespread utility in probing of specific interactions of proteins, enzymes and metals. Sulfur replacement for oxygen has also been carried out at the 2′-position of RNA (37-39) and in the 3′-5′-positions of RNA (40-45) and of DNA (46-56) Polyribonucleotide containing phosphorothioate linkages were obtained as early as 1967 by Eckstein et al. using DNA-dependent RNA polymerase from
E.coli
(57). DNA-dependent RNA polymerase is a complex enzyme whose essential function is to transcribe the base sequence in a segment of DNA into a complementary base sequence of a messenger RNA molecule. Nucleoside triphosphates are the substrates that serve as the nucleotide units in RNA. In the polymerization of triphosphates, the enzyme requires a DNA segment that serves as a template for the base sequence in the newly synthesized RNA. In the original procedure, Uridine 5′-O-(1-thiotriphosphate), adenosine 5′-O-triphosphate, and only d (AT) as a template was used. As a result, an alternating copolymer [Ap (S) UpAp (S) Up] is obtained, in which every other phosphate was replaced by a phosphorothioate group. Using the same approach and uridine 5′-O-(1-thiotriphosphate) and adenosine 5′-O-(1-thiotriphosphate), polyribonucleotide containing an all phosphorothioate backbone was also synthesized (58). In both cases, nucleoside 5′-O-(1-thiotriphosphates) as a mixture of two diastereomers were used.
The stereo chemical course of polymerization catalyzed by
Escherichia coli
polymerase was determined by Eckstein et al using the 1-thio-analog of adenosine 5′-O-triphosphate as a substrate (58,59). The Sp isomer was found to serve as an enzyme substrate and was further used with Uridine 5′-O-triphosphate and a DNA template of poly-d (AT). The resulting RNA polymer was the complementary alternating copolymer of adenosine and uridine linked by alternating 3′-5′-phosphodiester and 3′-5′-phosphorothioate bonds. Other polymerases useful in the synthesis of the phosphorothioate backbone bearing polyribonucleotides and polyrdeoxyibonucleotides have been described (60-68).
In addition, Single Nucleotide Polymorphisms (SNPs) have been characte

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