Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-29
2003-09-09
Souaya, Jehanne (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091500, C536S023100, C536S025300, C536S025320
Reexamination Certificate
active
06617106
ABSTRACT:
Statement of rights to inventions made under Federally-sponsored research: None
INTRODUCTION
1. Field of the Invention
The field of this invention is nucleic acids and their analogs, more specifically nucleoside building blocks for preparing oligonucleotide analogs capable of forming non-standard Watson-Crick base pairs, joined by non-standard patterns of hydrogen bonding, those different from hydrogen bonding patterns joining the standard adenine-thymine and cytosine-guanine base pairs.
2. Background of the Invention
Natural oligonucleotides bind to complementary oligonucleotides according to the well-known rules of base pairing first elaborated by Watson and Crick, where adenine (A) pairs with thymine (T) or uracil (U), and guanine (G) pairs with cytosine (C), with the complementary stands anti-parallel to one another. These pairing rules allow for the specific hybridization of an oligonucleotide with complementary oligonucleotides, making oligonucleotides valuable as probes in the laboratory, in diagnostic applications, as messages that can direct the synthesis of specific proteins, and in a wide range of other applications well known in the art. Further, the pairing is the basis by which enzymes are able to catalyze the synthesis of new oligonucleotides that are complementary to template nucleotides. In this synthesis, building blocks (normally the triphosphates of ribo or deoxyribo derivatives of A, T, U, C, or G) are directed by a template oligonucleotide to form a complementary oligonucleotide with the correct sequence. This process is the basis for replication of all forms of life, and also serves as the basis for all technologies for enzymatic synthesis and amplification of specific heterosequence nucleic acids by enzymes such as DNA and RNA polymerase, and in the polymerase chain reaction.
The Watson-Crick pairing rules can be understood chemically in terms of the arrangement of hydrogen bonding groups on the heterocyclic bases of the oligonucleotide, groups that can either be hydrogen bond donors or acceptors (FIG.
1
). In the standard Watson-Crick geometry, a large purine base pairs with a small pyrimidine base thus, the AT base pair is the same size as a GC base pair. This means that the rungs of the DNA ladder, formed from either AT or GC base pairs, all have the same length.
Further recognition between bases is determined by hydrogen bonds between the bases. Hydrogen bond donors are heteroatoms (nitrogen or oxygen in the natural bases) bearing a hydrogen; hydrogen bond acceptors are heteroatoms (nitrogen or oxygen in the natural bases) with a lone pair of electrons. In the geometry of the Watson-Crick base pair, a six membered ring (in natural oligonucleotides, a pyrimidine) is juxtaposed to a ring system composed of a fused six membered ring and a five membered ring (in natural oligonucleotides, a purine), with a middle hydrogen bond linking two ring atoms, and hydrogen bonds on either side joining functional groups appended to each of the rings, with donor groups paired with acceptor groups (FIG.
1
).
Derivatized oligonucleotide building blocks, where a side chain has been appended to one of the nucleoside bases A, T, U, G, or C (the “normal” bases), have application because of their combination of Watson-Crick base pairing and special reactivity associated with the chemical properties of the side chain. For example, oligonucleotides containing a T to which is appended a side chain bearing a biotin residue can first bind to a complementary oligonucleotide, and the hybrid can then be isolated by virtue of the specific affinity of biotin to avidin (Langer, P. R.; Waldrop, A. A.; Ward, D. C. (1981)
Proc. Nat. Acad. Sci
. 78, 6633-6637), and finds application in diagnostic work. Oligonucleotides containing special functional groups (e.g., thiols or hydrazines) can be immobilized to solid supports more readily than those composed solely of the five “natural” bases. Additionally, radiolabeled nucleotide bases can be included in an oligonucleotide, for example,
3
H or
32
P labeled nucleotide bases.
Often, derivatized building blocks can be incorporated into oligonucleotides by enzymatic transcription of natural oligonucleotide templates in the presence of the triphosphate of the derivatized nucleoside, the substrate of the appropriate (DNA or RNA) polymerase. In this process, a natural nucleoside is placed in the template, and standard Watson-Crick base pairing is exploited to direct the incoming modified nucleoside opposite to it in the growing oligonucleotide chain.
However, the presently available base pairs are limited in that there are only two mutually exclusive hydrogen bonding patterns available in natural DNA. This means that should one wish to introduce a modified nucleoside based on one of the natural nucleosides into an oligonucleotide, it would be incorporated wherever the complementary natural nucleoside is found in the template. For many applications, this is undesirable.
Further, in many applications, it would be desirable to have base pairs that behave as predictably as the AT and GC base pairs, but that do not pair with natural oligonucleotides, which are built from A, T, G, and C.
Many of the limitations that arise from the existence of only four natural nucleoside bases, joined in only two types of base pairs via only two types of hydrogen bonding schemes, could be overcome were additional bases available that could be incorporated into oligonucleotides, where the additional bases presented patterns of hydrogen bond donating and accepting groups in a pattern different from those presented by the natural bases, and therefore could form base pairs exclusively with additional complementary bases. The purpose of this invention is to provide compositions of matter containing these additional bases, and methods for using them to recognize complementary oligonucleotide strands also containing non-standard bases. A “non-standard” nucleobase is one that presents to a nucleobase on a complementary strand, placed in a Watson-Crick geometry, a pattern of hydrogen bond donor and acceptor groups different from those found in adenine or 2-aminoadenine, guanine, cytosine, and thymine or uracil.
SUMMARY OF THE INVENTION
The compositions of this invention are precursors and building blocks for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) containing non-standard nucleobases, including ribo and deoxynucleosides bearing non-standard nucleobases, ribo and deoxynucleosides having protected exocyclic amino group functionality, protected and activated ribo and deoxynucleosides suitable for solid phase DNA and RNA synthesis, and ribo and deoxynucleoside phosphates suitable for enzymatic synthesis of DNA and RNA.
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Pletta Gregory T.
Souaya Jehanne
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