Dendritic nucleic acids exhibiting maximal self-assembly

Details Dendritic nucleic acids exhibiting maximal self-assembly Dendritic nucleic acids exhibiting maximal self-assembly

2000-01-27

2001-08-14

Riley, Jezia (Department: 1656)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S002000, C536S023100, C536S025300, C435S006120, C435S091100, C435S091200

Reexamination Certificate

active

06274723

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to methods for the detection of nucleic acids, and more particularly to labeled carriers for probes that hybridize to the nucleic acid of interest.
BACKGROUND OF THE INVENTION
Dendritic molecules are highly-branched arborescent structures and have found applications as chemical reagents, lubricants, contrast media for magnetic resonance, and others. See, e.g., Barth et al., Bioconjugate Chemistry 5:58-66 (1994); Gitsov & Frechet, Macromolecules 26:6536-6546 (1993); Hawker & Frechet, J. Amer. Chem. Soc. 112:7638-7647 (1990a); Hawker & Frechet, Macromolecules 23:4726-4729 (1990b); Hawker et al., J. Chem. Soc. Perkin Trans. 1:1287-1297 (1993); Lochmann et al. J. Amer. Chem. Soc. 115:7043-7044 (1993); Miller et al., J. Amer. Chem. Soc. 114:1018-1025 (1992); Mousy et al., Macromolecules 25:2401-2406 (1992); Naylor et al., J. Amer. Chem. Soc. 111:2339-2341 (1989); Spindler & Frechet, Macromolecules Macromolecules 26:4809-4813 (1993); Turner et al., Macromolecules 26:4617-4623 (1993); Wiener et al., Magnetic Resonance Med. 31(1):1-8 (1994); Service, 267:458-459 (1995); Tomalia, Sci. Amer. 62-66 (1995); and U.S. Pat. Nos. 4,558,120; 4,507,466; 4,568,737; 4,587,329; 4,857,599; 5,527,524; and 5,338,532 to Tomalia. Dendritic molecules offer several advantages over other molecular architectures. First, dendrimers contact the maximum volume or area with a minimum of structural elements. Second, the growth of dendritic molecules can be highly controlled to yield molecules of ideal size and molecular weight. Finally, the large number of defined “ends” can be derivatized to yield highly labeled molecules with defined spacing between the labels.
Nucleic acid dendrimers have been constructed following the technology that Tomalia applied to conventional organic polymers. See Hudson et al., “Nucleic Acid Dendrimers: Novel Biopolymer Structures,” Am. Chem. Soc. 115:2119-2124 (1993); and U.S. Pat. No. 5,561,043 to Cantor.
DNA detection is typically achieved with an absorption measurement where the quantity of DNA is directly proportional to the absorbance of solution. Though this technique is moderately sensitive, it provides no information about the specific sequence of DNA, only how much DNA is present. DNA can also be labeled with fluorescent, radioactive or chemiluminescent molecules. Labeling offers the advantages of increased detection sensitivity and specificity. The specificity comes from only labeling the desired pieces of DNA. The greatest use of this labeling for DNA detection is by labeling a complementary probe to the target DNA. If the labeled probe hybridizes to its target and can be detected, one can infer that the target is present. The most common use of this technique is called Southern blotting where DNA-DNA targets are detected after hybridization to a labeled probe. Dendritic nucleic acids are useful for the development of nucleic acid diagnostics as signal amplification tools. Due the relatively large size of nucleic acid molecules, nucleic acid dendrimers are readily labeled with numerous fluorescent compounds and/or protein moieties with limited steric hindrance and/or quenching. They also show potential as drug (antisense) delivery vehicles.
Perhaps the most commonly used detection method for DNA relies on amplification of the DNA rather than adding a label. This technique for amplification is called the polymerase chain reaction (PCR). In this technique, the target strand is amplified in situ by the addition of deoxynucleotide triphosphates of all four bases, a thermally stable polymerase and a primer DNA which is a short strand complementary to target. The primer (or primers) mark the point of duplication (and the point of termination). If a single primer is used the complementary strand will be copied to the termination, but more commonly two primers are used to indicate the beginning and end of the amplified sequence. Detection of the amplified DNA can be achieved by any of a number of techniques. Perhaps the most common method entails separating the amplified DNA by electrophoresis (typically in agarose or polyacrylamide, though capillary electrophoresis has also been used) and detecting it by staining with a fluorescent intercalating reagent (usually ethidium bromide). The PCR technique therefore allows for both increased sensitivity and specificity.
Nucleic acid matrices assembled substantially upon nucleic acid hybridization and which are characterized by dentritic-type architecture, but yet structurally distinct from the aforementioned organic and nucleic acid dendrimers, are taught in U.S. Pat. Nos. 5,175,270; 5,484,904; and 5,487,973 to Nilsen et al., and assigned to Polyprobe, Inc. The unique molecular design of Polyprobe's matrices accommodates a large number of labels, resulting in more than a 100-fold amplification of the signal compared to various prior art methods—target nucleic acids can be detected even when present in the sample in extremely small (e.g., femptogram (10
−15
)) amounts. Thus, Polyprobe's technology represents a significant improvement over state-of-the-art nucleic acid detection methods, most notably PCR.
SUMMARY OF THE INVENTION
The present invention is directed to an improvement of the nucleic acid dendrimers described in the aforementioned Nilsen patents, and which exhibit maximal self-assembly. One aspect of the present invention provides a dendritic polynucleotide having a plurality of single stranded hybridization arms; said polynucleotide comprising a plurality of polynucleotide monomers bonded together by hybridization; each polynucleotide monomer having an intermediate region comprising a linear, double stranded waist region having a first end and a second end, said first end terminating with two single stranded hybridization regions, each from one strand of the waist region, and said second end terminating with one or two single stranded hybridization regions, each from one strand of the waist region; and in said dendritic polynucleotide each polynucleotide monomer is hybridization bonded to at least one other polynucleotide monomer at at least one such hybridization region;
and wherein each of said hybridization regions and said waist regions of said plurality of monomers comprise sequences containing no repeats of subsequences having X nucleotides, wherein X is an integer of at least 2. In preferred embodiments, X is an integer from 2 to about 7; in more preferred embodiments, X is 3, 4 or 5.
The nature and constitution of the DNAs that comprise the monomers allow for extremely precise and controlled assembly, e.g., maximal self-assembly, of the nucleic acid dendritic matrices of the invention. That is, the hybridization regions of a given monomer hybridize substantially only with a substantially complementary hybridization region of another monomer. Therefore, self-hybridization is reduced, preferably to the extent that it is negligible. The advantages are that interference with the assay is reduced, leading to greater accuracy and sensitivity.
Compositions, kits and methods of making and using the polynucleotides are also described.


REFERENCES:
patent: 5175270 (1992-12-01), Nilsen et al.
patent: 5484904 (1996-01-01), Nilsen et al.
patent: 5487973 (1996-01-01), Nilsen et al.
patent: 5561043 (1996-10-01), Cantor et al.
Hudson, et al., Am. Chem. Soc., 115:2119-2124 (1993).

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