Provided herein are reagents and methods for incorporating modified nucleotides into DNA and detecting DNA synthesis. In particular, haloalkyl-modified nucleobases are provided for incorporation into nucleic acids for detection by bioluminescent binding agents.

Embodiments for the synthesis of sensitive oligonucleotides as well as insensitive oligonucleotides are provided. Sulfur-based groups are used for the protection of exo-amino groups of nucleobases, phosphate groups and 2′—OH groups, and as cleavable linker for linking oligonucleotides to a support. Oligonucleotide syntheses are achieved under typical conditions using phosphoramidite chemistry with important modifications. To prevent replacing sulfur-based protecting groups by acyl groups via cap-exchange, special capping agents are used. To retain hydrophobic tag to assist RP HPLC purification, special phosphoramidites are used in the last synthetic cycle. With the sulfur-based groups for protection and linking, oligonucleotide deprotection and cleavage are achieved via oxidation followed by beta-elimination under mild conditions. Therefore, besides for insensitive oligonucleotide synthesis, the embodiments of the invention are capable for the synthesis of oligonucleotide analogs containing sensitive functional groups that cannot survive the harsh conditions used in prior art oligonucleotide synthesis technologies.

Nucleoside or nucleotide analog compounds having the structure shown below, a method for preparing them, and applications in nucleic acid sequencing are disclosed. The compounds have formula (I): ##STR00001## wherein L.sub.1, L.sub.2, and L.sub.3 are each independently a covalent bond or a covalently linked group; B is a base or a base derivative selected from purines, pyrimidines, or analogs thereof; R.sub.1 is —OH, a phosphate group, or a nucleotide; R.sub.2 as H or a cleavable group; R.sub.3 is a detectable group or a targeting group; R.sub.5 is an inhibitory group; R.sub.4 is H, —NH.sub.2, or —OR.sub.6, wherein R.sub.6 is H or a cleavable group; and C is a cleavable group or a cleavable bond.

Aspects of the present disclosure include compositions that make use of phosphorus and/or nucleobase protecting groups which find use in the synthesis of long polynucleotides. Phosphorus protecting groups are provided that help increase the stepwise coupling yield and/or phosphorous protecting groups that can be removed during the oxidation step. Amidine nucleobase protecting groups are provided that find use in the subject compositions and methods which provides for e.g., increased resistance to depurination during polynucleotide synthesis. In some instances, the methods and compositions disclosed herein utilize a combination of the phosphorus and amidine nucleobase protecting groups in the synthesis of polynucleotides having a sequence of 200 or more monomeric units in length. Also provided are methods for synthesizing a polynucleotide (e.g., a DNA) using one or more compounds disclosed herein.

Aspects of the present disclosure include compositions that make use of phosphorus and/or nucleobase protecting groups which find use in the synthesis of long polynucleotides. Phosphorus protecting groups are provided that help increase the stepwise coupling yield and/or phosphorous protecting groups that can be removed during the oxidation step. Amidine nucleobase protecting groups are provided that find use in the subject compositions and methods which provides for e.g., increased resistance to depurination during polynucleotide synthesis. In some instances, the methods and compositions disclosed herein utilize a combination of the phosphorus and amidine nucleobase protecting groups in the synthesis of polynucleotides having a sequence of 200 or more monomeric units in length. Also provided are methods for synthesizing a polynucleotide (e.g., a DNA) using one or more compounds disclosed herein.

An automated method and system for determining the risk of developing a cancer in a subject, the method comprising preparing a tissue sample obtained from the subject for visually identifying at least one biological marker associated with the cancer, digitally scanning the prepared tissue sample, analyzing the scanned image of the tissue sample to identify regions of interest, quantifying at least one parameter associated with the marker, and executing an algorithm using the quantified parameter to calculate a risk score, wherein the risk score is representative of the risk of the individual developing the cancer.

An automated method and system for determining the risk of developing a cancer in a subject, the method comprising preparing a tissue sample obtained from the subject for visually identifying at least one biological marker associated with the cancer, digitally scanning the prepared tissue sample, analyzing the scanned image of the tissue sample to identify regions of interest, quantifying at least one parameter associated with the marker, and executing an algorithm using the quantified parameter to calculate a risk score, wherein the risk score is representative of the risk of the individual developing the cancer.

The present disclosure, among other things, provides technologies for synthesis, including reagents and methods for stereoselective synthesis. In some embodiments, the present disclosure provides compounds useful as chiral auxiliaries. In some embodiments, the present disclosure provides reagents and methods for oligonucleotide synthesis. In some embodiments, the present disclosure provides reagents and methods for chirally controlled preparation of oligonucleotides. In some embodiments, technologies of the present disclosure are particularly useful for constructing challenging internucleotidic linkages, providing high yields and stereoselectivity.

Embodiments of the present application relate to N-acetylgalactosamine-conjugated nucleosides. In particular, the N-acetylgalactosamine is installed on the nucleobase of the nucleosides through a wide variety of linkers. Methods of making N-acetylgalactosamine-conjugated nucleosides are also disclosed herein. N-acetylgalactosamine is a well-defined liver-targeted moiety and N-acetylgalactosamine-conjugated nucleosides may be used in the preparation of targeted delivery of oligonucleotide-based therapeutics.

The invention relates to a method of treating a mitochondrial DNA depletion syndrome, comprising administering to a patient a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.