Systems and methods of coding for and isolating a fusion protein are provided. The fusion protein may be expressed by a DNA sequence and comprise an adenylate kinase linked by its carboxy-terminus to a biologically active polypeptide or protein. Later processing steps include isolating the biologically active polypeptide or protein via affinity elution of the fusion proteins with substrate analog of adenylate kinase.

The invention provides compositions and methods for ligating single stranded nucleic acids wherein the ligation is based on fast, efficient, and low-sequence bias hybridization of an acceptor molecule with a donor molecule. In one embodiment, the structure of the donor molecule comprises a stem-loop intramolecular nucleotide base pairing (i.e., hairpin) and a 3′-overhang region such that the overhang is able to hybridize to nucleotides present in the 3′ end of the acceptor molecule.

Formation of a modified oligonucleotide by treating four or more oligonucleotide raw material fragments in total in the presence of an oligonucleotide ligase; the four or more oligonucleotide raw material fragments in total corresponding to oligonucleotide raw material fragments that are obtained by dividing the modified oligonucleotide at a fragment linking site that satisfies following conditions (i) to (v): (i) one or more fragment linking sites are present in the complementary portion in each strand side, and two or more fragment linking sites in total are present in the modified oligonucleotide; (ii) when the modified oligonucleotide is divided at the fragment linking site, a sticky end is formed in the complementary portion, in which the sticky end has 1 to 10 nucleotide length; (iii) at least one oligonucleotide raw material fragment has a modified nucleotide; (iv) four oligonucleotide raw material fragments out of the four or more oligonucleotide raw material fragments in total include the complementary portion having 5 to 25 nucleotide length; and (v) total nucleotide length of the oligonucleotide raw material fragments corresponding to the complementary portions in each strand side is 11 to 27, is useful for efficiently producing an oligonucleotide comprising a complementary portion, such as an siRNA and a heteroduplex oligonucleotide.

The present invention provides a recombinant eukaryotic cell expressing one or more heterologous double strand break (DSB) repair proteins in an amount effective for enhancing DSB repair in the cell. The recombinant eukaryotic cell may express a recombinant product of interest. Also provided are methods for enhancing double strand break (DSB) repair in eukaryotic cells, establishing host cells for production of a recombinant product of interest, producing a recombinant product of interest, improving production of a recombinant product of interest by eukaryotic cells, and/or investigating suitability of eukaryotic cells as host cells for producing a recombinant product of interest.

The tissue microenvironment is a critical factor to disease mechanism and therapeutic efficiency. Provided here is an imaging platform, methods, and kits which enable measurement of tens of parameters simultaneously within a single tissue, with the potential to reveal unique and important biological associations related to the spatial dimension. The sensitivity of the platform, methods, and kits described herein extends to measure low level markers which can be important for tracking disease progression, diagnosing disease, or both.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

The present disclosure describes a method of adaptor ligation to the ends of the fragmented double-stranded DNA molecules.

The current inventive technology includes systems, methods, and compositions for directly synthesizing sticky ended DNA fragments for subsequent gene assembly. In a preferred embodiment, the inventive technology includes strategies for the direct synthesis of sticky ended DNA with 5′ overhangs that have any desired length and base composition, using typical PCR protocols with no additional manipulation. In another embodiment, the inventive technology includes the direct synthesis of sticky ended DNA using chemically modified oligonucleotide primers in a polymerase chain reaction (PCR). In certain embodiments, the inventive technology allows for the generation of larger DNA constructs formed by the sticky-ended assemblies generally described herein compared to traditional synthesis and ligation applications.

A number of T4 DNA ligase mutants exhibiting enhanced ligation activity at temperatures above 37° C. compared to the wild-type ligase were engineered, characterized, and selected via gel electrophoresis of ligation products from a standard ligation assay. T4 Ligase catalyzes the formation of phosphodiester bonds between the 5′ and 3′ ends of complementary cohesive ends or blunt ends of duplex DNA, a process that is vital to numerous molecular biology processes including cloning and sequencing.

The disclosure provides, for example, single stranded, covalently closed DNA that does not form a double stranded structure longer than 100 base pairs. The ssDNA may encode an effector sequence, for instance a therapeutic protein. The ssDNA may comprise a nuclear targeting sequence (NTS). In some embodiments, the ssDNA shows decreased activation of the innate immune system compared to an otherwise similar dsDNA.