Synthetic long single-stranded and circular DNA

Synthetic long single-stranded DNA (ssDNA) as well as circular DNA allow functional studies in vitro and in vivo. Improvements in DNA and RNA oligonucleotide synthesis methods enabled the production of long single-stranded DNA (ssDNA) and circular DNA, such as catenanes, chain-like molecules. Long synthetic ssDNA are valuable tools for studying their interactions with other molecules. The recombinases (RecA/Rad51), helicases, and translocases bind ssDNA to function as motor proteins. These proteins play a central role in genome maintenance. Long synthetic ssDNA may also be of use for the study of DNA repair mechanisms. Understanding the physicochemical properties of ssDNA and their exact conformations will help to elucidate their biological roles.

Single-stranded DNA (ssDNA) occurs naturally as a transient intermediate during genome maintenance processes. Genome maintenance is vital during DNA replication, repair, and recombination. Increased accumulation of ssDNA spells trouble for cells, for example, in autoimmune diseases.

Single-stranded DNA is known to occur in high incidence and concentrations in the sera of lupus patients (called systemic lupus erythematosus or SLE) at levels as high as 250 μg/ml. ssDNA is known as an immunogen for anti-ssDNA antibodies present in lupus patients. Complexes formed between ssDNA and the antibodies play a role in the pathogenesis of the acute inflammation of the kidney (glomerulonephritis) as found in lupus patients.

Scleroderma patients also have antibodies against ssDNA. Scleroderma is a disease of the connective tissues that cause scar tissue to form, usually in the skin but sometimes also in other organs.

Another example is the protein Trex1 which is the major 3’-DNA exonuclease in mammalian cells. Trex1 binds to ssDNA in mammalian cells, where it removes mismatched 3’-terminal deoxyribonucleotides at DNA strand breaks. The protein appears to play a DNA-editing role in DNA replication or gap-filling during DNA repair.

Compared to double-stranded DNA (dsDNA), the structure of ssDNA is very flexible and usually does not form well-defined secondary structures. If no internal base-pairing occurs, ssDNA is a random polymer.

Single molecules of ssDNA can be studied using biophysical methods such as single-molecule fluorescence resonance transfer (smFRET), molecular force spectroscopy, or optical tweezers. In most studies of single molecules, DNA or protein molecules are immobilized to a surface and are often mobilized with fluorophores or tethers to allow observations.

The synthesis of DNA catenanes, chain-like DNA molecules, allows the study of the secondary structure of these molecules. DNA catenanes are topoisomers of circular DNA molecules. Two or more DNA rings held together noncovalently such that one DNA circle or ring encircles the DNA strand of another to form DNA catenanes. The replication of circular DNA without the presence of topoisomerases produces DNA catenanes. A DNA gyrase also interlocks duplex DNA circles to form catenanes and resolves them as well into monomers.

When investigating DNA catenanes, Liang et al. found that secondary structures of ssDNA do form much easier than expected. Two strands of an internal loop in the folded ssDNA structure prefer to braid around instead of starting a separate circle. Also, a duplex containing only mismatched base pairs can form under physiological conditions.

Several methods are available for the formation of DNA catenanes. First, ligation of a linear oligonucleotide forms a circular one. Hybridization of another oligonucleotide on a circular one and sealing nicks using T4 DNA allows the synthesis of catenanes with specific linking numbers.The topology of these DNA products is studied using gel electrophoresis.

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