Completely Chemically Synthesized Long DNA Can be Transcribed in Human Cells.

Area of Science:

• Synthetic biology
• Molecular biology
• Chemical synthesis

Background:

• Chemical ligation is a key method for constructing long DNA molecules from smaller synthesized fragments.
• Unnatural linkages formed during DNA ligation can hinder the biological functionality of the synthesized DNA.

Purpose of the Study:

• To achieve complete chemical synthesis of long DNA molecules using multiple chemical ligation reactions.
• To demonstrate the biological function of chemically synthesized DNA, specifically gene silencing.
• To overcome the limitations of unnatural linkages in chemically ligated DNA.

Main Methods:

• Utilized multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments.
• Synthesized 78 and 258 base pair (bp) synthetic DNA molecules.
• Constructed a DNA molecule encoding shRNA for luciferase genes with a truncated SV promoter.

Main Results:

• Successfully achieved complete chemical synthesis of 78 and 258 bp DNA molecules.
• The 258 bp synthetic DNA successfully induced gene silencing of luciferase genes in HeLa cells.
• Demonstrated that chemically synthesized DNA can retain biological function despite ligation.

Conclusions:

• Multiple chemical ligation reactions with modified DNA fragments are effective for synthesizing functional long DNA.
• This approach overcomes the biological impairment typically caused by unnatural linkages in ligated DNA.
• Chemically synthesized DNA, including gene-silencing constructs, can be biologically active.