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Protein and DNA co-crystals are most commonly prepared to reveal structural and functional details of DNA-binding proteins when subjected to X-ray diffraction. However, biomolecular crystals are notoriously unstable in solution conditions other than their native growth solution. To achieve greater application utility beyond structural biology, biomolecular crystals should be made robust against harsh conditions. To overcome this challenge, we optimized chemical DNA ligation within a co-crystal. Co-crystals from two distinct DNA-binding proteins underwent DNA ligation with the carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) under various optimization conditions: 5′ vs. 3′ terminal phosphate, EDC concentration, EDC incubation time, and repeated EDC dose. This crosslinking and DNA ligation route did not destroy crystal diffraction. In fact, the ligation of DNA across the DNA–DNA junctions was clearly revealed via X-ray diffraction structure determination. Furthermore, crystal macrostructure was fortified. Neither the loss of counterions in pure water, nor incubation in blood serum, nor incubation at low pH (2.0 or 4.5) led to apparent crystal degradation. These findings motivate the use of crosslinked biomolecular co-crystals for purposes beyond structural biology, including biomedical applications.
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Electro-hydrodynamic extraction of DNA from mixtures of DNA and bovine serum albumin
We report separation of genomic DNA (48 kbp) from bovine serum albumin (BSA) by the electro-hydrodynamic coupling between a pressure-driven flow and a parallel electric field. Electro-hydrodynamic extraction exploits this coupling to trap DNA molecules at the entrance of a microfluidic contraction channel, while allowing proteins and salts to be flushed from the device. Samples (10 μL) containing λ-DNA (1 ng) and BSA (0.3 mg) were injected directly into the device and convected to the contraction channel entrance by a flowing buffer solution. The DNA remains trapped in this region essentially indefinitely, while proteins and salts are eluted. The effectiveness of the concept has been assessed by fluorescence measurements of DNA and BSA concentrations. Electro-hydrodynamic extraction in a single-stage device was found to enhance the concentration of DNA 40-fold, while reducing the BSA concentration by four orders of magnitude. The relative concentrations of DNA to BSA at the contraction channel entrance can be as large as 1.5 : 1, corresponding to an A260/280 ratio of 1.9. The maximum yield of DNA from a salt-free solution is 50%, while salted (150 mM) solutions have a lower yield (38%).
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- Award ID(s):
- 1804302
- PAR ID:
- 10233003
- Date Published:
- Journal Name:
- The Analyst
- Volume:
- 145
- Issue:
- 16
- ISSN:
- 0003-2654
- Page Range / eLocation ID:
- 5532 to 5538
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D0AN00961J
