The identification of small molecule protein interactions is essential for the research and development of drugs as well as fostering our understanding of the pathologic mechanisms underlying virus DTCs. This method facilitates the high throughput biopanning of drug recognizing peptides and global validation of drug binding sites on proteins for small molecule drugs of interest. To prepare a QCM sensor chip attach a ceramic sensor chip onto the oscillator of a 27 megahertz QCM apparatus and record the intrinsic frequency in the air phase before small molecule immobilization.
After the recording detach the chip and carefully add a 20 microliter drop of a one millimolar small molecule derivative solution in 70%ethanol to create a self assembled mono layer on the gold electrode of the sensor chip. Place the chip into a Petri dish lined with moistened tissue protect it from light for one hour at room temperature before gently washing the electrode surface with ultra pure water. Dry the chip with a gentle application of air and load the chip onto the QCM apparatus.
After one hour record the reduction in frequency in the air phase to measure the amount of the small molecule that has been immobilized. For T7 Phage Library Biopanning, place a cuvette with a dedicated magnetic stirrer onto the QCM biosensor set to 1000 revolutions per minute and add eight milliliters of reaction buffer to the cuvette While the buffer is being stirred attach the QCM sensor chip to the oscillator Hold down the arm of the oscillator to immerse the chip into the buffer and begin monitoring the QCM frequency. When the sensor gram equilibrates to around three Hertz per minute of frequency drift inject eight microliters of a T7 phage library into the cuvette and mark the injection point on the sensor.
Monitor the frequency reduction caused by the T7 phages binding to the small molecule immobilized on the gold electrode surface. After 10 minutes, stop the QCM frequency monitor and quickly lift the oscillator to remove the sensor chip from the batch, detach the sensor chip from the oscillator and remove the buffer from the chip. Place the dried sensor chip into a humid Petri dish and add a 20 microliter drop of log phase E-coli host cells onto the gold electrode.
Incubate the dish onto a 96 well microplate mixer at 37 degrees Celsius and 1000 to 1500 revolutions per minute for 30 minutes, protected from light to enhance the recovery of the bound T7 seven phages. At the end of the incubation transfer the 20 microliters of E-coli suspension into 200 microliters of LB medium. According to the general procedure conduct the phage plaque isolation in the medium and DNA sequencing that encodes the drug recognizing peptide sequences displayed on each phage capsid.
As an after maintenance of the sensor chip use a 1%sodium dodecyl sulfate solution soaked cotton swab to clean the electrode surface wash the gold surface with ultra pure water and dry the electrode with air. Then treat the electrode surface with five microliters of freshly prepared Parana solution for five minutes followed by an ultra pure water wash and air drying two times. To perform bioinformatics analysis using RELIC unzip the standalone RELIC program on a PC with a windows operating system and use the amino acid sequences of drug selected 15 mer peptides or single or multiple proteins in each text file with fast A format.
Place the required text files into the folder of AA-div, info, motif, match, hetero align, fast A con, or fast A scan. Click each executable file in the independent folder to open the personal version of FTN 95 and enter the appropriate file name and extension in the command line to execute each program and to obtain the resulting text format file. The resulting text format files obtained are shown here.
Then export the resulting text file to a spreadsheet to generate a plot of information content or cumulative similarity scores calculated using a blow some 62. Using this strategy, single and multiple small molecule binding sites on the target proteins have been successfully identified for six small molecule drugs. For example, 29 peptides that recognize the clinically approved drug Irinotecan immobilized as a self-assembled monolayer were identified by QCM biosensor based one cycle biopanning Subsequent pairwise alignment of the 29 peptides and acetylcholinesterase yielded maximal scores for specific amino acid residues that were consistent with those making up the Irinotecan binding site.
This same subset of peptides was also successfully identified in the vicinity of the catalytic triad in Carboxylesterase, indicating that these amino acids form a scaffold for Irinotecan recognition during de-esterification. The 27 peptides that recognize the anti-flu drug Oseltamivir covering the QCM sensor chip gold electrode surface successfully detected the oseltamivir binding site in neuraminidase. This binding site consists of unstructured peptide loops that potentially undergo dynamic movement while docking with oseltamivir.
Drugs, regions, chemicals, recombinant bacteriophages, and bacteria are biological hearses and should be handled according to the culture gain protocol. Remember to always wear gloves, goggles and a lab coat for safety. Following this procedure, target proteins for various drugs can be globally validated in humans, pathological viruses, even plants to understand the molecular mechanisms and potential therapeutic efficacy of drugs of interest.
