Experiment 8: Nuclease survival selection assay
Wednesday, July 5, 2000
Location: You will meet in room Z 744 for this week's experiment
(Verfügungsgebäude, 7th floor level).
Teaching Assistant: Your teaching assistant for this experiment is Charles
Information on the experiment.
The experiment planned for today is arguably the central one in this small project you have undertaken this semester. Its goal is to identify those oligonucleotide hybrids whose affinity for a complementary target strand is substantially higher than that of the control compound. In our case, the acetylated amino-oligonucleotide serves as the control compound, since we have good reason to believe that the acetyl group has a negligible effect on binding the target.
The experiment is one that evolved over time in these laboratories. Initially, UV-melting experiments of individual, purified oligonucleotide duplexes were performed. These are the standard experiment in the field of modified oligonucleotides. They involve equimolar amounts of oligonucleotides as a micromolar aqueous solution whose change in the UV absorbance is monitored in a UV spectrophotometer as the temperature is increased (typically by 1 °C/min). The disadvantage of this experiment is that it requires pure strands in nanomole quantities. - It was shown that several melting curves of all compounds of a very small library can be acquired by monitoring their depletion from a solution to which target DNA permanently attached on controlled pore glass is added (Bleczinski & Richert, Rapid Comm. Mass Spectrom. 1998, 12, 1737-1743). Unfortunately, the kinetics of the hybridization to the solid support were very slow, making the assay laborious, and surface effects, most probably due to non-specific adsorption were encountered. Further, relatively large error bars were measured for the data points of the curves. This experience led to the development of the current assay, which is being performed entirely in solution, is relatively quick, and only requires small quantities of reasonably pure oligonucleotide hybrids.
On the first of the attached sheets a schematic of the selection assay is shown. A simplified version of this schematic is Figure 1 in Altman, Schwope, Sarracino, Tetzlaff, Bleczinski & Richert, J. Combin. Chem. 1999, 1, 493-508, the paper in which the assay is described in detail. The oligonucleotide derivatives have the same core DNA portion and vary only in their 5'-appendage. When exposed to one equivalent of a target strand, a binding equilibrium is established, in which the highest affinity oligonucleotide hybrid is in the duplex form to the greatest extent. When treating the mixture with a single-strand specific nuclease, the unbound competitors are degraded preferentially, leading to a selective survival of the higher affinity compound(s). The prolonged survival can be detected via quantitative MALDI-TOF mass spectrometry. Under the MALDI matrix conditions the duplex between oligonucleotide hybrid and target strand dissociate, allowing for the detection of the high affinity binder. This is schematically shown in the second of the attached sheets.
There are several things that one should note when performing this assay. One is that the result depends on temperature, salt concentration, and strand concentration. Low temperature and high salt concentration (ammonium salts only, to prevent adduct formation in the MALDI) increase duplex stability and thus facilitate the kinetic read-out of the binding equilibrium. A low concentration of the strands shifts the binding equilibrium to the unbound side, decreasing the likelihood of selecting out the most tightly binding compound. So, it is planned to perform the assay in a concentrated solution of small volume in the cold.
Unfortunately, last week's experiment of preparing a balanced mix of your oligonucleotide probes was not a success. Some components disappeared since their isolation from the pre-purification. Others were not properly adjustable or adjusted. As a result, it was decided to pool those oligonucleotide hybrids looking most promising based on the MALDI spectra of the stock solutions. One library is being prepared from them this way for this experiment. The library will also contain the acetylated control compound. The actual nuclease experiment will be started by your teaching assistant, who will also acquire the MALDI spectra.
Some recommendations for what will largely be a demo experiment follow below.
Nuclease Selection Assay. A new balanced mix for the current nuclease experiment (additional ones may be performed later) will have been prepared by the teaching assistants. This will be annealed with the target strand and then treated with the nuclease. A typical protocol for such a selection assay is given below. It is adapted from the paper in J. Combin Chem. cited above.
The solution of the library (4.8 x 10-11 mol/µl of
each oligonucleotide) were pipetted into a polyethylene reaction vessel.
To this was added 2.5 µL (1.2 x 10-10 mol) of a solution
of the target, followed by addition of 1.75 µl of 500 mM ammonium
acetate buffer in water. The resulting solution was heated to 90°C
for 1 min, followed by centrifugation at room temperature to recover condensation
water. The reaction solution was then cooled to 0 °C and kept
at this temperature for 5 min. The sample was then brought to 23
°C and the t=0 aliquot was withdrawn (1 µL). Then, a solution
of phosphodiesterase I (E.C. 126.96.36.199) (1.5 µL of a solution of 1
x 10-4 u/µL) was added and aliquots of 1 µL were
taken at the desired time points. The aliquots withdrawn from the
reaction mixture were immediately mixed thoroughly with 5 µL of the
matrix mixture containing the internal standard. Immediately after
the mixing, 1 µL of the solution of analytes and matrix was applied
to a stainless steel MALDI target plate. The plate had earlier been
prepared by coating with silicone oil, followed by removal of excess oil
by wiping with wipes soaked in ethanol and ammonium hydroxide. The
matrix/analyte mixture was let drying at room temperature and residual
water was evaporated with a hair dryer and then the prechamber vacuum of
the mass spectrometer. Spectra were acquired in negative, linear
mode at 20 kV with a delayed extraction voltage of 18.2 kV and 180 ns delays
for laser shots at 60-70 µJ/shot. A total of 100 shots at 2
Hz laser frequency was accumulated for every spectrum. At least four
spectra were acquired per time point. Analyte and internal standard
peaks were integrated and the relative intensities of the analyte peaks
calculated and expressed as fractions of the t = 0 min value. Spectra
with less than 500 ion counts for the internal standard peak were not used
for data analysis.