Progress Report #1 of N50 Selections against Hemagglutinin

Shaan Patel

October 18, 2011

Fall 2011

N50 RNA Pool

Hemagglutinin (H1N1) – 6X His-Tagged (Bead-based selection)

Link to the proposal can be found here: http://dl.dropbox.com/u/33021630/Shaan%20Patel%20Hemagglutinin%20Proposal.docx

Progress, Results, Discussion

Round 1 of N50 nucleic acid aptamer selection against the H1N1 strain of the Hemagglutinin protein began on October 14^th. It was initially calculated that, given the molecular weight of the protein, the tube had a rather dilute concentration of 14.7 uM HA. Given this low concentration, 200 pmol of target will be used for every round of selection at the most. As a result, each 200 pmol aliquot made consisted of 13.6 uL of the protein solution. According to Table 1 below, Round 1 began with 200 pmol : 200 pmol target to pool ratio by following a general bead-based protocol with specifications and modifications outlined below. The PBS buffer is used due to the fact that HA was isolated from cell cultures and suspended in a PBS solution. Thus far, Reverse Transcription on the W0, W3, and E1 products, and ccPCR has been performed in order to determine the major Round 1 results, such as any negative template control amplifications, how many cycles of PCR will the E1 product undergo, and what kind of pool/target ratios and other modifications should be considered as the pool progresses into later rounds of bead-based selection.

Table 1 below only specifies various parameters for the first three rounds of selection due to the absence of results for the current and initial Round 1 of selection against HA. Since this is an independent protein target, it is especially important to analyze ccPCR and other gels in order to provide a guideline and direction as to how the pool of RNA at the end of each selection round is representing more of the tightly bound species and less of the weakly bound species. For example, in the table below, some changes that could be followed in Round 3 would be to increase the wash volume by 1, decrease the body temperature incubation time, and reduce the concentration of target by half.

Parameters Table 1 of R 1, 2, 3	Selection Buffer	Incubation Time	Incubation Temperature	Wash Volume	Pool #	Target : Pool Ratio	Beads	Special Protocols
Round 1	10X PBS with MgCl2 Salts	25 minutes	37 C Body	3 Washes, 2 volumes	N 50	200 : 200	Nickel NTA	N / A
Round 2	10X PBS with MgCl2 Salts	25 minutes	37 C Body	3 Washes, 2 volumes	N 50	200 : 200	Nickel NTA	(-) Selection
Round 3	10X PBS with MgCl2 Salts	12 minutes	37 C Body	TBA	N 50	100 : 200	Nickel NTA	(-) Selection

Furthermore, when R1/R2/R3 ccPCR results are obtained, this table can be modified with varying conditions for future rounds, and updated with the addition of Rounds 4, 5, and 6 with radio-binding assays in Round 5. The goal of bead-based selection with Hemagglutinin is perform close to 6 rounds and complete a binding assay to assess future rounds of selection in a long-term sense

The R1 ccPCR image below represents a successful round thus far. There was no amplification in in the NTC product, and the bands below the washes and elution represent only primer-dimers. According to the image, 12 cycles of PCR should be performed on the reverse-transcription product for lsPCR.

Problems Encountered

Thus far, no problems have been encountered, other than taking some time to calculate the concentration and molecular weight of Hemagglutinin given the amino-acid sequence of the protein in order to make aliquots. No problems have been noticed in the technique of the initial part of the bead and Reverse Transcription protocols. The R1 ccPCR image above shows good technique, and that the very short structured bands below the washes and the elution represent only primer-dimers and do not affect the results. Parameters can be changed appropriately once the final concentration of RNA has been yielded after lsPCR and a PAGE gel.

Conclusion and Future Work

Performing 12 cycles of PCR according to the ccPCR results above, as well as running a PAGE and quantifying the transcribed RNA will be the next steps in order to complete the first round of selection. Since nothing had amplified in the N50 pool that was used, it will continue to be used for all future rounds of selection. Furthermore, parameters in the table above, such as wash volumes, incubation times, and target : pool ratios can be altered from the bands that show up in the first R1 ccPCR gel image, as well as future ccPCR results. If W3 wash bands are amplifying more than E1 product bands, then wash volumes should be increased, based on how much visible difference there is in amplification. In addition, if excessively high concentration of RNA is yielded in the next round, then parameters should be much more stringent.

Ideally, by the first round of selection, a fairly high concentration of RNA should yield due to the fact that stringency of R1 parameters was low. This is favored, because high stringency in early rounds could lead to a premature decrease in the diversity of the binding species. Parameters should gradually be made stringent, the third wash is depicting a lot of background binding, which could be rectified with larger wash volumes. Currently, little is known as to how much of the pool should bind to the target, however ideally a fairly high concentration in excess of 1000 ng / uL is expected. Ultimately, as the concentration of the binding species start to decrease, many of these species will be biologically significant in preventing the HA mechanism.