Hcp1 Binding Assay Update: Corollary to Current Progress

Within the initial post addressing my current progress with Hcp1 SELEX, I concluded with a statement foreshadowing an eventual identification and exposition of binding assay data. Finally, considering all of you are so painstakingly curious, the very forces which undergird your lives pausing briefly upon my every syllable, here it is!

Reproduced below is a composite image, identifying the data yielded by the recent binding assay in three distinct formats representing the analytical progression of the unfashioned radioactive imprints (a) unto the final graphical depiction (c):

Upon first glance, in this case an eminently accurate perspective of the data offered, the binding assay is particularly inconclusive, in may ways highly similar to the initial binding assay casting rounds two, four, six, and eight performed in late June. Like this binding assay, the antecedent assay featured such characteristics as: comparatively uniform degrees of nonspecific vs. specific binding, nonspecific binding superseding that of specific binding on all occasions, and generally, high quantities of both binding types. Unfortunately, this confers virtually no sentiments of success to my attempts over the past several months, as the RNA pool I have evolved via sixteen round of SELEX seeming bears effectually equivalent proportions of nonspecific and specific binding species, differing by an approximate mean of 0.74%.

A potential conclusion which, I feel, may be equitably gleaned from this data, upholds the speculation virtually pervading the progress section of October 11th; that is, assuming a pool possessing inherently tenacious, sticky specifies, one would anticipate a certain amount of nonspecific filter-binding, in addition to the specific binding observed. Naturally, this postulate fails to address the exact quantitative relationships among the two types of binding or the extent to which nonspecific filter-binding would occur with a pool intentionally engineered for sticky properties.

Extant possibilities for future selection include the following:

1. Reinstatement of early round (potentially R3) coupled with highly stringent selection techniques: i.e. negative selections, wash volumes, etc. which will hopefully function for the elimination of nascent nonspecific binding, after which a greater amount of target-binding sequences will be amplified at a comparatively greater rate

2. Complete selective restart with R0 N34 RNA

o However, featuring the following methodological manipulation: initial round selecting solely for beads, lsPCR amplification of W0 for elimination of initial bead-binding species, R2 initial target round with R1, W0 RNA.

3. Some other outlet suggested by incredibly kind aptamate/Brad/Gwen/mentor… :)

1 comment:

Katherine Li said...

Well, if you wanted, i can do a parallel selection with you for this, using my format and we can simultaneously attempt to get results and see if my method makes for a more stringent selection process? Your choice. also, you could use the Nickle Beads from the ellington lab if there are anymore and perhaps see if it works. But everyone else that has Hcp-1 that has used those has said that it makes their selections fail...if you start at round 3 again, i'd be happy to help.