As-opposed to providing a mind-numbingly exhaustive account of the current target progress I have protracted throughout the summer unto now, I find it more apt to refer all to the “Experimental Design and Current Progress” section of my complete proposal for a necessary and succinct review of pre-fall semester progress (in an effort to both legitimate my laziness and obviate encumbering most of this post with gratuitous data). Thus, below is a brief exposition of the current state of my research with Hcp1.
Initially, upon returning to lab in early September, a round of selection (specifically, Round 15) was performed in order to assess the resultant R14 RNA pool for latent degradation. Following successful completion, in preparation for a binding assay, a (+)/(-) protein selection was performed for the determination of the degree of specific (i.e. target-specific) and nonspecific binding (i.e. bead-specific binding).
Referring directly to Figure 6 of the complete proposal, and its apparent signification therein of increasingly specific binding independently of stringency manipulations, three conclusions were reached, and are as follows: specific binding, unwarranted nonspecific binding, and inherent pool “stickiness.” Specific binding, obviously the most desirable, could be examined against nonspecific binding via a simple (+)/(-) selection as mentioned before. In the context of pool tenacity however, a separate negative selection was performed on streptavidin beads. Now, the N34 pool is not intrinsically sticky, however, it is an extant possibility that, through various round of selection, strands featuring high concentrations of inherently sticky adenine/thymine bases may have been evolved. Each of these potentialities, and the extent to which they applied to the pool, were to be completely elucidated via the composite gel, depicted below.
As a pretext for a binding assay, this (+)/(-) selection was rather inconclusive, is it confers equivalent binding capacity for both (+) target and (-) target selection, decisively contradicting the definition of aptamer, i.e. specific nucleic acid binding agent. However, despite this ostensible failure, I have proceeded with the binding assay, as it may more conclusively establish the nature of select RNA pools derived via past rounds of selection, namely 8, 12, and 16. Additionally, assuming the pool primarily features nonspecifically tenacious or Ni-NTA-binding sequences, isolating them would offer at least one respectable use, reverse engineering them out of R0 pools for the prevention of bead-binding background.
At the moment, the binding assay is in progress, and should afford results at the end of the week, prompting a forthcoming addition to this report.