Modified Nucleotides

Larry Gold et al, just published an article about a single selection against 800 protein targets for the purposes of proteomic diagnostics. Pretty interesting paper that we will read for journal club this semester. The cool figure for our stream though is plastered in Table 1 of the Supplementary data. In summary, they attempted selection against 14 targets with 4 different base modifications and showed the general success (with binding affinity) for each of the selections. The conclusion is that regular dT only produced viable aptamers for 2 of the 14 targets. 5-benzylaminocarbonyl-dU (BndU) and 5-isobutylaminocarbonyl-dU (iBudU) worked for about 50% of the targets. However, the real news is 5-tryptaminocarbonyl-dU (TrpdU) worked for all of the targets attempted. Figure below from the paper:

Figure legend from the paper: Nucleotide triphosphate analogs modified at the 5-position (R) of uridine (dUTP). The 5-position modifications include: (A) 5-benzylaminocarbonyl-dU (BndU); (B) 5-naphthylmethylaminocarbonyl-dU (NapdU): (C) 5-tryptaminocarbonyl-dU (TrpdU); and (D) 5-isobutylaminocarbonyl-dU (iBudU).

Let me reiterate.

In our lab we have a lot of failures. Maybe we could increase our hit rate by using modified nucleotides. Now, the thoughts that should be floating in your mind are: great(!) but how do I get TrpdU and how do I incorporate it into my pool and where does it get incorporated and...does this mean I'm not a failure?

TrpdU must be made in house and is not particularly easy. I don't think anyone is selling it currently, but you should do a thorough search at Tri-Link and other nucleotide suppliers. Shawn, our friendly organic chemist may be able to help out, or we may be able to ask one of the Organic streams to help out. The paper describing the method is by Vaught et al. The compound we want is 6d in figure 1 and the method for producing it is in Schema 1 (shown below). Bruce Eaton has also published two papers explaining alternate synthesis of these modifications. "The Joys of in vitro selection..." and "New uridine derivatives for systematic evolution...".

Primer extension efficiency is around 83% with KOD XL. We will likely want to amplify

template with normal nucleotides and then peform one primer extension with the following mixture: (nucleotide incorporation and amplification) they used 0.5 mM each dNTP (dATP, dGTP, 5'-methyl-dCTP, 5'-tryptaminocarbonyl-dU), 4.8nmol of forward primer, 4nmol template with 1X Thermococcus kodakaraensis (KOD) XL DNA Polymerase Buffer (EMD) and 0.125U/uL KOD enzyme.

During the selection, they also used dextran sulfate (a polyanion) as a nonspecific inhibitor.