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Why use 0.1N HCl to measure Morpholino concentration by UV spec?

Here is an excerpt from a letter Jim Summerton recently wrote in which he described the reason why Morpholinos are quantitated by measuring UV absorbance at 265 nm in 0.1N HCl.

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Morpholinos are unusual in having exceptionally good base stacking - better than single-stranded DNA and much better than single-stranded RNA. Most likely this exceptional base stacking is a consequence of the lack of ionic charges on the Morpholino backbone and so no electrostatic repulsions act to counter the base stacking - in contrast to the case for nucleic acids.

Because of Morpholinos’ exceptional base stacking they exhibit an exceptional level of hypochromic shift (reduced absorbance values due to base stacking) which varies as a function of the sequence of the nucleobases in any given oligo sequence. At first glance this variability of spectral absorbance as a function of sequence might be expected to preclude precise quantitation of such compounds by their UV absorbance. However, we have overcome this apparent limitation by thoroughly disrupting the base stacking by assessing absorbance in 0.1 N HCl. At this pH cytosines, adenosines, and guanosines are fully ionized - thereby precluding virtually all stacking of the bases. Thus, taking the UV scan in 0.1 N HCl precludes the sequence-dependent hypochromic shifts common to Morpholinos at pH values closer to neutrality where the nucleobases are not ionized.

Further, we picked a specific wavelength, 265 nm, which for each unstacked nuceobase in 0.1 N HCl has a unique absorbance value which is independent of sequence of the Morpholino oligo. By using the 265 nm absorbance values for each of the four different nucleobases we can precisely and reliably calculate the extinction coefficient of any Morpholino oligo independent of its sequence, dependent only on its nucleobase composition.

Therefore, by using 0.1 N HCl as the solvent and reading the absorbance at 265 nm wavelength you can get a precise quantitation of your product independent of nucleobase sequence and without the variable effects of hypochromism arising from sequence-dependent base stacking. That absorbance value precisely reflects the number of each of the four bases in the Morpholino oligo - and does so essentially independent of sequence.

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