Research Update
Capped RNA Fever
Capped RNA is a useful tool in the research of nuclear export regulation, degradation, translation promotion and intron excision. Capped RNA is precursor mRNA, which is capped at the 5’ end with an altered nucleotide. Capping makes mRNA stable thereby allowing it to undergo translation. In a recent study by Kroschewski et al, sequential methylation of viral mRNA cap formation was investigated using a custom synthesized 3’ biotinylated capped RNA from TriLink. Kroschewski and her team are searching for mutations in viral replication that could be used as targets in the development of antiviral agents against the dengue virus. TriLink has done extensive research on modified polyphosphorylated oligonucleotides. We have made significant advancements in the synthesis of 5’ triphosphate oligonucleotides, 5’ adenylate and 2’-5’ adenylate triphosphate oligonucleotides, as well as capped and 7-Me capped RNA.
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Technology Spotlight
Expanding Applications:
2’ Fluoro Oligonucleotides
2’ Fluoro substituted RNA oligonucleotides have the useful property of increasing Tm by 1.3 ˚C per substitution relative to RNA. When hybridized to an RNA target, oligonucleotides increase in stability as follows: DNA < RNA < 2’ OMe RNA< 2’ Fluoro RNA. These unique monomers are being introduced in more and more applications, including stabilizing siRNA, aptamers and antagomirs. TriLink continues to optimize protocols for highly substituated 2' Fluoro RNA. We have successfully synthesized 100mg of a 60bp RNA oligonucleotide with 40 2' Fluoro insertions. Methodology is being developed to produce even larger quantities. 2' Fluoro modifications can now be introduced at any base, A, C, G or U.
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Question of the Month
Q - What is Tm?
A - Tm is shorthand for melting temperature. The melting temperature of a nucleic acid duplex is the temperature at which half of the strands are in duplex form, and the other half are single stranded. This is determined by taking advantage of a spectral phenomenon called hypochromicity, which is the reduction in UV absorbance that is observed when nucleic acid bases form a duplex. The act of hydrogen pairing with the opposing strand and the change in electron patterns results in the observed reduction in the spectral reading at 260 nm. By cooling a sample containing complimentary strands in a cuvette until the strands are fully hybridized (absorbance is stable), then heating the sample slowly and recording the absorbance during the process, one obtains a sigmoidal shaped plot when temperature is plotted against absorbance. The transition temperature at the inflection point is the Tm.
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