A modified guanosine phosphoramidite for click functionalization of RNA on the sugar edge

Seidu-Larry, Salifu; Krieg, Bettina; Hirsch, Markus; Helm, Mark; Domingo, Olwen

doi:10.1039/c2cc34015a

Cited by 23 publications

(21 citation statements)

References 32 publications

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“…After hybridization of clicked sense-strand derivatives to the appropriate antisense strand, cells were incubated with a concentration series of siRNA double strands and eGFP fluorescence emission measured by FACS 24 h later. In keeping with our previously reported identification of a permissive attachment site on the 3′-end of the sense strand (70), conjugation of various azides did not significantly increase the IC 50 values (Figure 6D). Indeed, the *PDI derivative (pink in Figure 6) showed an IC 50 value improved by ~3-fold.…”

Section: Resultssupporting

confidence: 92%

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7

et al. 2017

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A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern-recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single-stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccines have been reported to exhibit synergistic effects. Here, we describe a concept to chemically combine three therapeutic functions in one RNA bioconjugate. This consists in the simultaneous TLR7 stimulation by ssRNA and smTLRa as well as the therapeutic function of the RNA itself, e.g., as a vaccinating or knockdown agent. We have hence synthesized bioconjugates of mRNA and siRNA containing covalently attached smTLRa and tested their function in TLR7 stimulation. Strikingly, the bioconjugates displayed decreased rather than synergistically increased stimulation. The decrease was distinct from the antagonistic action of an siRNA bearing a Gm motive, as observed by direct comparison of the effects in the presence of otherwise stimulatory RNA. In summary, these investigations showed that TRL7 activation can be impeded by bioconjugation of small molecules to RNA.

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Section: Resultssupporting

confidence: 92%

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7

et al. 2017

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“…Earlier reports on alkylation of the exocyclic guanosine amine had suggested minimal impact on duplex stability for single substitutions, and had also reported that significant differences in melting temperature of modified duplexes were only evident upon a double methylation ( 17 – 19 ). In agreement with this finding, our results showed that mono-labeling on the sugar edge of guanosine via its exocyclic amine had little impact on structure and RNAi activity of an siRNA labeled on the 5′ of the antisense strand ( 16 ). However, data on the impact of propargyl groups on exocyclic amines inside helices are still amiss.…”

Section: Introductionsupporting

confidence: 88%

“…The DNA strand corresponds in sequence to the passenger strand of an anti-eGFP siRNA which we had used in previous studies ( 16 ). To investigate the stability of helices containing propargylated cytidines, the respective oligodeoxynucleotides were thus hybridized (i) to an oligoribonucleotide identical to the antisense strand of the corresponding siRNA, and (ii) to an oligodeoxynucleotide of identical sequence.…”

Section: Resultsmentioning

confidence: 99%

“…A previous effort to introduce clickable units onto nucleosides was focused on N 2 -propargylated guanosine in RNA ( 16 ). Earlier reports on alkylation of the exocyclic guanosine amine had suggested minimal impact on duplex stability for single substitutions, and had also reported that significant differences in melting temperature of modified duplexes were only evident upon a double methylation ( 17 – 19 ).…”

Section: Introductionmentioning

confidence: 99%

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Intermolecular 'cross-torque': the N4-cytosine propargyl residue is rotated to the 'CH'-edge as a result of Watson-Crick interaction

Domingo

Hellmuth

Jäschke

et al. 2015

Nucleic Acids Research

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Propargyl groups are attractive functional groups for labeling purposes, as they allow CuAAC-mediated bioconjugation. Their size minimally exceeds that of a methyl group, the latter being frequent in natural nucleotide modifications. To understand under which circumstances propargyl-containing oligodeoxynucleotides preserve base pairing, we focused on the exocyclic amine of cytidine. Residues attached to the exocyclic N4 may orient away from or toward the Watson–Crick face, ensuing dramatic alteration of base pairing properties. ROESY-NMR experiments suggest a uniform orientation toward the Watson–Crick face of N4-propargyl residues in derivatives of both deoxycytidine and 5-methyl-deoxycytidine. In oligodeoxynucleotides, however, UV-melting indicated that N4-propargyl-deoxycytidine undergoes standard base pairing. This implies a rotation of the propargyl moiety toward the ‘CH’-edge as a result of base pairing on the Watson–Crick face. In oligonucleotides containing the corresponding 5-methyl-deoxycytidine derivative, dramatically reduced melting temperatures indicate impaired Watson–Crick base pairing. This was attributed to a steric clash of the propargyl moiety with the 5-methyl group, which prevents back rotation to the ‘CH’-edge, consequently preventing Watson–Crick geometry. Our results emphasize the tendency of an opposing nucleic acid strand to mechanically rotate single N4-substituents to make way for Watson–Crick base pairing, providing no steric hindrance is present on the ‘CH’-edge.

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“…We have previously validated several such FRET-siRNA systems qualitatively ( 37 ) and quantitatively ( 38 ) using EGFP as a reporter gene. The knockdown was routinely measured 24 h after transfection, and reached close to perfect (>95%) knockdown levels.…”

Section: Introductionmentioning

confidence: 99%

Live cell imaging of duplex siRNA intracellular trafficking

Hirsch

Helm

2015

Nucleic Acids Research

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Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.

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A modified guanosine phosphoramidite for click functionalization of RNA on the sugar edge

Cited by 23 publications

References 32 publications

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7

Intermolecular 'cross-torque': the N4-cytosine propargyl residue is rotated to the 'CH'-edge as a result of Watson-Crick interaction

Live cell imaging of duplex siRNA intracellular trafficking

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