Chemical synthesis of RNA with site-specific methylphosphonate modifications

Flür, Sara; Micura, Ronald

doi:10.1016/j.ymeth.2016.03.024

Cited by 9 publications

(5 citation statements)

References 39 publications

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“…Both separations closely followed previous reports. 38,39 As previously observed, the Rp-PSO isomer eluted first. 39 Ultraviolet (UV) Thermal Melting Experiments with PO2-, PSO-, and PS2-SRL RNAs.…”

Section: ■ Experimental Proceduressupporting

confidence: 83%

“…The dAMePO-SRL was synthesized according to previously published protocols. 38 After completion of the automated synthesis, oligonucleotides were manually released from support and deprotected using 30% NH 4 OH and 5% diethylamine for 6 h at 55 °C. After filtration through a nylon syringe filter (0.45 μm), 2′hydroxyl groups were deprotected by treatment with Et 3 N• 3HF at 60 °C for 10 min.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

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Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost

Pallan¹,

Lybrand²,

Schlegel

et al. 2020

Biochemistry

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GNRA (N = A, C, G, or U; R = A or G) tetraloops are common RNA secondary structural motifs and feature a phosphate stacked atop a nucleobase. The rRNA sarcin/ricin loop (SRL) is capped by GApGA, and the phosphate p stacks on G. We recently found that regiospecific incorporation of a single dithiophosphate (PS2) but not a monothiophosphate (PSO) instead of phosphate in the backbone of RNA aptamers dramatically increases the binding affinity for their targets. In the RNA:thrombin complex, the key contribution to the 1000-fold tighter binding stems from an edge-on contact between PS2 and a phenylalanine ring. Here we investigated the consequences of replacing the SRL phosphate engaged in a face-on interaction with guanine with either PS2 or PSO for stability. We found that PS2···G and Rp-PSO···G contacts stabilize modified SRLs compared to the parent loop to unexpected levels: up to 6.3 °C in melting temperature T m and −4.7 kcal/mol in ΔΔG°. Crystal structures demonstrate that the vertical distance to guanine for the closest sulfur is just 0.05 Å longer on average compared to that of oxygen despite the larger van der Waals radius of the former (1.80 Å for S vs 1.52 Å for O). The higher stability is enthalpy-based, and the negative charge as assessed by a neutral methylphosphonate modification plays only a minor role. Quantum mechanical/molecular mechanical calculations are supportive of favorable dispersion attraction interactions by sulfur making the dominant contribution. A stacking interaction between phosphate and guanine (SRL) or uracil (U-turn) is also found in newly classified RNA tetraloop families besides GNRA.

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Section: ■ Experimental Proceduressupporting

confidence: 83%

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost

Pallan¹,

Lybrand²,

Schlegel

et al. 2020

Biochemistry

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“…Among these, the methylphosphonates (MPO) are the most important ( Figure 10 ). Their synthesis is simple by using methylphosphonoamidites in conventional oligonucleotide synthesis [ 128 , 129 ]. The MPOs are very resistant against nucleases.…”

Section: Artificial Nucleic Acidsmentioning

confidence: 99%

The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic Oligonucleotides

Bege

Borbás

2022

Pharmaceuticals

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Nucleic acids play a central role in human biology, making them suitable and attractive tools for therapeutic applications. While conventional drugs generally target proteins and induce transient therapeutic effects, nucleic acid medicines can achieve long-lasting or curative effects by targeting the genetic bases of diseases. However, native oligonucleotides are characterized by low in vivo stability due to nuclease sensitivity and unfavourable physicochemical properties due to their polyanionic nature, which are obstacles to their therapeutic use. A myriad of synthetic oligonucleotides have been prepared in the last few decades and it has been shown that proper chemical modifications to either the nucleobase, the ribofuranose unit or the phosphate backbone can protect the nucleic acids from degradation, enable efficient cellular uptake and target localization ensuring the efficiency of the oligonucleotide-based therapy. In this review, we present a summary of structure and properties of artificial nucleic acids containing nucleobase, sugar or backbone modifications, and provide an overview of the structure and mechanism of action of approved oligonucleotide drugs including gene silencing agents, aptamers and mRNA vaccines.

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“…Abeydeera et al [ 94 ] modified the single nucleotide of aptamer with phosphorodithioate, and the X-ray co-crystal structure of the α-thrombin: aptamer complex revealed a locally induced fitting rearrangement of this nucleotide, which significantly enhanced the affinity of aptamer by 1000-fold. However, some studies have shown that if the modified site and configuration of the modification are not appropriate, the formation of double strands with complementary DNA/RNA may be affected [ 95 ].…”

Section: Post-selex Optimization Of Aptamersmentioning

confidence: 99%

Systematic bio-fabrication of aptamers and their applications in engineering biology

Cai

Chen

Zhang

et al. 2022

Syst Microbiol and Biomanuf

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Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets. Compared to antibodies, aptamers are easy to in vitro synthesize with low cost, and exhibit excellent thermal stability and programmability. With these features, aptamers have been widely used in biology and medicine-related fields. In the meantime, a variety of systematic evolution of ligands by exponential enrichment (SELEX) technologies have been developed to screen aptamers for various targets. According to the characteristics of targets, customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity. In this review, we first summarize the latest research on the systematic bio-fabrication of aptamers, including various SELEX technologies, post-SELEX optimization, and aptamer modification technology. These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers. The latter provides a new perspective for the systems bio-fabrication of aptamers. Furthermore, on this basis, we review the applications of aptamers, particularly in the fields of engineering biology, including industrial biotechnology, medical and health engineering, and environmental and food safety monitoring. And the encountered challenges and prospects are discussed, providing an outlook for the future development of aptamers.

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Chemical synthesis of RNA with site-specific methylphosphonate modifications

Cited by 9 publications

References 39 publications

Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost

Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost

The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic Oligonucleotides

Systematic bio-fabrication of aptamers and their applications in engineering biology

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