Polymerase-directed synthesis of C5-ethynyl locked nucleic acids

Veedu, Rakesh N.; Burri, Harsha Vardhan Reddy; Kumar, Pawan; Sharma, Pawan K.; Hrdlicka, Patrick J.; Vester, Birte; Wengel, Jesper

doi:10.1016/j.bmcl.2010.09.044

Cited by 21 publications

(11 citation statements)

References 46 publications

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“…Historically, the first key modification of therapeutic SSOs was the introduction of a phosphorothioated backbone, and more recent developments included modifications of the sugar moieties, eg, locked nucleic acids (LNAs). These modifications were shown to improve stability, target affinity and potency, and to reduce non-specific toxicities (Veedu et al , 2010; Vester and Wengel, 2004). Although numerous SSOs are currently being evaluated in clinical trials for various therapeutic indications, up to now only 2 SSO drugs have been approved (FDA Briefing Document, 2012; Perry and Balfour, 1999).…”

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confidence: 99%

The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides

et al. 2017

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Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.

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The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides

et al. 2017

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“…First, we synthesized UNA-C nucleoside 5 0 -triphosphates in small scale according to a procedure published for nucleoside triphosphate synthesis, 9 after which all four UNA triphosphates ( Fig. 1) were prepared in larger scale.…”

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Stepping towards highly flexible aptamers: enzymatic recognition studies of unlocked nucleic acid nucleotides

et al. 2012

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Enzymatic recognition of unlocked nucleic acid (UNA) nucleotides was successfully accomplished. Therminator DNA polymerase was found to be an efficient enzyme in primer extension reactions. Polymerase chain reaction (PCR) amplification of a 81 mer UNA-modified DNA library was efficiently achieved by KOD DNA polymerase.Development of nucleic acid therapeutics has attracted significant interest for the treatment of many diseases. With one aptamerbased drug, Macugen (Pegaptanib sodium), 1 on the market for the treatment of age-related macular degeneration (AMD), this class of nucleic acid constructs are an emerging attractive class of therapeutic molecules.2 Highly specific DNA or RNA aptamers with high binding affinity to their targets are normally selected from a large pool of oligonucleotides by in vitro selection processes.3 Chemically modified aptamers are used to improve pharmacodynamic, as well as pharmacokinetic properties. The application of modified nucleotides in the aptamer selection processes is rather limited due to their poor substrate specificities to polymerases. However, there are a few reports that describe the selection of aptamers in a single step with only one enzymatic protocol that involves polymerase chain reaction (PCR) amplification. 4 Establishing an efficient PCR method for a library containing modified nucleotides is a key step prior to successful selection of chemically modified aptamers.Unlocked nucleic acid (UNA) is an RNA mimic in which the bond between the C2 0 and C3 0 atoms of the ribose ring is cleaved, which results in high flexibility relative to the parent RNA monomer (Scheme 1).5 Thermodynamic stability of i-motif structures can be modulated by introducing UNA nucleotide monomers.6 Furthermore, UNA was found to be a useful modification in siRNA-based gene-silencing technology as it can protect the siRNAs from serum degradation and offer reduced off-target effects while retaining gene silencing potency. 7Most importantly, the applicability of UNA nucleotides in aptamer development was recently studied by systematically incorporating them in a known DNA aptamer against thrombin, whereby it was demonstrated that introduction of UNA monomers at specific positions significantly increased the binding affinity. 8 These remarkable findings clearly substantiate the need to develop UNA-modified aptamers from a large library by in vitro selection processes. Herein, for the first time, we report enzymatic recognition studies involving UNA nucleotides.We investigated UNA nucleotides as substrates for DNA polymerases in line with our current focus on one-step selection of UNA-modified DNA aptamers. First, we synthesized UNA-C nucleoside 5 0 -triphosphates in small scale according to a procedure published for nucleoside triphosphate synthesis, 9 after which all four UNA triphosphates (Fig. 1) were prepared in larger scale. Primer extension assays were carried out to screen six different DNA polymerases from both A-and B-families, including Therminator, KOD, Phusion, Pfu, Klenow, and Taq...

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“…Locked nucleic acids (LNAs) are nucleic acid analogues in which the sugar ring is locked by an O2'ÀC4' methylene linkage that conformationally restricts LNA monomers into an Ntype sugar puckering. [42][43][44] Veedu et al [45][46][47][48][49][50][51] and others [45,52] have synthesized LNA NTPs (9, Scheme 1) and studied their ability to serve as enzymatic substrates for the production of LNA-modified DNA encoded by a native or an LNA-modified DNA template. Veedu et al [46] first reported that LNA-TTP and LNA-ATP could serve as substrates for Phusion High-Fidelity DNA polymerase, which could incorporate up to three LNA-T and up to eight consecutive LNA-A nucleotides.…”

Section: Locked Nucleoside-5'-triphosphatesmentioning

confidence: 99%

Enzymatic Recognition of 2′‐Modified Ribonucleoside 5′‐Triphosphates: Towards the Evolution of Versatile Aptamers

2011

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The quest for effective, selective and nontoxic nucleic-acid-based drugs has led to designing modifications of naturally occurring nucleosides. A number of modified nucleic acids have been made in the past decades in the hope that they would prove useful in target-validation studies and therapeutic applications involving antisense, RNAi, aptamer, and ribozyme-based technologies. Since their invention in the early 1990s, aptamers have emerged as a very promising class of therapeutics, with one drug entering the market for the treatment of age-related macular degeneration. To combat the limitations of aptamers containing naturally occurring nucleotides, chemically modified nucleotides have to be used. In order to apply modified nucleotides in aptamer drug development, their enzyme-recognition capabilities must be understood. For this purpose, several modified nucleoside 5'-triphosphates were synthesized and investigated as substrates for various enzymes. Herein, we review studies on the enzyme-recognition of various 2'-sugar-modified NTPs that were carried out with a view to their effective utilization in SELEX processes to generate versatile aptamers.

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Polymerase-directed synthesis of C5-ethynyl locked nucleic acids

Cited by 21 publications

References 46 publications

The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides

The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides

Stepping towards highly flexible aptamers: enzymatic recognition studies of unlocked nucleic acid nucleotides

Enzymatic Recognition of 2′‐Modified Ribonucleoside 5′‐Triphosphates: Towards the Evolution of Versatile Aptamers

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