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

Abstract

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...

Therminator DNA Polymerase: Modified Nucleotides and Unnatural Substrates

“…Locked nucleic acids (LNAs) contain a ribofuranose ring in a locked, single conformation leading to duplex stabilization (Vester and Wengel, 2004). Alternatively, “unlocked” nucleic acids (UNAs) synthesized by Therminator DNA Polymerase offer additional substrate functionalization (Dubois et al, 2012). Other examples of functionalized DNAs made by Therminator DNA Polymerase using modified nucleotides include 2′-fluoro-NTPs (Kasuya et al, 2014), glyceronucleotides (gNTPs) (Chen et al, 2009), 7′,5′-Bicyclo-NTPs (Diafa et al, 2017), 3-phosphono-L-Ala-dNMPs (Yang and Herdewijn, 2011; Giraut et al, 2012), 3′-2′-phosphonomethyl-threosyl-NTPs (Renders et al, 2007, 2008), 5′-3′-phosphonomethyl-dNTPs (Renders et al, 2007, 2008), 2′-deoxy-2′-isonucleoside (iNTPs) (Ogino et al, 2010), 3′-deoxyapionucleotide 3′-triphosphates (apioNTPs) (Kataoka et al, 2008, 2011), 5-trifluoromethyl-dUTP (Holzberger and Marx, 2009) and 4′-C-aminomethyl-2′-O-methyl-TTP (Nawale et al, 2012).…”

PCR Amplification of 4′-ThioDNA Using 2′-Deoxy-4′-thionucleoside 5′-Triphosphates

“…Since this method affords much longer sequences from readily available natural DNA templates, if successful, it would be a versatile method for use in a variety of applications. Therefore, intense efforts are currently underway toward efficient enzymatic synthesis of modified ONs by using not only primer extension and PCR amplification using DNA polymerases but also in vitro transcription with RNA polymerases. − A typical example of this research area is the investigation of the evolution of aptamers. − To the best of our knowledge, there are very few examples in which PCR proceeds in the presence of all four kinds of chemically modified dNTPs instead of natural dNTPs. Andreola et al reported PCR in the presence of all four kinds of α-phosphorothioate dNTPs (α-PS-dNTPs); however, the incorporation efficiency of the full length product was very low .…”

Unlocked nucleic acid modified primer-based enzymatic polymerization assay: towards allele-specific genotype detection of human platelet antigens

“…9,10 In addition, UNA nucleotides can also be recognised by polymerases, offering great potential in developing novel polymerase-based diagnostic assays involving UNA. 11 Single nucleotide polymorphisms (SNPs) refer to a specic genetic variation occurring in the whole genome, and there are several genetic disorders associated with SNPs. 12 Thus, SNPs detection or mismatch discrimination technology plays a key role in identifying the root cause of such diseases for the appropriate therapeutic interventions.…”

“… 9,10 In addition, UNA nucleotides can also be recognised by polymerases, offering great potential in developing novel polymerase-based diagnostic assays involving UNA. 11 …”