Kazuhiro Furukawa

PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside 5'-triphosphates.

2-Deoxy-4-thioribonucleic acid (4-thioDNA) having a sulfur atom instead of an oxygen atom in the furanose ring has a nuclease resistance and hybridization ability higher than that of natural DNA. Despite its great potential for various biological applications, a long 4-thioDNA having all four kinds of 2-deoxy-4-thionucleosides has not been reported. In this study, we describe systematic analysis of the incorporation of 2-deoxy-4-thionucleoside 5-triphosphates (dSNTPs) using various DNA polymerases. We found that family B DNA polymerases, which do not have 3→5 exonuclease activity, could efficiently incorporate dSNTPs via single nucleotide insertion and primer extension. Moreover, 104-mer PCR product was obtained even under the conditions in the presence of all four kinds of dSNTPs when KOD Dash DNA polymerase was used. The resulting PCR product was converted into a natural dsDNA by using PCR with dNTPs, and sequencing of the natural dsDNA revealed that the PCR cycle successfully proceeded without losing the sequence information of the template. To the best of our knowledge, this is the first example of accurate PCR amplification of highly modified DNA in the presence of only unnatural dNTPs.

Allosteric control of a DNA-hydrolyzing deoxyribozyme with short oligonucleotides and its application in DNA logic gates

Allosteric control of deoxyribozymes is useful for a broad range of practical applications, such as nucleic acid sensing and DNA-computing. We found that the catalytic activity of a DNA-hydrolyzing deoxyribozyme could be allosterically regulated by adding short oligonucleotides. We used this technique to construct deoxyribozyme-based logic gates.

Chemistry, Properties, and in Vitro and in Vivo Applications of 2′‐O‐Methoxyethyl‐4′‐thioRNA, a Novel Hybrid Type of Chemically Modified RNA

We report the synthesis, properties, and in vitro and in vivo applications of 2′‐O‐methoxyethyl‐4′‐thioRNA (MOE‐SRNA), a novel type of hybrid chemically modified RNA. In its hybridization with complementary RNA, MOE‐SRNA showed a moderate improvement of Tm value (+3.4u2009°C relative to an RNA:RNA duplex). However, the results of a comprehensive comparison of the nuclease stability of MOE‐SRNA relative to 2′‐O‐methoxyethylRNA (MOERNA), 2′‐O‐methyl‐4′‐thioRNA (Me‐SRNA), 2′‐O‐methylRNA (MeRNA), 4′‐thioRNA (SRNA), and natural RNA revealed that MOE‐SRNA had the highest stability (t1/2>48 h in human plasma). Because of the favorable properties of MOE‐SRNA, we evaluated its in vitro and in vivo potencies as an anti‐microRNA oligonucleotide against miR‐21. Although the in vitro potency of MOE‐SRNA was moderate, its in vivo potency was significant for the suppression of tumor growth (similar to that of MOERNA).

Faithful PCR Amplification of an Unnatural Base‐Pair Analogue with Four Hydrogen Bonds

In vitro replication of an unnatural imidazopyridopyridine:naphthyridine base pair, (i.e., ImN(N):NaO(O)), having four hydrogen bonds was investigated. Kinetic studies of single-nucleotide insertion revealed that ImN(N) and NaO(O) were recognized as complementary bases by an exonuclease-deficient Klenow fragment with higher specificity and efficiency than two previously described pairs (ImN(O):NaO(N) and ImO(N):NaN(O)) because of higher thermal and thermodynamic stabilities and the DAAD:ADDA (D=donor, A=acceptor) hydrogen-bonding pattern of the ImN(N):NaO(O) pair. Faithful polymerase chain reaction (PCR) amplification of a DNA fragment containing the ImN(N):NaO(O) pair was achieved by using DNA polymerases possessing 3→5 exonuclease activity (≈99.5u2009% per doubling).

Gene Silencing Using 4′-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4′-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4′-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4′-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response.

First Synthesis of Fully Modified 4′-SelenoRNA and 2′-OMe-4′-selenoRNA Based on the Mechanistic Considerations of an Unexpected Strand Break

This study investigated oligonucleotide (ON) synthesis containing 4-selenoribonucleoside(s) under standard phosphoramidite conditions. Careful operation using a manual ON synthetic system revealed that an unexpected strand break occurred to afford a C2-symmetric homodimer as a byproduct. In addition, this side reaction occurred during I2 oxidation. On the basis of these findings, the first synthesis of fully modified 4-selenoRNA and 2-OMe-4-selenoRNA was achieved using tert-butyl hydroperoxide (TBHP) as the alternative oxidant.

Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2'-modified-4'-thionucleosides.

Gene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a target domain and a U1 domain, we prepared adaptor ONs using 2-modified-4-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2-fluoro-4-thionucleoside and 2-fluoronucleoside units as well as only 2-fluoronucleoside units, while those prepared as combination of 2-OMe nucleoside/2-OMe-4-thionucleoside and 2-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity.

Synthesis and evaluation of c-di-4′-thioAMP as an artificial ligand for c-di-AMP riboswitch

Cyclic-di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that binds to an RNA receptor called riboswitch and regulates its downstream genes involving cell wall metabolism, ion transport, and spore germination. Therefore, the c-di-AMP riboswitch can be a novel target of antibiotics. In this study, we synthesized c-di-4-thioAMP (1), which possesses a sulfur atom instead of an oxygen atom in the furanose ring, as a candidate of a bioisoster for natural c-di-AMP. The resulting 1 bound to the c-di-AMP riboswitch with a micromolar affinity (34.8μM), and the phosphodiesterase resistance of 1 was >12-times higher than that of c-di-AMP. Thus, 1 can be considered to be a stable ligand against a c-di-AMP riboswitch.