Junji Kawakami

In vitro selection of aptamers that act with Zn2

An in vitro selection was carried out with Zn2+ to isolate novel RNA molecules, zinc-dependent aptamers, that bind to HIV-1 Tat protein. RNAs bound to Tat were collected by using a nitrocellulose filter from a library of random RNAs and regenerated to the next generation of the RNA library by subsequent reverse transcription, polymerase chain reaction, and transcription. Sequences of the selected RNAs were determined after 6 and 12 rounds of the selection. The control clones after normal selection procedure with Mg2+ had a consensus UUG that resembled essential sequences of TAR or Arg aptamers. On the other hand, many unique sequences were revealed from a library selected with Zn2+ and the RNA with most abundant sequence (clone 31) bound to Tat tightly only when Zn2+ existed. The secondary structure of clone 31 RNA was predicted by using a computational prediction with our thermodynamic parameters and enzymatic scission of the RNA. Several model RNAs were prepared and the binding property of these RNAs to Tat were investigated. As a result, all the model RNAs did not reproduce the binding property of clone 31. Therefore, the Tat aptamer that acts with Zn2+ should require a relatively longer region of the sequence which is able to offer tertiary cooperation of several motifs for the binding.

Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro

Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing.

Self-assembling polymer micelle/clay nanodisk/doxorubicin hybrid injectable gels for safe and efficient focal treatment of cancer.

The purpose of this study was to fabricate a safe and effective doxorubicin (DOX)-delivery system for focal cancer chemotherapy. A novel biodegradable injectable gel was developed through self-assembly of poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (PLGA-PEG-PLGA) copolymer micelles, clay nanodisks (CNDs), and DOX. We discovered that DOX loaded in the hybrid gels acts as an anticancer drug and as a building block to organize new gel networks. Accordingly, long-term sustained release of DOX from hybrid injectable gels without initial burst release was achieved. Moreover, it was revealed that the DOX incorporated into gel networks controls its own release profile. This hybrid injectable gel is a self-controlled drug release system, which is a novel concept in controlled drug release. Importantly, a single injection of PLGA-PEG-PLGA/CND/DOX hybrid gel provides long-term sustained antitumor activity in vivo against human xenograft tumors in mice, suggesting the potential of hybrid gels as a valuable local DOX-delivery platform for cancer focal therapy.

Discovery of a new function of curcumin which enhances its anticancer therapeutic potency

Curcumin has received immense attention over the past decades because of its diverse biological activities and recognized as a promising drug candidate in a large number of diseases. However, its clinical application has been hindered due to extremely low aqueous solubility, chemical stability, and cellular uptake. In this study, we discovered quite a new function of curcumin, i.e. pH-responsive endosomal disrupting activity, derived from curcumin’s self-assembly. We selected anticancer activity as an example of biological activities of curcumin, and investigated the contribution of pH-responsive property to its anticancer activity. As a result, we demonstrated that the pH-responsive property significantly enhances the anticancer activity of curcumin. Furthermore, we demonstrated a utility of the pH-responsive property of curcumin as delivery nanocarriers for doxorubicin toward combination cancer therapy. These results clearly indicate that the smart curcumin assemblies act as promising nanoplatform for development of curcumin-based therapeutics.

In vitro selection of aptamers that recognize a monosaccharide

Abstract Attempts, were made to obtain RNAs that can bind to monosaccharides (galactose, glucose, and mannose) by in vitro selection. As a result, an RNA library that can bind to galactose with outstanding affinity has been obtained. Furthermore, the library was able to distinguish galactose from other monosaccharides, though they have closely similar structures. After another selection in order to obtain fast binders, a selected library tightly bound to galactose was obtained but it lost selectivity to galactose. This selectivity seemed to be derived from hydrogen bond geometry with the epimeric hydroxyl groups of monosaccharides. The results show an ability of RNAs to recognize any small molecule which consists of only carbon, hydrogen and oxygen.

Recognition of a Flipped Base in a Hairpinloop DNA by a Small Peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258–259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 × 106 variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use π–π stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 × 106 M−1 at 25°C and the resulting binding free energy change at 25°C (ΔG°25) was −8.2 kcal mol−1. The binding of the peptide to AP2 was also analyzed and the resulting binding constant and ΔG°25 were about 4.2 × 104 M−1 and −6.3 kcal mol−1, respectively. The difference in the binding free energy changes (ΔΔG°25) of 1.9 kcal mol−1 was comparable to the values reported in other systems and was considered a consequence of the loss of π–π stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.

A selection of short peptides that interact with a porphyrin as a small target by immobilized phage display

Porphyrin H2TMpyP binding peptides were selected from a phage displayed pentapeptide library that was immobilized onto a solid phase and, as a result, a binding motif that binds to the porphyrin in the same way as a motif obtained by our previous combinatorial chemistry was revealed.

Minimization of genomic human hepatitis delta virus ribozyme

In order to determine the minimum structure of the human hepatitis delta virus (HDV) ribozyme, several variants were constructed by replacing stem IV with UU, CG, A, C, G or U (MTS-N). Cleavage assays of these cis- and trans-acting shortened HDV ribozymes (cis- and trans-MTS-N) showed that almost all had cleavage activity except MTS-CG and MTS-G. Trans-MTS-CG/G seem to have different stable secondary structures from others by RNA folding program. Among them, both cis- and trans-MTS-U showed the most efficient activity. As a result of point mutations of trans-MTS-U in single-stranded regions, there are more strict base requirements at that position than that of cis-acting HDV ribozyme (HDV88). Therefore, stem IV plays a role to support an active conformation, and it is not directly involved in the catalytic core of the HDV ribozyme. Both cis- and trans-acting MTS-U are, to our knowledge, the smallest HDV ribozyme thus reported.

A Novel Stable RNA Pentaloop that Interacts Specifically with A Motif Peptide of Lambda-N Protein

To achieve a novel specific peptide–nucleic acid binding model, we designed an in vitro selection procedure to decrease the energetic contribution of the electrostatic interaction in the total binding energy and to increase the contribution of hydrogen bonding and π–π stacking. After the selection of hairpin-loop RNAs that specifically bound to a model peptide of lambda N protein (N peptide), a new thermostable pentaloop RNA motif (N binding thermostable RNA hairpin: NTS RNA) was revealed. The obtained NTS RNA was able to bind to the N peptide with superior specificity to the boxB RNA, which is the naturally occurring partner of the lambda N protein.

Quantitative relationship between chemical properties and bioactivities of anti-microRNA oligonucleotides targeted to tumor-associated microRNA-21.

Synthetic anti-microRNA oligonucleotides (AMOs) are promising drug candidates to inactivate disease-related microRNAs because of their sequence-specific binding to their targets and the variety of chemical modifications available. Over the last decade, the qualitative relationships between the chemical properties of AMOs and bioactivity (inactivation of their target miRNAs) have been studied to enhance their bioactivity. On the other hand, in real-world drug development, drugs must be designed case-by-case, taking many factors into account. Thus, in order to design AMOs that target specific miRNA, understanding the quantitative relationship between the chemical properties of AMOs and inactivation of their target miRNA is necessary. Here, we aimed to find the specific quantitative relationship of AMOs targeted to tumor-associated miR-21 through direct comparison of their inactivation efficacies with systematically varied chemical properties, including sequence-specific binding affinity, nuclease resistance, and RNase H activation. As a result, we newly found the quantitative relationships; (1) sequence-specific binding affinity of AMOs against miR-21 is the main determining factor for inactivation efficacy, (2) nuclease resistance of AMOs impacts their miR-21 inactivation efficacy acting cooperatively with the binding affinity, although nuclease resistance alone does not affect the miRNA inactivation efficacy, and (3) RNase H activation is unnecessary. This study also demonstrates the utility of the obtained relationship for the design of AMO-based drugs targeted to miR-21, through cell-based analyses. Thus, the obtained quantitative relationship would make it possible to predict the miR-21 inactivation efficacy of AMOs which are newly designed.