Breaking Down Barriers in Drug Delivery by Stromal Remodeling Approaches in Pancreatic Cancer.

Pancreatic cancer remains a formidable challenge in oncology due to its aggressive nature and limited treatment options. The dense stroma surrounding pancreatic tumors not only provides structural support but also presents a formidable barrier to effective therapy, hindering drug penetration and immune cell infiltration. This review delves into the intricate interplay between stromal components and cancer cells, highlighting their impact on treatment resistance and prognosis. Strategies for stromal remodeling, including modulation of cancer-associated fibroblasts (CAFs), pancreatic stellate cells (PSCs) activation states, and targeting extracellular matrix (ECM) components, are examined for their potential to enhance drug penetration and improve therapeutic efficacy. Integration of stromal remodeling with conventional therapies, such as chemotherapy and immunotherapy, is discussed along with the emerging field of intelligent nanosystems for targeted drug delivery. This comprehensive overview underscores the importance of stromal remodeling in pancreatic cancer treatment and offers insights into promising avenues for future research and clinical translation.

Repolarizing neutrophils via MnO2 nanoparticle-activated STING pathway enhances Salmonella-mediated tumor immunotherapy.

Engineered Salmonella has emerged as a promising microbial immunotherapy against tumors; however, its clinical effectiveness has encountered limitations. In our investigation, we unveil a non-dose-dependent type of behavior regarding Salmonella's therapeutic impact and reveal the regulatory role of neutrophils in diminishing the efficacy of this. While Salmonella colonization within tumors recruits a substantial neutrophil population, these neutrophils predominantly polarize into the pro-tumor N2 phenotype, elevating PD-L1 expression and fostering an immunosuppressive milieu within the tumor microenvironment. In order to bypass this challenge, we introduce MnO2 nanoparticles engineered to activate the STING pathway. Harnessing the STING pathway to stimulate IFN-ß secretion prompts a shift in neutrophil polarization from the N2 to the N1 phenotype. This strategic repolarization remodels the tumor immune microenvironment, making the infiltration and activation of CD8+ T cells possible. Through these orchestrated mechanisms, the combined employment of Salmonella and MnO2 attains the synergistic enhancement of anti-tumor efficacy, achieving the complete inhibition of tumor growth within 20 days and an impressive 80% survival rate within 40 days, with no discernible signs of significant adverse effects. Our study not only unveils the crucial in vivo constraints obstructing microbial immune therapy but also sets out an innovative strategy to augment its efficacy. These findings pave the way for advancements in cell-based immunotherapy centered on leveraging the potential of neutrophils.

Evaluation of bioequivalence and pharmacokinetic profiles for topical desonide cream using Chinese skin.

INTRODUCTION: Skin blanching assay has been established as a surrogate method for assessing bioequivalence of topical corticosteroids. This study aimed to apply the skin blanching assay to evaluate the bioequivalence of a test desonide cream (T) compared with the reference Desonide® (R) using Chinese skins. Additionally, the pharmacokinetics and safety profiles were also assessed. METHODS: By detecting the degree of skin blanching under different dose duration in a pilot dose-duration-response study, the area under the observed effect-time curve (AUEC) and half of the maximum effect (ED50) was calculated. Based on this, the skin color of different time points after a dose duration of ED50, D1 (0.5×ED50) and D2 (2×ED50) were detected as a pharmacodynamic indicator to compare between test and reference creams. A single-center, single-dose, randomized, open-label, two-cycle crossover pharmacokinetic studies were designed to make sure the exposure of tested formulations was not higher than that of the reference formulations. Subjects experiencing adverse events (AEs) were monitored and utilized for safety analysis. RESULTS: These studies involved twelve subjects for the dose-duration-response study, 100 subjects for the bioequivalence study, and twelve subjects for pharmacokinetic study. The results showed that the population ED50 was 0.88±0.45 h, the mean ratio of area under effective curve (AUEC0-24h) of test and reference preparations was 0.95, with a 90% confidence interval as 88.09%-101.72%, indicating the bioequivalence of the test formulation and Desonide®. The maximum concentration (Cmax) and exposure (AUC0-t) of T and R were 20.8 ± 11.5 pg/mL versus 19.7 ± 10.1 pg/mL, respectively, and 451.04 ± 363.65 pg∙h/mL versus 541.47 ± 581.41 pg∙h/mL, respectively. The systemic exposure of a single dose of the test cream was not higher than that of the reference preparation. All of the volunteers experienced grade 1 adverse events (AEs), suggesting that single administration of the test desonide cream is well tolerated in the Chinese healthy population. CONCLUSIONS: This study demonstrated the applicability of skin blanching assay in Chinese skins and established the bioequivalence of test and reference desonide creams.

Radicals Scavenging MOFs Enabling Targeting Delivery of siRNA for Rheumatoid Arthritis Therapy.

Macrophages play essential roles in the progression of rheumatoid arthritis (RA), which are polarized into the pro-inflammatory M1 phenotype with significant oxidative stress and cytokines excretion. Herein, an active targeting nanomedicine based on metal-organic frameworks (MOFs) to re-educate the diseased macrophages for RA therapy is reported. The MOFs are prepared via coordination between tannic acid (TA) and Fe3+ , and anti-TNF-α siRNA is loaded via a simple sonication process, achieving high loading capacity comparable to cationic vectors. The MOFs show excellent biocompatibility, and enable rapid endo/lysosome escape of siRNA via the proton-sponge effect for effective cytokines down-regulation. Importantly, such nanomedicine displays intrinsic radicals scavenging capability to eliminate a broad spectrum of reactive oxygen and nitrogen species (RONS), which in turn repolarizes the M1 macrophages into anti-inflammatory M2 phenotypes for enhanced RA therapy in combination with siRNA. The MOFs are further modified with bovine serum albumin (BSA) to allow cascade RA joint and diseased macrophages targeted delivery. As a result, an excellent anti-RA efficacy is achieved in a collagen-induced arthritis mice model. This work provides a robust gene vector with great translational potential, and offers a vivid example of rationally designing MOF structure with multifunctionalities to synergize with its payload for enhanced disease treatment.

A double-blind, randomized, placebo and positive-controlled study in healthy volunteers to evaluate pharmacokinetic and pharmacodynamic properties of multiple oral doses of cetagliptin.

AIMS: This study investigated the pharmacokinetics and pharmacodynamics properties, safety and tolerability of cetagliptin. METHODS: Forty-eight healthy subjects were enrolled in this study. Three cohorts were investigated in sequential order: 50, 100 and 200 mg cetagliptin. Positive control (sitagliptin 100 mg) was designed as open label. Blood samples were collected and analysed for pharmacokinetic and pharmacodynamic properties. Safety and tolerability were assessed throughout the study. RESULTS: Following multiple oral doses, cetagliptin was rapidly absorbed and reached peak plasma concentrations after approximately 1.0-1.5 hours. Plasma cetagliptin concentrations increased at a rate greater than dose. Accumulation of cetagliptin was modest, and steady state was generally achieved at day 5. Doses ≥50 mg of cetagliptin administered once daily will result in sustained dipeptidyl peptidase-4 (DPP-4) inhibition (≥80%). The plasma concentration giving 50% of maximum drug effect of DPP-4 inhibition for cetagliptin (5.29 ng/mL) was lower than that of sitagliptin (7.03 ng/mL). Active glucagon-like-1 peptide (GLP-1) concentrations were significantly increased in the cetagliptin groups by 2.3- to 3.1-fold at day 1 and 3.1- to 3.6-fold at steady state compared with that of placebo, and active GLP-1 concentrations were increased with increasing dose. Compared with sitagliptin, doses ≥100 mg once daily of cetagliptin produced postprandial increases in active GLP-1 level and induced to long-lasting glucose-lowering efficacy. Cetagliptin was well tolerated across all doses studied. CONCLUSION: Cetagliptin demonstrates the great potential for treatment with type 2 diabetes patients based on the inhibition of DPP-4, the increase in GLP-1 and insulin, the decrease in glucose, and might be more effective in DPP-4 inhibition than sitagliptin.

The metabolism and excretion of the dipeptidyl peptidase 4 inhibitor [14C] cetagliptin in healthy volunteers.

The metabolism and excretion of cetagliptin were investigated in healthy male subjects after a single oral dose of 100mg/50µCi [14C] cetagliptin.The mean concentration-time profile of cetagliptin was similar to that of total radioactivity in plasma after oral administration of [14C] cetagliptin in healthy male subjects. Cetagliptin was rapidly absorbed after oral administration. Unchanged cetagliptin was the most abundant radioactive component in all matrices investigated. Approximately 53.13% of plasma AUC of total radioactivity was accounted for by cetagliptin. Each metabolite plasma AUC was not higher than 2.93% of plasma AUC of total radioactivity. By 336 h after administration, 91.68% of the administered radioactivity was excreted, and the cumulative excretion in the urine and faeces was 72.88% and 18.81%, respectively. The primary route of excretion of radioactivity was via the kidneys.Four metabolites were detected at trace levels, and it involved hydroxylated (M436-1 and M436-3), N- sulphate (M500), and N-carbamoyl glucuronic acid conjugates (M640B) of cetagliptin. These metabolites were detected also in plasma, urine, and faeces at low levels, except that metabolite M640B was not detected in faeces. All metabolites were observed with <10% of parent compound systemic exposure after oral administration.

"Trojan Horse" Salmonella Enabling Tumor Homing of Silver Nanoparticles via Neutrophil Infiltration for Synergistic Tumor Therapy and Enhanced Biosafety.

Salmonella selectively colonizes into the hypoxic tumor region and exerts antitumor effects via multiple mechanisms, while the tumor colonized Salmonella recruits host neutrophils into the tumor, presenting a key immunological restraint to compromise the Salmonella efficacy. Here, we develop a combinatorial strategy by employing silver nanoparticles (AgNPs) to improve the efficacy and biosafety of Salmonella. The AgNPs were decorated with sialic acid (SA) to allow selective recognition of L-selectin on neutrophil surfaces, based on which the tumor-homing of AgNPs was achieved by neutrophil infiltration in the Salmonella colonized tumor. The tumor-targeting AgNPs exert the functions of (1) local depletion of neutrophils in tumors to boost the efficacy of Salmonella, (2) direct killing tumor cells via L-selectin-mediated intracellular delivery, and (3) clearing the residual Salmonella after complete tumor eradication to minimize the side effects. With a single tail vein injection of such combination treatment, the tumor was eliminated with high biosafety, resulting in a superior therapeutic outcome.

A cyclic nano-reactor achieving enhanced photodynamic tumor therapy by reversing multiple resistances.

BACKGROUND: Photodynamic therapy (PDT) is a clinically implemented modality to combat malignant tumor, while its efficacy is largely limited by several resistance factors from tumor microenvironment (TME), such as hypoxia, anti-oxidant systems, and ATP-dependent tumor adaptive resistances. The aim of this work is to construct a multifunctional nanoplatform to remodel multiple resistant TME for enhanced PDT. RESULTS: Here, a targeting nano-reactor was facilely constructed to reverse the multiple resistances of PDT by incorporating glucose oxidase (GOx) and chlorin e6 (Ce6) into poly (D, L-lactic-co-glycolic acid) (PLGA)/ metal-organic framework (MOF) core-shell nanoassembly, with surface deposition of hyaluronic acid (HA) stabilized MnO2. The nano-reactor could selectively target tumor cells by virtue of surface HA modification, and once internalization, a few reactions were initiated to modulate TME. Glucose was consumed by GOx to inhibit ATP generation, and the produced H2O2 was catalyzed by MnO2 to generate O2 for tumor hypoxia alleviation and photodynamic sensitization, and glutathione (GSH) was also effectively depleted by MnO2 to suppress the tumor antioxidant defense. Consequently, the nano-reactor achieved robust PDT with amplified tumor therapy via intravenous injection. CONCLUSIONS: This nano-reactor offers a multifunctional nanoplatform to sensitize TME-limited tumor treatment means via reversing multiple resistances.

In vitro study of the drug-drug interaction potential of cetagliptin and clinical study of pharmacokinetic interaction of cetagliptin and metformin in healthy volunteers.

Cetagliptin is an oral, potent, and newly developed selective inhibitor of dipeptidyl peptidase-4 (DPP-4). We evaluated the in vitro drug-drug interaction (DDI) potential of cetagliptin, as well as the pharmacokinetics of cetagliptin and metformin and the interaction between cetagliptin and metformin.Cetagliptin did not inhibit CYP1A2, CYP2C8, CYP2B6, CYP2C9, CYP2C19, and CYP3A4, only has a moderate inhibitory effect on CYP2D6, and did not induce CYP1A2, CYP2B6, and CYP3A4. Plasma protein binding of cetagliptin didn't have species differences or concentration dependence. Cetagliptin was a substrate for P-glycoprotein (P-gp).The 34 healthy subjects enrolled were randomly divided into two sequences (A and B) with 17 subjects in each sequence. Coadministration with metformin had no effect on cetagliptin AUC0-120 (GMR, 99.25%; 90% CI, 95.96%-102.65%). There was a slightly increase in cetagliptin Cmax (GMR, 117.33%; 90% CI, 102.54%-134.25%). Coadministration with cetagliptin did not affect the metformin's AUC0-24 (GMR, 108.54%; 90% CI, 101.41%-116.17%) or Cmax (GMR, 97.67%; 90% CI, 90.96%-104.89%).Based on in vitro study results, cetagliptin is unlikely to cause CYP-mediated, clinically relevant DDI. Although the possibility of transporter-mediated, clinically relevant DDI cannot be ruled out, there is little or no risk of side effects. Coadministration of cetagliptin and metformin had no clinically meaningful effect on the pharmacokinetics of each drug. There was no drug-drug interaction between cetagliptin and metformin. Both monotherapies and combination therapy were well tolerated. No serious AEs and hypoglycaemia was reported.

Nanoscale Copper(II)-Diethyldithiocarbamate Coordination Polymer as a Drug Self-Delivery System for Highly Robust and Specific Cancer Therapy.

Disulfiram (DSF), an old alcohol-aversion drug, has been repurposed for cancer therapy, and mechanistic studies reveal that it needs to be metabolized to diethyldithiocarbamate (DTC) and subsequently coordinates with copper(II) to form the DTC-copper complex (CuET) for anticancer activation. Here, we utilized this mechanism to construct a CuET self-delivery nanosystem based on the metal coordination polymer for highly robust and selective cancer therapy. In our design, the nanoparticles were facilely prepared under mild conditions by virtue of the strong coordination between Cu2+ and DTC, yielding 100% CuET loading capacity and allowing for further hyaluronic acid (HA) modification (CuET@HA NPs). The CuET@HA NPs could selectively deliver into cancer cells and release the active component of CuET in response to both endo/lysosome acidic pH and intracellular abundant GSH, which induces strong cytotoxicity toward cancer cells over normal cells taking advantage of the p97 pathway interference mechanism. Upon intravenous injection, the self-assembled system could passively accumulate into a tumor and elicit potent tumor growth inhibition at a dose of 1 mg/kg without any noticeable side effects. Given the cost-effective and easily scaled-up preparation, our designed nanosystem provides a promising strategy to pave the way for clinical translation of DSF-based cancer chemotherapy.

Polyarginine-Mediated siRNA Delivery: A Mechanistic Study of Intracellular Trafficking of PCL-R15/siRNA Nanoplexes.

As a cell-penetrating peptide, polyarginine is widely used in drug delivery systems based on its membrane permeation ability. Previously, we developed the mPEG-PLA-b-polyarginine(R15) triblock copolymer, which exhibited a high siRNA delivery efficiency both in vitro and in vivo. As a continued effort, here the amphiphilic diblock polymer PCL-R15 was synthesized as a simplified model to further elucidate the structure-activity relationship of arginine-based amphiphilic polymers as siRNA delivery systems, and the cellular trafficking mechanisms of the PCL-R15/siRNA nanoplexes were investigated to understand the interaction patterns between the nanoplexes and cells. Compared to the R15/siRNA complexes, the introduction of PCL moiety was found to result in the stronger interactions with cells and the enhanced transfection efficiency after the formation of condensed nanoplexes. Caveolae-mediated endocytosis and clathrin-mediated endocytosis were major routes for the internalization of PCL-R15/siRNA nanoplexes. The intracellular release of siRNA from nanoplexes was confirmed by fluorescence resonance energy transfer assay. It was also noticed that the internalized PCL-R15/siRNA nanoplexes were transported through digestive routes and trapped in lysosomes, which may be the bottleneck for efficient siRNA delivery of PCL-R15/siRNA nanoplexes. This study investigated the relationship between the polymer structure of PCL-R15 and the cellular interaction patterns, which may render implications on the rational design of polyarginine-based siRNA delivery systems.

Design, synthesis and evaluation of quinolinone derivatives containing dithiocarbamate moiety as multifunctional AChE inhibitors for the treatment of Alzheimer's disease.

A series of novel quinolinone derivatives bearing dithiocarbamate moiety were designed and synthesised as multifunctional AChE inhibitors for the treatment of AD. Most of these compounds exhibited strong and clearly selective inhibition to eeAChE. Among them, compound 4c was identified as the most potent inhibitor to both eeAChE and hAChE (IC50 = 0.22 µM for eeAChE; IC50 = 0.16 µM for hAChE), and it was also the best inhibitor to AChE-induced Aß aggregation (29.02% at 100 µM) and an efficient inhibitor to self-induced Aß aggregation (30.67% at 25 µM). Kinetic and molecular modelling studies indicated that compound 4c was a mixed-type inhibitor, which could interact simultaneously with the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 4c had good ability to cross the BBB, showed no toxicity on SH-SY5Y neuroblastoma cells and was well tolerated in mice at doses up to 2500 mg/kg (po).

An aptamer-tethered, DNAzyme-embedded molecular beacon for simultaneous detection and regulation of tumor-related genes in living cells.

Simultaneous detection and regulation of tumor-related genes presents a promising strategy for early diagnosis and treatment of cancer, but achieving this has been a huge challenge for both chemical and biomedical communities. Towards this objective, we have devised a novel aptamer-tethered, DNAzyme-embedded molecular beacon (MB) for multiple functions in cancer cells. In this design, a tumor targeting aptamer was employed to specifically deliver the sensor into cancer cells for target gene detection, and an RNA-cleaving DNAzyme was embedded to realize gene regulation. Both aptamer-tethering and DNAzyme-embedding had little influence on the sensor performance, with a detection limit of ∼2 nM and high specificity. After delivering into tumor cells, our device could monitor the tumor-related genes by producing detectable fluorescence signals, and regulate the gene expression at both mRNA and protein levels as evidenced by the RT-PCR and western blot analyses. This study provides a simple and efficient strategy to rationally combine various functional nucleic acids for multi-functional applications in living cells, which hold great potential for cancer diagnosis and therapy.

A tetrahedral DNA nanoflare for fluorometric determination of nucleic acids and imaging of microRNA using toehold strands.

The authors describe a tetrahedral DNA nanostructure loaded with SYBR Green (SG-TDN) for fluorometric determination of nucleic acids. After intercalating into the TDN, fluorescence of SG is enhanced by 260-fold (exc 480 nm, em 524 nm), and the resulting SG-TDN nanoflare displays >7-fold stronger fluorescence than that of FAM-labeled TDN. The SG-TDNs were coupled to magnetic microparticles and polydopamine nanoparticles to construct multi-functional nanoprobes through sequence hybridization using a toehold strand. The method was applied to detect a stretch of microRNA sequence (20 bp) in buffer and in undiluted serum with excellent selectivity, over a wide linear range and with a low limit of detection (0.2 nM). The probe was also applied for visualization of tumor-related microRNA in living cells via fluorescence imaging. Graphical abstract Schematic representation of tetrahedron-based DNA nanoflare for fluorometric nucleic acid determination in undiluted blood serum and living cells.

A Facile Way to Increase the Cellular Uptake Efficiency of Hybrid Nanoparticles.

Lipid-polymer hybrid nanoparticles composed of polymer cores and lipid shells have been intensively studied as cancer drug delivery systems. The aim of the present study was to investigate the effect of phosphatidylcholine (PC) on physicochemical properties, stability and cellular uptake of lipid-poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticles. Coumarin-6 (cou-6) loaded hybrid nanoparticles (NPs) were prepared using PC with different alkyl chain lengths from C12 to C18, and were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and encapsulation efficiency (EE). The quality and quantity of cellular uptake of NPs were carefully assessed. The NPs were 140-180 nm in size, negatively charged of 7-12 mV and with EE values higher than 80%. NPs remained stable in storage at 4 °C for 28 d. Cell viability rates of NPs were above 90%, and the as-prepared nanoparticles showed excellent biocompatibility by MTT assay. Interestingly, the uptake order was as follows: C12 < C14 < C16-C18. As the alkyl chain length of PC increased, the cellular uptake efficiency of hybrid nanoparticles was enhanced. C16 to C18 saturated PC exhibited the highest cellular uptake efficiency and did not significantly differ. PC had little or no effect on physicochemical properties and stability but did affect cellular uptake of hybrid nanoparticles. The obtained findings could provide a fundamental basis for rational design of hybrid nanoparticles and a facile way to improve the cellular uptake of hybrid nanoparticles.

A highly specific sodium aptamer probed by 2-aminopurine for robust Na+ sensing.

Sodium is one of the most abundant metals in the environment and in biology, playing critical ecological and physiological roles. Na+ is also the most common buffer salt for nucleic acids research, while its specific interaction with DNA has yet to be fully studied. Herein, we probe a highly selective and robust Na+ aptamer using 2-aminopurine (2AP), a fluorescent adenine analog. This aptamer has two DNA strands derived from the Ce13d DNAzyme. By introducing a 2AP at the cleavage site of the substrate strand, Na+ induces ∼40% fluorescence increase. The signaling is improved by a series of rational mutations, reaching >600% with the C10A20 double mutant. This fluorescence enhancement suggests relaxed base stacking near the 2AP label upon Na+ binding. By replacing a non-conserved adenine in the enzyme strand by 2AP, Na+-dependent fluorescence quenching is observed, suggesting that the enzyme loop folds into a more compact structure upon Na+ binding. The fluorescence changes allow for Na+ detection. With an optimized sequence, a detection limit of 0.4 mM Na+ is achieved, reaching saturated signal in less than 10 s. The sensor response is insensitive to ionic strength, which is critical for Na+ detection.

A DNAzyme requiring two different metal ions at two distinct sites.

Most previously reported RNA-cleaving DNAzymes require only a single divalent metal ion for catalysis. We recently reported a general trivalent lanthanide-dependent DNAzyme named Ce13d. This work shows that Ce13d requires both Na(+) and a trivalent lanthanide (e.g. Ce(3+)), simultaneously. This discovery is facilitated by the sequence similarity between Ce13d and a recently reported Na(+)-specific DNAzyme, NaA43. The Ce13d cleavage rate linearly depends on the concentration of both metal ions. Sensitized Tb(3+) luminescence and DMS footprinting experiments indicate that the guanines in the enzyme loop are important for Na(+)-binding. The Na(+) dissociation constants of Ce13d measured from the cleavage activity assay, Tb(3+) luminescence and DMS footprinting are 24.6, 16.3 and 47 mM, respectively. Mutation studies indicate that the role of Ce(3+) might be replaced by G23 in NaA43. Ce(3+) functions by stabilizing the transition state phosphorane, thus promoting cleavage. G23 competes favorably with low concentration Ce(3+) (below 1 µM). The G23-to-hypoxanthine mutation suggests the N1 position of the guanine as a hydrogen bond donor. Together, Ce13d has two distinct metal binding sites, each fulfilling a different role. DNAzymes can be quite sophisticated in utilizing metal ions for catalysis and molecular recognition, similar to protein metalloenzymes.

Two Completely Different Mechanisms for Highly Specific Na+ Recognition by DNAzymes.

Our view of the interaction between Na+ and nucleic acids was changed by a few recently discovered Na+ -specific RNA-cleaving DNAzymes. In addition to nonspecific electrostatic interactions, highly specific recognition is also possible. Herein, two such DNAzymes, named EtNa and Ce13d, are compared to elucidate their mechanisms of Na+ binding. Mutation studies indicate that they have different sequence requirements. Phosphorothioate (PS) substitution at the scissile phosphate drops the activity of EtNa 140-fold, and it cannot be rescued by thiophilic Cd2+ or Mn2+ , whereas the activity of PS-modified Ce13d can be rescued. Na+ -dependent activity assays indicate that two Na+ ions bind cooperatively in EtNa, and each Na+ likely interacts with a nonbridging oxygen atom in the scissile phosphate, whereas Ce13d binds only one Na+ ion in a well-defined Na+ aptamer, and this Na+ ion does not directly interact with the scissile phosphate. Both DNAzymes display a normal pH-rate profile, with a single deprotonation reaction required for catalysis. For EtNa, Na+ fails to protect the conserved nucleotides from dimethyl sulfate attack, and no specific Na+ binding is detected by 2-aminopurine fluorescence, both of which are different from those observed for Ce13d. This work suggests that EtNa binds Na+ mainly through its scissile phosphate without significant involvement of the nucleotides in the enzyme strand, whereas Ce13d has a well-defined aptamer for Na+ binding. Therefore, DNA has at least two distinct ways to achieve highly selective Na+ binding.

An Exceptionally Selective DNA Cooperatively Binding Two Ca2+ Ions.

Ca2+ is a highly important metal ion in biology and in the environment, and thus there is extensive work in developing sensors for Ca2+ detection. Although many Ca2+ -binding proteins are known, few nucleic acids can selectively bind Ca2+ . DNA-based biosensors are attractive for their high stability and excellent programmability. We report a RNA-cleaving DNAzyme, EtNa, cooperatively binding two Ca2+ ions but to only one Mg2+ . Four DNAzymes with known Ca2+ -dependent activity were compared, and the EtNa had the best selectivity for Ca2+ . The EtNa is 90 times more active in Ca2+ than in Mg2+ . Phosphorothioate (PS) modification showed that both non-bridging oxygen atoms at the scissile phosphate contribute equally to Ca2+ binding. The pH-rate profile suggests two concurrent deprotonation reactions. EtNa was further engineered for Ca2+ sensing, and found to have a detection limit of 17 µm Ca2+ and excellent selectivity. The detection of Ca2+ in tap water was performed, and the result was comparable with that by ICP-MS. This study offers new fundamental insights into Ca2+ binding by nucleic acids and improved metal selectivity by having multiple cooperative metal binding sites.

Splitting a DNAzyme enables a Na+-dependent FRET signal from the embedded aptamer.

Recently, a few Na+-specific RNA-cleaving DNAzymes have been reported, and a Na+ aptamer was identified from the NaA43 and Ce13d DNAzymes. These DNAzymes and the embedded aptamer have been used for Na+ detection. In this work, we studied the Na+-dependent folding of the Ce13d DNAzyme using fluorescence resonance energy transfer (FRET). When a FRET donor and an acceptor were respectively labeled at the ends of the DNAzyme, Na+ failed to induce an obvious end-to-end distance change, suggesting a rigid global structure. To relax this rigidity, the Ce13d DNAzyme was systematically split at various sites on both the enzyme and the substrate strands. The Na+ binding activity of the split structures was characterized by 2-aminopurine fluorescence, enzymatic activity, Tb3+-sensitized luminescence, and DMS footprinting. Among the various constructs, the only one that retained Na+ binding was the split at the cleavage site, and this construct was further labeled with two dyes near the split site. This FRET result showed Na+-dependent folding with a Kd of 26 mM Na+. This study provides important structural information related to Na+ binding to this new aptamer, which might also be useful for future work in biosensor design.