Investigation of metal–organic frameworks (MOFs) for biomedical applications has attracted much attention in recent years. MOFs are regarded as a promising class of nanocarriers for drug delivery owing to well-defined structure, ultrahigh surface area and porosity, tunable pore size, and easy chemical functionalization. In this review, the unique properties of MOFs and their advantages as nanocarriers for drug delivery in biomedical applications were discussed in the first section. Then, state-of-the-art strategies to functionalize MOFs with therapeutic agents were summarized, including surface adsorption, pore encapsulation, covalent binding, and functional molecules as building blocks. In the third section, the most recent biological applications of MOFs for intracellular delivery of drugs, proteins, and nucleic acids, especially aptamers, were presented. Finally, challenges and prospects were comprehensively discussed to provide context for future development of MOFs as efficient drug delivery systems.

Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson-Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS-SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS-SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS-SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS-SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.

The next goals in the development of a synthetic biology that use artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and non-sequential non-standard nucleotides. This amplification must ensure that the non-standard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional non-standard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence “six-letter” GACTZP DNA. These are all not yet reported for any other synthetic genetic system.

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add additional functional groups, not found in natural DNA but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via strict Watson-Crick geometry. These were added to lies in a ibrarlaboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection systems, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Z’s and P’s. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system, and that GACTZP libraries are richer reservoir of functionality than standard libraries.

To support efforts to develop a ‘synthetic biology’ based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson–Crick complement, the purine analog 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where ‘py’ indicates a pyrimidine analog and ‘pu’ indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA.

DNA polymerases are identified that copy a non-standard nucleotide pair joined by a hydrogen bonding pattern different from the patterns joining the dA:T and dG:dC pairs. 6-Amino-5-nitro-3-(1′-β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) implements the non-standard ‘small’ donor–donor–acceptor (pyDDA) hydrogen bonding pattern. 2-Amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP) implements the ‘large’ acceptor–acceptor–donor (puAAD) pattern. These nucleobases were designed to present electron density to the minor groove, density hypothesized to help determine specificity for polymerases. Consistent with this hypothesis, both dZTP and dPTP are accepted by many polymerases from both Families A and B. Further, the dZ:dP pair participates in PCR reactions catalyzed by Taq, Vent (exo−) and Deep Vent (exo−) polymerases, with 94.4%, 97.5% and 97.5%, respectively, retention per round. The dZ:dP pair appears to be lost principally via transition to a dC:dG pair. This is consistent with a mechanistic hypothesis that deprotonated dZ (presenting a pyDAA pattern) complements dG (presenting a puADD pattern), while protonated dC (presenting a pyDDA pattern) complements dP (presenting a puAAD pattern). This hypothesis, grounded in the Watson–Crick model for nucleobase pairing, was confirmed by studies of the pH-dependence of mismatching. The dZ:dP pair and these polymerases, should be useful in dynamic architectures for sequencing, molecular-, systems- and synthetic-biology.

BackgroundZika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives.MethodsProcedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration.ResultsThe tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India.ConclusionsA kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-017-2382-0) contains supplementary material, which is available to authorized users.

We report the design of a single-molecule nanomotor driven by photons. The nanomotor is a DNA hairpin-structured molecule incorporated with azobenzene moieties to facilitate reversible photocontrollable switching. Upon repeated UV-vis irradiation, this nanomotor displayed 40-50% open-close conversion efficiency. This type of nanomotor displays well-regulated responses and can be operated under mild conditions with no output of waste. In contrast to multiple-component DNA nanomachines, the intramolecular interaction in this single-molecule system offers unique concentration-independent motor functionality. Moreover, the hairpin structure of the motor backbone can significantly improve the efficiency of light-to-movement energy conversion. These results suggest that azobenzene-incorporated, hairpin-structured single-molecule DNA nanomotors have promising potential for applications which require highly efficient light-driven molecular motors.Nucleic acid (DNA) self-assembly based on spontaneous hybridization between complementary strands is an effective way to construct multidimensional nano-objects such as macromolecular architecture, 1-6 biochip, 7 photonic wires, 8 enzyme assemblies, 9 and functional DNA probes. 10,11 Similar to RNA or protein-based nanostructures, 12-14 DNAbased nanomachines have been developed with the ability to change their conformation upon external stimuli. 15,16 Of special interest is a single DNA molecular motor developed by us a few years ago. 17 The single-molecule nanomotor can switch between two conformations and perform extending-shrinking motion with stable, convenient operation and high efficiency. However, this single-DNA nanomotor requires addition and removal of fuel and waste strands for motor function. 17,18 Although some artificial nanomotors can utilize alternative energy sources, including hydrolysis of the DNA backbone 19 and ATP, 20 applying an electromagnetic field as a convenient energy source is highly desired. As an alternative, a photon-driven single-DNA nanomotor would be of great scientific interest as well as contribute to applications in the nanosciences.Azobenzene molecules have been extensively studied due to their reversible isomerization between planar trans form and nonplanar cis form under UV and visible light irradiation. [21][22][23] The first light-powered molecular device using azobenzene polymer has successfully proved the concept of optomechanical energy conversion in a single device. 24 Several DNA-based nanostructures incorporated with azobenzene moiety have also been constructed and