Non-invasive single cell aptasensing in live cells and animals.

We report a genetically encoded aptamer biosensor platform for non-invasive measurement of drug distribution in cells and animals. We combined the high specificity of aptamer molecular recognition with the easy-to-detect properties of fluorescent proteins. We generated six encoded aptasensors, showcasing the platform versatility. The biosensors display high sensitivity and specificity for detecting their specific drug target over related analogs. We show dose dependent response of biosensor performance reaching saturating drug uptake levels in individual live cells. We designed our platform for integration into animal genomes; thus, we incorporated aptamer biosensors into zebrafish, an important model vertebrate. The biosensors enabled non-invasive drug biodistribution imaging in whole animals across different timepoints. To our knowledge, this is the first example of an aptamer biosensor-expressing transgenic vertebrate that is carried through generations. As such, our encoded platform addresses the need for non-invasive whole animal biosensing ideal for pharmacokinetic-pharmacodynamic analyses that can be expanded to other organisms and to detect diverse molecules of interest.

Disrupting Protein Expression with Double-Clicked sgRNA-Cas9 Complexes: A Modular Approach to CRISPR Gene Editing.

CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, the Cas9 endonuclease is guided toward its DNA target sequence by the guide RNA (gRNA). Chemical synthesis of a functional single gRNA (sgRNA) is nontrivial because of the length of the RNA strand. Recently we demonstrated that a sgRNA can be stitched together from three smaller fragments through a copper-catalyzed azide-alkyne cycloaddition, making the process highly modular. Here we further advance this approach by leveraging this modulator platform by incorporating chemically modified nucleotides at both ends of the modular sgRNA to increase resistance against ribonucleases. Modified nucleotides consisted of a 2'-O-Me group and a phosphorothioate backbone in varying number at both the 5'- and 3'-ends of the sgRNA. It was observed that three modified nucleotides at both ends of the sgRNA significantly increased the success of Cas9 in knocking out a gene of interest. Using these chemically stabilized sgRNAs facilitates multigene editing at the protein level, as demonstrated by successful knockout of both Siglec-3 and Siglec-7 using two fluorophores in conjunction with fluorescence-activated cell sorting. These results demonstrate the versatility of this modular platform for assembling sgRNAs from small, chemically modified strands to simultaneously disrupt the gene expression of two proteins.

CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs.

Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.

Enhanced mismatch selectivity of T4 DNA ligase far above the probe: Target duplex dissociation temperature.

T4 DNA ligase is a widely used ligase in many applications; yet in single nucleotide polymorphism analysis, it has been found generally lacking owing to its tendency to ligate mismatches quite efficiently. To address this lack of selectivity, we explored the effect of temperature on the selectivity of the ligase in discriminating single base pair mismatches at the 3'-terminus of the ligating strand using short ligation probes (9-mers). Remarkably, we observe outstanding selectivities when the assay temperature is increased to 7 °C to 13 °C above the dissociation temperature of the matched probe:target duplexes using commercially available enzyme at low concentration. Higher enzyme concentration shifts the temperature range to 13 °C to 19 °C above the probe:target dissociation temperatures. Finally, substituting the 5'-phosphate terminus with an abasic nucleotide decreases the optimal temperature range to 7 °C to 10 °C above the matched probe:target duplex. We compare the temperature dependence of the T4 DNA ligase catalyzed ligation and a nonenzymatic ligation system to contrast the origin of their modes of selectivity. For the latter, temperatures above the probe:target duplex dissociation lead to lower ligation conversions even for the perfect matched system. This difference between the two ligation systems reveals the uniqueness of the T4 DNA ligase's ability to maintain excellent ligation yields for the matched system at elevated temperatures. Although our observations are consistent with previous mechanistic work on T4 DNA ligase, by mapping out the temperature dependence for different ligase concentrations and probe modifications, we identify simple strategies for introducing greater selectivity into SNP discrimination based on ligation yields.

Quick Click: The DNA-Templated Ligation of 3'-O-Propargyl- and 5'-Azide-Modified Strands Is as Rapid as and More Selective than Ligase.

The copper(I)-mediated azide-alkyne cycloaddition (CuAAC) of 3'-propargyl ether and 5'-azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate-sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA-templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a kobs value of 1.1 min-1 , comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3'-propargyl ether and 5'-azide-terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.

Correction: The presence of a 5'-abasic lesion enhances discrimination of single nucleotide polymorphisms while inducing an isothermal ligase chain reaction.

Correction for 'The presence of a 5'-abasic lesion enhances discrimination of single nucleotide polymorphisms while inducing an isothermal ligase chain reaction' by Abu Kausar et al., Analyst, 2016, 141, 4272-4277.

The presence of a 5'-abasic lesion enhances discrimination of single nucleotide polymorphisms while inducing an isothermal ligase chain reaction.

Lesion-induced DNA amplification (LIDA) has been employed in the detection of single nucleotide polymorphisms (SNPs). Due to the presence of the proximal abasic lesion, T4 DNA ligase exhibits greater intolerance to basepair mismatches when compared with mismatch ligation in the absence of the abasic lesion. Moreover the presence of the abasic group also results in an isothermal ligase chain reaction enabling SNP detection with great discrimination and sensitivity. Specifically, at forty minutes, the ratio of amplified product from the matched and mismatched initiated reactions are 7-12 depending on the mismatch. The ease of implementation of our method is demonstrated by real-time analysis of DNA amplification using a fluorescent plate reader.