New realm of precision multiplexing enabled by massively-parallel single molecule UltraPCR.

PCR has been a reliable and inexpensive method for nucleic acid detection in the past several decades. In particular, multiplex PCR is a powerful tool to analyze many biomarkers in the same reaction, thus maximizing detection sensitivity and reducing sample usage. However, balancing the amplification kinetics between amplicons and distinguishing them can be challenging, diminishing the broad adoption of high order multiplex PCR panels. Here, we present a new paradigm in PCR amplification and multiplexed detection using UltraPCR. UltraPCR utilizes a simple centrifugation workflow to split a PCR reaction into ∼34 million partitions, forming an optically clear pellet of spatially separated reaction compartments in a PCR tube. After in situ thermocycling, light sheet scanning is used to produce a 3D reconstruction of the fluorescent positive compartments within the pellet. At typical sample DNA concentrations, the magnitude of partitions offered by UltraPCR dictate that the vast majority of target molecules occupy a compartment uniquely. This single molecule realm allows for isolated amplification events, thereby eliminating competition between different targets and generating unambiguous optical signals for detection. Using a 4-color optical setup, we demonstrate that we can incorporate 10 different fluorescent dyes in the same UltraPCR reaction. We further push multiplexing to an unprecedented level by combinatorial labeling with fluorescent dyes - referred to as "comboplex" technology. Using the same 4-color optical setup, we developed a 22-target comboplex panel that can detect all targets simultaneously at high precision. Collectively, UltraPCR has the potential to push PCR applications beyond what is currently available, enabling a new class of precision genomics assays.

Real World SB4 (Etanercept Biosimilar) Use in Patients With Psoriasis: Data from the British Association of Dermatologists Biologic Interventions Register (BADBIR).

Psoriasis is a chronic, systemic, inflammatory skin disease with a risk of comorbidities and a potential high impact on patients’ quality of life.

DNA-based delivery vehicles: pH-controlled disassembly and cargo release.

Non-Watson-Crick base pairing provides an in situ approach for actuation of DNA nanostructures through responses to solution conditions. Here we demonstrate this concept by using physiologically-relevant changes in pH to regulate DNA pyramid assembly/disassembly and to control the release of protein cargo.

The role of defects on the assembly and stability of DNA nanostructures.

Defects are known to underlie the mechanical properties of materials, especially so at the nanoscale. Using four compositionally identical DNA triangles, defect density is found to be inversely correlated with assembly efficiency and melting temperature. These findings are supported by a series of experiments with more complex DNA pyramids. Because they are naturally responsive to stresses, defects present an attractive opportunity as design elements for responsive DNA materials.

Expression screening of fusion partners from an E. coli genome for soluble expression of recombinant proteins in a cell-free protein synthesis system.

While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes.

Design, assembly, and activity of antisense DNA nanostructures.

Discrete DNA nanostructures allow simultaneous features not possible with traditional DNA forms: encapsulation of cargo, display of multiple ligands, and resistance to enzymatic digestion. These properties suggested using DNA nanostructures as a delivery platform. Here, DNA pyramids displaying antisense motifs are shown to be able to specifically degrade mRNA and inhibit protein expression in vitro, and they show improved cell uptake and gene silencing when compared to linear DNA. Furthermore, the activity of these pyramids can be regulated by the introduction of an appropriate complementary strand. These results highlight the versatility of DNA nanostructures as functional devices.

Controlling forces and pathways in self-assembly using viruses and DNA.

The ability of both viruses and DNA to self-assemble in solution has continues to enable numerous applications at the nanoscale. Here we review the relevant interactions dictating the assembly of these structures, as well as discussing how they can be exploited experimentally. Because self-assembly is a process, we discuss various strategies for achieving spatial and temporal control. Finally, we highlight a few examples of recent advances that exploit the features of these nanostructures.

Enhanced resistance of DNA nanostructures to enzymatic digestion.

The ability of nucleases to perform their catalytic functions depends on the sequence and structural features of target DNA substrates. Due to their size and shape, several DNA tetrahedra are resistant to the action of specific and non-specific nucleases. Such enhanced stability is a key requirement for DNA nanostructures to be useful as delivery vehicles.

Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system.

Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell-free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN-mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti-antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti-antisense ODNs enabled the stop-and-go expression of protein molecules. Through the on-demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components.

High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins.

We describe a high-throughput strategy for tuning the expression of recombinant proteins through engineering their early nucleotide sequences. After randomizing the +2 and +3 codons of the target genes, each of the variant genes was isolated in vivo and subsequently expressed using in vitro protein synthesis techniques. When several hundreds of clones were examined in parallel, it was found that expression levels of target genes varied as much as 70-fold depending on the identity of the codons in the randomized region. This broad and continuous distribution of expression levels enabled the selection of specific codon arrangements for the expression of target genes at a desired level. Furthermore, codon-dependent variations in protein expression were reproduced when the same genes were expressed in vivo. Thus, we expect that the methodology reported here could be utilized as a versatile platform for rapid expression of protein molecules at modulated levels either in vitro or in vivo.

An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6-bisphosphate as an energy source.

In this study, we describe the development of a cost effective and highly productive cell-free protein synthesis system derived from Escherichia coli. Through the use of an optimal energy source and cell extract, approximately 1.3mg/mL of protein was generated from a single batch reaction at greatly reduced reagent costs. Compared to previously reported systems, the described method yields approximately 14-fold higher productivity per unit reagent cost making this cell-free synthesis technique a promising alternative for more efficient protein production.

Prolonged cell-free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate.

The accumulation of inorganic phosphate inhibits protein synthesis in cell-free protein synthesis reactions that are energized by high-energy-phosphate-containing compounds. This study developed a new scheme for supplying energy using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate (CP) and glucose were simultaneously used as the energy sources, the phosphate released from the CP was subsequently used in the glycolytic pathway for the utilization of the glucose, which enhanced the ATP supply and reduced the rate of inorganic phosphate accumulation. When tested against different proteins, the developed method produced 2-3 times more protein than the conventional ATP regeneration methods using single energy sources.

The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract.

In our experiments to produce different combinations of recombinant proteins in a cell-free protein synthesis system derived from Escherichia coli, we found that certain pairs of ORFs were not expressed equally. Instead, only a single DNA species was expressed dominantly, while the expression of the others was almost completely repressed. This bias during the co-expression of the DNA pairs was eliminated when an identical downstream box sequence was added to the 5'-ends of the template DNA pairs. By introducing identical nucleotide sequences of the his-tag or the downstream box of chloramphenicol acetyltransferase (CAT-DB) in front of the target genes that were otherwise not expressed compatibly, both of the encoded proteins were produced at similar productivities. Moreover, in the presence of a common downstream box, multiple genes were simultaneously expressed in the same reaction mixture. We expect that the proposed approach will offer a powerful tool for the preparation of unbiased protein libraries, as well as for studying the structure and functions of interacting proteins.

Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system.

In this study, as a part of our efforts to improve the robustness and economical feasibility of cell-free protein synthesis, we developed a simple method of preparing the cell extracts used for catalyzing cell-free protein synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support cell-free protein synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.