Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection

Hall, Bradley; Micheletti, John M.; Satya, Pooja; Ogle, Krystal; Pollard, Jack D.; Ellington, Andrew D.

doi:10.1002/0471142700.nc0902s39

Cited by 34 publications

(45 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A standard w/o emulsion composition of 4.5% (v/v) Span 80, 0.5% (v/v) Tween 80 in 0.95 mL of mineral oil 23 , and 50 μL of an ATPS consisting of 10% (w/w) dextran 10 kDa and 7% (w/w) PEG 8 kDa was chosen to evaluate the potential of w/o droplets in encapsulating multiple aqueous phases. 9 [Note: surfactant and polymer percent concentrations are given in (v/v) and (w/w), respectively.]…”

Section: Resultsmentioning

confidence: 99%

Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription–Translation

2014

View full text Add to dashboard Cite

The construction of genetically encoded cellular mimics in compartments containing organized synthetic cytosols is desirable for the development of artificial cells. Phase separated aqueous domains were placed within water-in-oil emulsion droplets in a manner compatible with transcription and translation machinery. Aqueous two-phase and three-phase systems (ATPS and A3PS) were assembled with dextran, poly(ethylene glycol), and Ficoll. Aqueous two-phase systems were capable of supporting the cell-free expression of protein within water droplets, whereas the aqueous three-phase-based system did not give rise to detectable protein synthesis. The expressed protein preferentially partitioned to the dextran-enriched phase. The system could serve as a foundation for building cellular mimics with liquid organelles.

show abstract

Section: Resultsmentioning

confidence: 99%

Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription–Translation

2014

View full text Add to dashboard Cite

show abstract

“…The N70 library was chemically synthesized by GenScript (Piscataway, NJ) with 26 and 24 nt constant regions flanking a 70 nt random region as described (18). NS1 and NS2 were selected from this library using the H3-C-unrelated targets Ublcp1 (Uniprot ID:Q8WVY7) and Chk2 (Uniprot ID:096017), respectively.…”

Section: Methodsmentioning

confidence: 99%

Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection

Ozer

White

Lis

et al. 2013

Nucleic Acids Research

View full text Add to dashboard Cite

The non-specific binding of undesired ligands to a target is the primary factor limiting the enrichment of tight-binding ligands in affinity selection. Solution-phase non-specific affinity is determined by the free-energy of ligand binding to a single target. However, the solid-phase affinity might be higher if a ligand bound concurrently to multiple adjacent immobilized targets in a cooperative manner. Cooperativity could emerge in this case as a simple consequence of the relationship between the free energy of binding, localization entropy and the spatial distribution of the immobilized targets. We tested this hypothesis using a SELEX experimental design and found that non-specific RNA aptamer ligands can concurrently bind up to four bead-immobilized peptide targets, and that this can increase their effective binding affinity by two orders-of-magnitude. Binding curves were quantitatively explained by a new statistical mechanical model of density-dependent cooperative binding, which relates cooperative binding to both the target concentration and the target surface density on the immobilizing substrate. Target immobilization plays a key role in SELEX and other ligand enrichment methods, particularly in new multiplexed microfluidic purification devices, and these results have strong implications for optimizing their performance.

show abstract

“…34 Following deprotection, libraries were gel purified by denaturing (7 mol/l urea) gel electrophoresis on an 8% polyacrylamide gel. The single-stranded DNA library was amplified by PCR to generate a double-stranded DNA bearing a T7 promoter and transcribed in vitro using the Y639F mutant of T7 RNA polymerase 35,36 or Durascribe kits and 2′-fluoro-pyrimidines.…”

Section: Methodsmentioning

confidence: 99%

A General RNA Motif for Cellular Transfection

et al. 2012

View full text Add to dashboard Cite

We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells.

show abstract

Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection

Cited by 34 publications

References 56 publications

Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription–Translation

Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription–Translation

Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection

A General RNA Motif for Cellular Transfection

Contact Info

Product

Resources

About