In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization

This unit describes the selection of aptamers from a pool of single‐stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially function as protein inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate a ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143643/1/cpnc0903.pd

Introduction to the Synthesis and Purification of Oligonucleotides

Modern nucleic acid synthesizers utilize phosphite triester chemistries that employ stable phosphoramidite monomers to build a growing polymer. These robust reactions allow easy generation of specific oligodeoxyribo‐ and oligoribonucleotides with a variety of labels, modified linkages, and nonstandard bases. Strategies are given for the maximization of synthetic yield, the generation of sequences containing site‐specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143751/1/cpnca03c.pd

Analyzing Machupo virus-receptor binding by molecular dynamics simulations

In many biological applications, we would like to be able to computationally predict mutational effects on affinity in protein-protein interactions. However, many commonly used methods to predict these effects perform poorly in important test cases. In particular, the effects of multiple mutations, non-alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here an existing method applied in a novel way to a new test case; we interrogate affinity differences resulting from mutations in a host-virus protein-protein interface. We use steered molecular dynamics (SMD) to computationally pull the machupo virus (MACV) spike glycoprotein (GP1) away from the human transferrin receptor (hTfR1). We then approximate affinity using the maximum applied force of separation and the area under the force-versus-distance curve. We find, even without the rigor and planning required for free energy calculations, that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Moreover, we show that this simple SMD scheme correlates well with relative free energy differences computed via free energy perturbation. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our approach provides a framework to compare the effects of multiple mutations, individually and jointly, on protein-protein interactions.Comment: 33 pages, 8 figures, 5 table

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

Preparation of a random‐sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer. Purification of a single‐stranded pool and conversion to a double‐stranded pool are presented as step‐by‐step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143624/1/cpnc0902.pd

A combined in vitro / in vivo selection for polymerases with novel promoter specificities

BACKGROUND: The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro / in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10(3) – 10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendent mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection. RESULTS AND CONCLUSIONS: A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified. A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity. This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides

Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies

It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation.This work was supported by grants from the United States National Institutes of Health, National Science Foundation, and Welch (F1515) and Packard Foundations to EMM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Cellular and Molecular Biolog

Proximity ligation assays with peptide conjugate ‘burrs’ for the sensitive detection of spores

The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide–oligonucleotide–phycoerythrin conjugates (‘burrs’) that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins

Engineering stochasticity in gene expression

Stochastic fluctuations (noise) in gene expression can cause members of otherwise genetically identical populations to display drastically different phenotypes. An understanding of the sources of noise and the strategies cells employ to function reliably despite noise is proving to be increasingly important in describing the behavior of natural organisms and will be essential for the engineering of synthetic biological systems. Here we describe the design of synthetic constructs, termed ribosome competing RNAs (rcRNAs), as a means to rationally perturb noise in cellular gene expression. We find that noise in gene expression increases in a manner proportional to the ability of an rcRNA to compete for the cellular ribosome pool. We then demonstrate that operons significantly buffer noise between coexpressed genes in a natural cellular background and can even reduce the level of rcRNA enhanced noise. These results demonstrate that synthetic genetic constructs can significantly affect the noise profile of a living cell and, importantly, that operons are a facile genetic strategy for buffering against noise

Deoxyribozymes that recode sequence information

Allosteric nucleic acid ligases have been used previously to transform analyte-binding into the formation of oligonucleotide templates that can be amplified and detected. We have engineered binary deoxyribozyme ligases whose two components are brought together by bridging oligonucleotide effectors. The engineered ligases can ‘read’ one sequence and then ‘write’ (by ligation) a separate, distinct sequence, which can in turn be uniquely amplified. The binary deoxyribozymes show great specificity, can discriminate against a small number of mutations in the effector, and can read and recode DNA information with high fidelity even in the presence of excess obscuring genomic DNA. In addition, the binary deoxyribozymes can read non-natural nucleotides and write natural sequence information. The binary deoxyribozyme ligases could potentially be used in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of short nucleic acids such as microRNAs