Biomedical subjects

Scott M Knudsen

Publications and source records attributed to Scott M Knudsen.

6 recordsLinked to original sources

PubMed2005

Arginine-rich motifs present multiple interfaces for specific binding by RNA.

A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.

Alanine↗

PubMed2004

Micromechanical detection of proteins using aptamer-based receptor molecules.

We report label-free protein detection using a microfabricated cantilever-based sensor that is functionalized with DNA aptamers to act as receptor molecules. The sensor utilizes two adjacent cantilevers that constitute a sensor/reference pair and allows direct detection of the differential bending between the two cantilevers. One cantilever is functionalized with aptamers selected for Taq DNA polymerase while the other is blocked with single-stranded DNA. We have found that the polymerase-aptamer binding induces a change in surface stress, which causes a differential cantilever bending that ranges from 3 to 32 nm depending on the ligand concentration. Protein recognition on the sensor surface is specific and has a concentration dependence that is similar to that in solution.

Base Sequence↗

PubMed2004

Ribozyme déjà vu.

Explore the source record for details and available documents.

5' Untranslated Regions↗

PubMed2004

In vitro selection of ribozymes dependent on peptides for activity.

A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide- and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).

Amino Acid Motifs↗

PubMed2003

Simultaneous detection of diverse analytes with an aptazyme ligase array.

Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection.

Biosensing Techniques↗

PubMed2002

Group I aptazymes as genetic regulatory switches.

BACKGROUND: Allosteric ribozymes (aptazymes) that have extraordinary activation parameters have been generated in vitro by design and selection. For example, hammerhead and ligase ribozymes that are activated by small organic effectors and protein effectors have been selected from random sequence pools appended to extant ribozymes. Many ribozymes, especially self-splicing introns, are known control gene regulation or viral replication in vivo. We attempted to generate Group I self-splicing introns that were activated by a small organic effector, theophylline, and to show that such Group I aptazymes could mediate theophylline-dependent splicing in vivo. RESULTS: By appending aptamers to the Group I self-splicing intron, we have generated a Group I aptazyme whose in vivo splicing is controlled by exogenously added small molecules. Substantial differences in gene regulation could be observed with compounds that differed by as little as a single methyl group. The effector-specificity of the Group I aptazyme could be rationally engineered for new effector molecules. CONCLUSION: Group I aptazymes may find applications as genetic regulatory switches for generating conditional knockouts at the level of mRNA or for developing economically viable gene therapies.

Allosteric Regulation↗