Molecular Recognition of cAMP by an RNA Aptamer

Koizumi, Makoto; Breaker, Ronald R.

doi:10.1021/bi000149n

Cited by 65 publications

(41 citation statements)

References 41 publications

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“…RzB is thus a logical starting point for the construction of new ribozymes for use in gene knockdown experiments for therapeutic applications or for studies of biological mechanisms. It may also be possible to achieve highly efficient, regulated cleavage by combining these tertiary stabilization motifs into an RNA that also carries modules for allosteric regulation by oligonucleotides (e.g., the TRAP design), by small molecules (e.g., cAMP or theophylline), or by proteins (Koizumi et al 1999;Koizumi and Breaker 2000;Sekella et al 2000;Burke et al 2002;Wang and Sen 2002;Saksmerprome andBurke 2003, 2004).…”

Section: Discussionmentioning

confidence: 99%

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Saksmerprome

Roychowdhury-Saha²,

Jayasena³

et al. 2004

RNA

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Tertiary stabilizing motifs (TSMs) between terminal loops or internal bulges facilitate folding of natural hammerhead ribozymes (hRz) under physiological conditions. However, both substrate and enzyme strands contribute nucleotides to the TSMs of trans-cleaving hRz, complicating the design of hRz that exploit TSMs to target specific mRNA. To overcome this limitation, we used SELEX to identify new, artificial TSMs that are less sensitive to sequence context. Nucleotides in loop II or in a bulge within the ribozyme strand of stem I were randomized, while the interaction partner was held constant. All nucleotides of the substrate pair with the ribozyme, minimizing their possible recruitment into the TSM, as such recruitment could constrain choice of candidate target sequences. Six cycles of selection identified cis-acting ribozymes that were active in 100 µM MgCl 2 . The selected motifs partially recapitulate TSMs found in natural hRz, suggesting that the natural motifs are close to optimal for their respective contexts. Ribozyme "RzB" showed enhanced thermal stability by retaining trans-cleavage activity at 80°C in 10 mM MgCl 2 and at 70°C in 2 mM MgCl 2 . A variant of ribozyme "RzB" with a continuously paired stem 1 rapidly lost activity as temperature was increased. The selected motifs are modular, in that they permit trans-cleavage of several substrates in submillimolar MgCl 2 , including two substrates derived from the U5 genomic region of HIV-1. The new, artificial tertiary stabilized hRz are thus nearly independent of sequence context and enable for the first time the use of highly active hRz targeting almost any mRNA at physiologically relevant magnesium concentrations.

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Section: Discussionmentioning

confidence: 99%

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Saksmerprome

Roychowdhury-Saha²,

Jayasena³

et al. 2004

RNA

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“…Aptamers are oligonucleotides that have been selected for specific binding to a variety of molecular targets, ranging from small organics to proteins [20,21]. These nucleic acidbinding species can consist of RNA or DNA and are typically 15-60 nucleotides long [22].…”

Section: Recognition Of Aptamer With Thrombinmentioning

confidence: 99%

FTIR-ATR detection of proteins and small molecules through DNA conjugation

Liao

Fang

Liú

et al. 2006

Sensors and Actuators B: Chemical

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“…(1,2) Targets to which aptamers have successfully been generated against include proteins, small molecules and RNA. (3)(4)(5)(6)(7)(8) Aptamers have been suggested as valuable alternatives to antibodies in biodetection and diagnostic applications due to their ease of discovery, their stability, and robust methods for their synthesis. (9)(10)(11)(12) A wealth of sensitive aptamer-based biodetection approaches have been reported in which aptamers were labeled with molecules such as redox probes, fluorescent dyes, or nanocrystals to be an integral part of signal transduction.…”

Section: Introductionmentioning

confidence: 99%

Protein detection via direct enzymatic amplification of short DNA aptamers

Fischer

Tarasow

Tok

2008

Analytical Biochemistry

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Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM).

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Molecular Recognition of cAMP by an RNA Aptamer

Cited by 65 publications

References 41 publications

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

FTIR-ATR detection of proteins and small molecules through DNA conjugation

Protein detection via direct enzymatic amplification of short DNA aptamers

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