Binding of a Structured <scp>d</scp>-RNA Molecule by an <scp>l</scp>-RNA Aptamer

Sczepanski, Jonathan T.; Joyce, Gerald F.

doi:10.1021/ja406634g

Cited by 66 publications

(61 citation statements)

References 39 publications

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“…(62) Reagents that inhibit the Tat/TAR complex can suppress the transcription of full-length HIV RNA, leading to suppression or abrogation of HIV protein expression and production of virus. (63)…”

Section: Resultsmentioning

confidence: 99%

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription

Crawford¹,

Blakeley²,

Chen

et al. 2016

ACS Chem. Biol.

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Potent and selective recognition and modulation of disease-relevant RNAs remains a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition, and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) Trans-Activation Response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here we use yeast display and saturation mutagenesis of established RNA binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA binding selectivity; it binds TAR with sub-nanomolar affinity (apparent dissociation constant ~0.5 nM), but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5′-untranslated region, inhibits the interaction between TAR RNA and an HIV Trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents – small molecule, nucleic acid, or protein - reported to date, and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition—and modulation—of a disease-relevant RNA.

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Section: Resultsmentioning

confidence: 99%

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription

Crawford¹,

Blakeley²,

Chen

et al. 2016

ACS Chem. Biol.

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“…8 0.1 nM [5′- 32 P]-labeled D-pre-miR RNA was incubated with various concentrations of L-aptamiR in the presence of either 0.5 or 5 mM MgCl 2 , 150 mM NaCl, 25 mM Tris (pH 7.6), and 0.1 mg/mL tRNA for 30 min at either 23 or 37 °C. Samples were loaded on a 10% non-denaturing polyacrylamide gel (29:1 acylamide:bis-acrylamide) that had been pre-heated to the incubation temperature and contained either 0.5 or 5 mM MgCl 2 , 50 mM NaOAc, and 10 mM Tris (pH 7.6).…”

Section: Methodsmentioning

confidence: 99%

Specific Inhibition of MicroRNA Processing Using l-RNA Aptamers

Sczepanski

Joyce

2015

J. Am. Chem. Soc.

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In vitro selection was used to obtain L-RNA aptamers that bind the distal stem-loop of various precursor microRNAs (pre-miRs). These L-aptamers, termed “aptamiRs”, bind their corresponding pre-miR target through highly specific tertiary interactions rather than Watson–Crick pairing. Formation of a pre-miR–aptamiR complex inhibits Dicer-mediated processing of the pre-miR, which is required to form the mature functional microRNA. One of the aptamiRs, which was selected to bind oncogenic pre-miR-155, inhibits Dicer processing under simulated physiological conditions, with an IC50 of 87 nM. Given that L-RNAs are intrinsically resistant to nuclease degradation, these results suggest that aptamiRs might be pursued as a new class of miR inhibitors.

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“…To date, L -RNA aptamers only have been selected against small molecules (Klussmann et al, 1996; Nolte et al, 1996), peptides (Maasch et al, 2010), chemokines (Eulberg, 2008), and small structured D -RNAs (Sczepanski and Joyce, 2013). None have been selected against a complete functional enzyme, largely because of the difficulty in synthesizing the full-length enantiomeric protein.…”

Section: Introductionmentioning

confidence: 99%

An L-RNA Aptamer that Binds and Inhibits RNase

Olea

Weidmann

Dawson

et al. 2015

Chemistry & Biology

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SUMMARY L-RNA aptamers were developed that bind to barnase ribonuclease and thereby inhibit the function of the enzyme. These aptamers were obtained by first carrying out in vitro selection of D-RNAs that bind to the full-length synthetic D-enantiomer of barnase, then reversing the mirror and preparing L-RNAs of identical sequence that similarly bind to natural L-barnase. The resulting L-aptamers bind L-barnase with an affinity of ~100 nM and function as competitive inhibitors of enzyme cleavage of D-RNA substrates. L-RNA aptamers are resistant to degradation by ribonucleases, thus enabling them to function in biological samples, most notably for applications in molecular diagnostics and therapeutics. In addition to the irony of using RNA to inhibit ribonuclease, L-RNA aptamers such as those described here could be used to measure the concentration or to inhibit the function of ribonuclease in the laboratory or in biological systems.

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Binding of a Structured d-RNA Molecule by an l-RNA Aptamer

Cited by 66 publications

References 39 publications

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription

Specific Inhibition of MicroRNA Processing Using l-RNA Aptamers

An L-RNA Aptamer that Binds and Inhibits RNase

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