Analysis of Protein–RNA Complexes Involving a RNA Recognition Motif Engineered To Bind Hairpins with Seven- and Eight-Nucleotide Loops

Blakeley, Brett D.; Shattuck, Jenifer E.; Coates, Melissa B.; Tran, Elizabeth; Laird-Offringa, Ite A.; McNaughton, Brian R.

doi:10.1021/bi400801q

Cited by 3 publications

(14 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on previous studies(38), including our own(39), we hypothesized that high-throughput screening would yield a privileged β2-β3 loop which selectively recognizes TAR RNA. Based on this hypothesis, we hypothesized that high-throughput screening would yield a privileged β2-β3 loop that is selective for TAR RNA recognition.…”

Section: Resultsmentioning

confidence: 99%

“…We previously showed how mutating the N-terminal U1A RRM (U1A, Figure 1A) alters its target RNA sequence selectivity. (39, 43) In particular, we probed how one or two mutations to putative RNA-binding residues within the β1-α1 and β2-β3 loops lower affinity for U1hpII and increase affinity for Trans-Activation Reporter (TAR) element RNA(44) (Figure 1D). TAR RNA has an extended stem-loop structure that is crucial for efficient transcription of the integrated HIV genome, and is an established therapeutic target.…”

mentioning

confidence: 99%

“…Additionally, the β2-β3 loop protrudes into the U1hpII RNA loop. Although no residue in the β2-β3 loop directly engages any nucleotide in U1hpII, we have previously shown that this loop acts as a “steric ruler”–compensatory mutations or deletions in this loop lead to U1A-derived proteins that bind U1hpII-derived RNA hairpins with smaller loops. , Sequence-selectivity in this protein–RNA interaction is largely achieved through highly defined hydrogen bonding networks involving U1A residues N15, N16, and E19 (in the β1-α1 loop) with U1hpII nucleotides U2, U3, and G4 and residues S91 and D92 in the C-terminal helix with nucleotides A6 and C7 . The structural importance of I94 in the C-terminal helix has been inferred from observations that its substitution affected structural dynamics of the helix, which dramatically changes conformation upon recognition of U1hpII …”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Crawford¹,

Blakeley²,

Chen

et al. 2016

ACS Chem. Biol.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Crawford¹,

Blakeley²,

Chen

et al. 2016

ACS Chem. Biol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Such adenine-adenine stacks are also of relevance for the oxidation of oligonucleotides [50]. Combinations of p-p interactions also occur in recognition reactions, e.g., between the tyrosine or phenylalanine residues of a protein and the cytosine or adenine moieties of an RNA, respectively [51]. Stack formation between an indole residue (red) of a proteintryptophan and guanine-RNA moieties (blue) in the SR-like protein ZRANB2 and the RNA in its single-stranded RNA-binding domain [52].…”

Section: Entry 3 Inmentioning

confidence: 99%

Comparison of the π-stacking properties of purine versus pyrimidine residues. Some generalizations regarding selectivity

2014

View full text Add to dashboard Cite

Aromatic-ring stacking is pronounced among the noncovalent interactions occurring in biosystems and therefore some pertinent features regarding nucleobase residues are summarized. Self-stacking decreases in the series adenine > guanine > hypoxanthine > cytosine ~ uracil. This contrasts with the stability of binary (phen)(N) adducts formed by 1,10-phenanthroline (phen) and a nucleobase residue (N), which is largely independent of the type of purine residue involved, including (N1)H-deprotonated guanine. Furthermore, the association constant for (phen)(A)(0/4-) is rather independent of the type and charge of the adenine derivative (A) considered, be it adenosine or one of its nucleotides, including adenosine 5'-triphosphate (ATP(4-)). The same holds for the corresponding adducts of 2,2'-bipyridine (bpy), although owing to the smaller size of the aromatic-ring system of bpy, the (bpy)(A)(0/4-) adducts are less stable; the same applies correspondingly to the adducts formed with pyrimidines. In accord herewith, [M(bpy)](adenosine)(2+) adducts (M(2+) is Co(2+), Ni(2+), or Cu(2+)) show the same stability as the (bpy)(A)(0/4-) ones. The formation of an ionic bridge between -NH3 (+) and -PO3 (2-), as provided by tryptophan [H(Trp)(±)] and adenosine 5'-monophosphate (AMP(2-)), facilitates recognition and stabilizes the indole-purine stack in [H(Trp)](AMP)(2-). Such indole-purine stacks also occur in nature. Similarly, the formation of a metal ion bridge as occurs, e.g., between Cu(2+) coordinated to phen and the phosphonate group of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA(2-)) dramatically favors the intramolecular stack in Cu(phen)(PMEA). The consequences of such interactions for biosystems are discussed, especially emphasizing that the energies involved in such isomeric equilibria are small, allowing Nature to shift such equilibria easily.

show abstract

“…Mutagenesis of residues within putative RNA recognition regions of U1A generally does not result in protein instability and unfolding. (32, 41-43, 46) In addition, certain mutations within the putative RNA-binding regions can lead to altered RNA binding selectivity. For example, researchers have shown that mutating residues within the b2-b3 loop lead to U1A-derived proteins with improved affinity for U1hpII.…”

Section: Introductionmentioning

confidence: 99%

Synthetic RNA Recognition Motifs That Selectively Recognize HIV-1 Trans-Activation Response Element Hairpin RNA

Blakeley

McNaughton

2014

ACS Chem. Biol.

Self Cite

View full text Add to dashboard Cite

A multitude of RNA hairpins are directly implicated in human disease. Many of these RNAs are potentially valuable targets for drug discovery and basic research efforts. However, very little is known about the molecular requirements for achieving sequence-selective recognition of a particular RNA sequence and structure. While a relatively modest number of synthetic small to medium-sized RNA-binding molecules have been reported, rapid identification of sequence-selective disease-relevant RNA-binding molecules remains a daunting challenge. RNA Recognition Motif (RRM) domains may represent unique privileged scaffolds for the generation of synthetic proteins that selectively recognize disease-relevant RNAs, including RNA hairpins. As a demonstration of this potential, we mutated putative RNA-binding regions within the U1A RRM, and a variant thereof, and screened these synthetic proteins for affinity to HIV-1 Trans-Activation Response (TAR) element hairpin RNA. Impressively, some of these U1A-derived proteins bind TAR with single-digit micromolar dissociation constants, and do so preferentially over the native protein's original target RNA (U1hpII) and a DNA TAR variant. For two TAR-binding proteins, binding affinity is not appreciably diminished by addition of 10 molar equivalents of cellular tRNAs from Escherichia coli. Taken together, our findings represent the first synthetic RRMs that selectively bind a disease-relevant RNA hairpin, and may represent a general approach for achieving sequence-selective recognition of RNA hairpins, which are the focus of therapeutic discovery and basic research

show abstract

Analysis of Protein–RNA Complexes Involving a RNA Recognition Motif Engineered To Bind Hairpins with Seven- and Eight-Nucleotide Loops

Cited by 3 publications

References 21 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

Comparison of the π-stacking properties of purine versus pyrimidine residues. Some generalizations regarding selectivity

Synthetic RNA Recognition Motifs That Selectively Recognize HIV-1 Trans-Activation Response Element Hairpin RNA

Contact Info

Product

Resources

About