scholarly journals Specific suppression of D-RNA G-quadruplex–protein interaction with an L-RNA aptamer

2020 ◽  
Vol 48 (18) ◽  
pp. 10125-10141 ◽  
Author(s):  
Mubarak I Umar ◽  
Chun Kit Kwok
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
In Vitro Selection ◽  
Rna Aptamer ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
G Quadruplex ◽  
Preferential Binding ◽  
Better Than

Abstract G-quadruplexes (G4s) are nucleic acid structure motifs that are of significance in chemistry and biology. The function of G4s is often governed by their interaction with G4-binding proteins. Few categories of G4-specific tools have been developed to inhibit G4–protein interactions; however, until now there is no aptamer tool being developed to do so. Herein, we present a novel L-RNA aptamer that can generally bind to D-RNA G-quadruplex (rG4) structure, and interfere with rG4–protein interaction. Using hTERC rG4 as the target for in vitro selection, we report the shortest L-aptamer being developed so far, with only 25 nucleotides. Notably, this new aptamer, L-Apt.4-1c, adopts a stem–loop structure with the loop folding into an rG4 motif with two G-quartet, demonstrates preferential binding toward rG4s over non-G4s and DNA G-quadruplexes (dG4s), and suppresses hTERC rG4–nucleolin interactions. We also show that inhibition of rG4–protein interaction using L-RNA aptamer L-Apt.4-1c is comparable to or better than G4-specific ligands such as carboxypyridostatin and QUMA-1 respectively, highlighting that our approach and findings expand the current G4 toolbox, and open a new avenue for diverse applications.

scholarly journals Mapping of the Hepatitis B Virus Reverse Transcriptase TP and RT Domains by Transcomplementation for Nucleotide Priming and by Protein-Protein Interaction

1999 ◽  
Vol 73 (3) ◽  
pp. 1885-1893 ◽  
Author(s):  
Robert E. Lanford ◽  
Young-Ho Kim ◽  
Helen Lee ◽  
Lena Notvall ◽  
Burton Beames
Keyword(s):  
Amino Acids ◽  
Reverse Transcriptase ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Rnase H ◽  
Genome Replication ◽  
Protein Protein Interactions ◽  
Stem Loop ◽  
Protein Protein Interaction

ABSTRACT Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction. We previously described a complementation assay for analysis of the roles of the TP and RT domains of HBV reverse transcriptase (pol) in the priming reaction. Independently expressed TP and RT domains form a complex functional for in vitro priming reactions. To map the minimal functional TP and RT domains, we prepared baculoviruses expressing amino- and carboxyl-terminal deletions of both the TP and RT domains and analyzed the proteins for the ability to participate in transcomplementation for the priming reaction. The minimal TP domain spanned amino acids 20 to 175; however, very little activity was observed without a TP domain spanning amino acids 1 to 199. The minimal RT domain spanned amino acids 300 to 775; however, little activity was observed unless the carboxyl end of the RT domain extended to amino acid 800. Thus, most of the RNase H domain was required. In previous studies, we observed a TP inhibitory domain between amino acids 199 and 344. The current analysis narrowed this domain to residues 300 to 334, which is a portion of the minimal RT domain. In addition, the ability of TP and RT deletion mutants to form stable TP-RT complexes was examined in coimmunoprecipitation assays. The minimal TP and RT domains capable of protein-protein interaction were considerably smaller than the domains required for functional interaction in the transcomplementation assays, and unlike priming activity, TP-RT interaction did not require the epsilon RNA stem-loop. These studies help to further define the complex protein-protein interactions required in HBV genome replication.

scholarly journals Novel Approach to Analyzing RNA Aptamer-Protein Interactions: Toward Further Applications of Aptamers

2006 ◽  
Vol 11 (6) ◽  
pp. 599-605 ◽  
Author(s):  
Joonsung Hwang ◽  
Satoshi Nishikawa
Keyword(s):  
Protein Interactions ◽  
In Vitro Selection ◽  
Selection Procedure ◽  
Rna Aptamers ◽  
Sensor Chip ◽  
Rna Aptamer ◽  
Surface Plasmon Resonance Analysis ◽  
Resonance Analysis ◽  
Novel Approach

Surface plasmon-resonance analysis using a Biacore biosensor is a powerful tool for the detailed study of biomolecular interactions. The authors examined the methods of immobilizing proteins on the surface of NTA, SA, and CM5 sensor chips to study RNA aptamer-protein interactions. RNA aptamers and their deletion variants were loaded onto a protein-immobilized sensor chip, and their binding affinities were analyzed. Immobilizing the protein on a CM5 sensor chip via an anti-His-tag antibody was the only strategy that clearly detected the kinetic parameters of the interactions. ΔNEO-III-14U, one of the deletion variants of the NS3 aptamer, had the highest binding affinity for the ΔNS3 protein in this study (KD = 4 × 10-8). Moreover, the 29-amino-acid spacer fragment was essential for protein immobilization using this strategy. This novel method will be useful in comparing the affinity of various RNA aptamers and selecting the most suitable candidates for a given target, as well as facilitating the in vitro selection procedure itself.

RNA—protein interactions in the 5′ untranslated region of preproinsulin mRNA

1992 ◽  
Vol 8 (3) ◽  
pp. 225-234 ◽  
Author(s):  
S. W. Knight ◽  
K. Docherty
Keyword(s):  
Protein Interactions ◽  
Untranslated Region ◽  
In Vitro Transcription ◽  
Specific Protein ◽  
Start Codon ◽  
Mobility Shift ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Rna Probes

ABSTRACT A comparison between species of the 5′ untranslated region of preproinsulin mRNA revealed conserved sequences associated with a potential stem—loop structure. The present study was undertaken to determine whether specific protein interactions exist with mRNA sequences involved in the formation or stabilization of this structure in the 5′ untranslated region. 32P-Labelled RNA probes corresponding to sequences from this region were synthesized by an in-vitro transcription reaction and used in electrophoretic mobility shift and u.v.-crosslinking studies with cytoplasmic protein extracts from a number of cell lines. Specific protein—RNA interactions were mapped to a sequence located between nucleotides –21 and –50 upstream of the AUG start codon. A number of proteins of molecular mass 25kDa, 40kDa, 46kDa, 58kDa, 69kDa, 97kDa, 110kDa and 160kDa were specifically crosslinked to this sequence. The observed specific protein—RNA interactions in the 5′ untranslated region may affect the activity of preproinsulin mRNA.

scholarly journals Locking up the AS1411 Aptamer with a Flanking Duplex: Towards an Improved Nucleolin-Targeting

2021 ◽  
Vol 14 (2) ◽  
pp. 121
Author(s):  
André Miranda ◽  
Tiago Santos ◽  
Eric Largy ◽  
Carla Cruz
Keyword(s):  
Loop Structure ◽  
Binding Properties ◽  
Stem Loop ◽  
Affinity Ligand ◽  
Stem Loop Structure ◽  
Topology Maintenance ◽  
G Quadruplex ◽  
Dimeric Form ◽  
Biophysical Techniques ◽  
Melting Experiments

We have designed AS1411-N6, a derivative of the nucleolin (NCL)-binding aptamer AS1411, by adding six nucleotides to the 5′-end that are complementary to nucleotides at the 3′-end forcing it into a stem-loop structure. We evaluated by several biophysical techniques if AS1411-N6 can adopt one or more conformations, one of which allows NCL binding. We found a decrease of polymorphism of G-quadruplex (G4)-forming sequences comparing to AS1411 and the G4 formation in presence of K+ promotes the duplex folding. We also studied the binding properties of ligands TMPyP4, PhenDC3, PDS, 360A, and BRACO-19 in terms of stability, binding, topology maintenance of AS1411-N6, and NCL recognition. The melting experiments revealed promising stabilizer effects of PhenDC3, 360A, and TMPyP4, and the affinity calculations showed that 360A is the most prominent affinity ligand for AS1411-N6 and AS1411. The affinity determined between AS1411-N6 and NCL denoting a strong interaction and complex formation was assessed by PAGE in which the electrophoretic profile of AS1411-N6 showed bands of the dimeric form in the presence of the ligands and NCL.

scholarly journals Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1517
Author(s):  
Rebecca S. Brown ◽  
Lisa Kim ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Rna Structure ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Specific Binding ◽  
Genomic Rna ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Labeled Rna

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

RNA binding by Sxl proteins in vitro and in vivo

1994 ◽  
Vol 14 (7) ◽  
pp. 4975-4990
Author(s):  
M E Samuels ◽  
D Bopp ◽  
R A Colvin ◽  
R F Roscigno ◽  
M A Garcia-Blanco ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Rna Binding ◽  
Polytene Chromosomes ◽  
Regulatory Sequences ◽  
Acceptor Site ◽  
Nuclear Extracts ◽  
Preferential Binding

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

scholarly journals Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA.

1990 ◽  
Vol 10 (3) ◽  
pp. 939-946 ◽  
Author(s):  
R Singh ◽  
S Gupta ◽  
R Reddy
Keyword(s):  
Cell Extract ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
Rna Sequence ◽  
U6 Snrna ◽  
Stem Loop Structure ◽  
Close Proximity ◽  
Nuclear Rna ◽  
Tightly Coupled

The cap structure of U6 small nuclear RNA (snRNA) is gamma-monomethyl phosphate and is distinct from other known RNA cap structures (R. Singh and R. Reddy, Proc. Natl. Acad. Sci. USA 86:8280-8283, 1989). Here we show that the information for capping the U6 snRNA in vitro is within the initial 25 nucleotides of the U6 RNA. The capping determinant in mammalian U6 snRNA is a bipartite element--a phylogenetically conserved stem-loop structure and an AUAUAC sequence, or a part thereof, following this stem-loop. Wild-type capping efficiency was obtained when the AUAUAC motif immediately followed the stem-loop and when the gamma-phosphate of the initiation nucleotide was in close proximity to the capping determinant. Incorporation of a synthetic stem-loop followed by an AUAUAC sequence is sufficient to covert a noncapped heterologous transcript into a capped transcript. Transcripts with the initial 32 nucleotides of Saccharomyces cerevisiae U6 snRNA are accurately capped in HeLa cell extract, indicating that capping machinery from HeLa cells can cap U6 snRNA from an evolutionarily distant eucaryote. The U6-snRNA-specific capping is unusual in that it is RNA sequence dependent, while the capping of mRNAs and other U snRNAs is tightly coupled to transcription and is independent of the RNA sequence.

In Vitro Selection of the RNA Aptamer against the Sialyl Lewis X and Its Inhibition of the Cell Adhesion

2001 ◽  
Vol 281 (1) ◽  
pp. 237-243 ◽  
Author(s):  
Sunjoo Jeong ◽  
Tae-Yeon Eom ◽  
Se-Jin Kim ◽  
Seong-Wook Lee ◽  
Jaehoon Yu
Keyword(s):  
Cell Adhesion ◽  
In Vitro Selection ◽  
Sialyl Lewis X ◽  
Rna Aptamer ◽  
Lewis X ◽  
Sialyl Lewis ◽  
Selection Of

In vitro selection and prediction of TIP47 protein-interaction interfaces

2004 ◽  
Vol 1 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Alondra Schweizer Burguete ◽  
Pehr B Harbury ◽  
Suzanne R Pfeffer
Keyword(s):  
Protein Interaction ◽  
In Vitro Selection
Sign in / Sign up

Export Citation Format

Share Document