In vitro evolution of functional nucleic acids: high-affinity RNA ligands of HIV-1 proteins

Gene ◽  
1993 ◽  
Vol 137 (1) ◽  
pp. 33-39 ◽  
Author(s):  
Craig Tuerk ◽  
Sheela MacDougal-Waugh
Keyword(s):  
Nucleic Acids ◽  
In Vitro Evolution ◽  
High Affinity ◽  
Rna Ligands ◽  
Functional Nucleic Acids ◽  
Hiv 1

scholarly journals Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
David Wensel ◽  
Yongnian Sun ◽  
Zhufang Li ◽  
Sharon Zhang ◽  
Caryn Picarillo ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
High Affinity ◽  
Glycosylation Sites ◽  
Spectrum Activity ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Broad Spectrum Activity

ABSTRACT A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

scholarly journals SARS-CoV-2 RBD in vitro evolution parrots and predicts contagious mutation spread

2021 ◽  
Author(s):  
Gideon Schreiber ◽  
Jiri Zahradník ◽  
Shir Marciano ◽  
Maya Shemesh ◽  
Eyal Zoler ◽  
...  
Keyword(s):  
South African ◽  
Convergent Evolution ◽  
Vaccine Development ◽  
Spike Protein ◽  
Structural Basis ◽  
Receptor Binding Domain ◽  
In Vitro Evolution ◽  
High Affinity ◽  
Future Drug

Abstract SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the “European” (20E-EU1), “British” (501.V1),”South African” (501.V2), and Brazilian variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9Å Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

scholarly journals The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo.

1997 ◽  
Vol 17 (6) ◽  
pp. 3194-3201 ◽  
Author(s):  
R J Buckanovich ◽  
R B Darnell
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Motor Dysfunction ◽  
High Affinity ◽  
Loop Region ◽  
Nuclear Rna ◽  
Rna Ligands

Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of approximately 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (Kd of approximately 2 nM). The loop region of these RNAs harbors a approximately 15-bp pyrimidine-rich element [UCAU(N)(0-2)]3 which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)]3 element as necessary for in vitro binding. Consensus [UCAU (N)(0-2)], elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha2 (GlyR alpha2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha2 pre-mRNA may underlie the motor dysfunction seen in POMA.

scholarly journals CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA

2000 ◽  
Vol 20 (18) ◽  
pp. 6958-6969 ◽  
Author(s):  
Mitchell E. Garber ◽  
Timothy P. Mayall ◽  
Eric M. Suess ◽  
Jill Meisenhelder ◽  
Nancy E. Thompson ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Immunodeficiency Virus ◽  
Tar Rna ◽  
Cdk9 Phosphorylation ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathioneS-transferase–C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat–P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1–728)], but not truncated CycT1(1–303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1–303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1–303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat–P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

scholarly journals Isolation of High-Affinity RNA Ligands to HIV-1 Integrase from a Random Pool

Virology ◽  
1995 ◽  
Vol 209 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Patrick Allen ◽  
Steve Worland ◽  
Larry Gold
Keyword(s):  
High Affinity ◽  
Rna Ligands ◽  
Hiv 1

scholarly journals Capacity of simian immunodeficiency virus strain mac Nef for high-affinity Src homology 3 (SH3) binding revealed by ligand-tailored SH3 domains

2002 ◽  
Vol 83 (12) ◽  
pp. 3147-3152 ◽  
Author(s):  
Marita Hiipakka ◽  
Kalle Saksela
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Binding Motif ◽  
Sh3 Domains ◽  
Src Homology 3 ◽  
High Affinity ◽  
Loop Region ◽  
Immunodeficiency Virus ◽  
Src Homology ◽  
Hiv 1

The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound in vitro to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.

Sign in / Sign up

Export Citation Format

Share Document