In Vitro Evolution of Functional Nucleic Acids: High-Affinity RNA Ligands of the HIV-1 rev Protein

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.

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

10.21203/rs.3.rs-183310/v1 ◽

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.

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High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication

Molecular Therapy — Nucleic Acids ◽

10.1038/mtna.2015.1 ◽

2015 ◽

Vol 4 ◽

pp. e228 ◽

Cited By ~ 21

Author(s):

Sonald Duclair ◽

Archana Gautam ◽

Andrew Ellington ◽

Vinayaka R Prasad

Keyword(s):

Viral Replication ◽

Protease Activity ◽

Rna Aptamers ◽

High Affinity ◽

Hiv 1

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

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3194 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3194-3201 ◽

Cited By ~ 157

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6958-6969.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6958-6969 ◽

Cited By ~ 109

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.

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