Molecular BioSystems This article was published as part of the 2009 Emerging Investigators Issue Highlighting the work of outstanding young scientists at the chemical- and systems-biology interfaces Please take a look at the full table of contents to access the other papers in this issue.
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MolecularBioSystems
This article was published as part of the
2009 Emerging Investigators IssueHighlighting the work of outstanding young scientists at the
chemical- and systems-biology interfaces
Please take a look at the full table of contents to access the other papers in this issue.
Tat is an 86 amino acid protein that contains an arginine rich
motif (ARM), which specifically interacts with the TAR
tri-nucleotide bulge (U23, C24, and U25). Disruption of
Tat–TAR interaction results in the blockade of viral
replication and thus represents a viable strategy in developing
new anti-HIV therapeutics. Several ligands (aminoglycosides,6
argininamide,7 peptides,8 peptidomimetics,9 small molecules,10
and others11) are continuously being developed to inhibit
Tat–TAR interaction (Fig. 1).
Owing to the complexity, the lack of designable ligands and
a growing interest in developing new molecules that can
selectively interact with RNA, we aimed to develop a strategy
that allows for the rapid screening of a short branched peptide
library and determine their capacity as possible ligands for
HIV-1 TAR RNA (Fig. 2). Branched peptides have found
extensive use in biological systems including synthetic peptide
vaccines (multiple antigen peptides), drug delivery vehicles and
therapeutics for a variety of disease states.12 To date, the
potential of using branched peptides for the selective targeting
of structured RNA targets has not been realized. Because
multivalency is often described to increase the affinity of a
ligand to a particular receptor,13 we wanted to determine
whether branched molecules are capable of forming
multivalent interactions with RNA and whether presentation
Fig. 1 Sequence and secondary structure of HIV-1 TAR RNA (A),
small molecule ligands (neomycin B (B) and argininamide (C)) and
11-mer Tat-derived peptide (D) that inhibit Tat–TAR interaction.
a Virginia Tech, Department of Chemistry, Blacksburg, VA 24061,USA. E-mail: [email protected]; Tel: +1 540-231-5742
bVirginia Tech, Department of Biochemistry, Blacksburg, VA 24061,USA
w This article is part of the 2009 Molecular BioSystems ‘EmergingInvestigators’ issue: highlighting the work of outstanding youngscientists at the chemical- and systems-biology interfaces.z Electronic supplementary information (ESI) available: Experimentaldetails including the synthesis and characterization of branchedpeptides; protocols for on-bead screening and fluorescence polariza-tion measurements. See DOI: 10.1039/b904304g
1070 | Mol. BioSyst., 2009, 5, 1070–1073 This journal is �c The Royal Society of Chemistry 2009
PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems
of structurally diverse functional groups can enhance selectivity
with the target RNA. Branched peptides are a good starting
point because naturally occurring amino acids contain a wide
array of substituents that can display unique molecular
architectures, and their synthetic accessibility is straight-
forward. An attractive feature of this strategy is the synthesis
of the library on solid support, allowing for rapid generation
of a large number of peptides using the split and pool
technique, and subsequent on-bead screening against
TAR.8,14,15 We designed our initial branched peptide library
with biased parameters that enhance binding and selectivity to
RNA (Fig. 2). These interactions include electrostatics
(positively charged residues interacting with the negatively
charged phosphate), p–p interactions and hydrogen bonding.
In principle, the diversity of possible peptide structures can be
immense because of the commercial availability of unnatural
amino acid monomers.
Results and discussion
The branched peptide library was prepared such that each
variable position contained one of four possible amino acid
monomers (Fig. 2). Branches A1–A3 had an identical sequence
in order to simplify the de novo sequencing by MALDI/mass
spectrometry (MS) and were branched through the a- and
e-amino groups on lysine. Using orthogonal protecting
groups, 4096 compounds were generated by the split and pool
technique in a few peptide coupling steps (ESIz). It was
important that the peptides were not released from the beads
after deprotection of the side chain residues because the
Fig. 5 Fluorescence polarization binding curves of branched peptides
with HIV-1 TARRNA and mutant (U24- C24) TAR RNA. T15 is a
linear peptide (RRAGVRD) version of BP15. Each experiment was
done in triplicate.
1072 | Mol. BioSyst., 2009, 5, 1070–1073 This journal is �c The Royal Society of Chemistry 2009
Conclusion
In conclusion, we discovered selective binders for HIV-1 TAR
RNA using an on-bead screening of a focused library of
branched peptides. The use of branched structures with diverse
functional groups and capacity to form multivalent inter-
actions is important in defining new molecular entities that
can be selective for the tertiary structure of target RNA. In
addition to synthesizing a more complex branched peptide
library, future work in our laboratory will focus on extensive
characterization including mapping of TAR–branched peptide
interactions, cell permeability and in vitro inhibition of
Tat–TAR RNA interactions.
Acknowledgements
We thank Prof. Richard Helm at VA Tech mass spectrometry
incubator and Jason Crumpton for help with sequencing
branched peptides. We also thank Dr Phillippe Bissel for
synthesizing Fmoc-ANP and Dr Kristi DeCourcy at the Fralin
Life Science Institute for assistance with confocal microscopy.
Support for this project was provided by the Department of
Chemistry at Virginia Tech. This material is based upon work
supported in part by the Macromolecular Interfaces with Life
Sciences (MILES) Integrative Graduate Education and
Research Traineeship (IGERT) of the National Science
Foundation under Agreement No. DGE-0333378.
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