The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 10, 2012, is named 116290_SEQ_ST25.txt and is 25,144 bytes in size.
Aptamers are nucleic or amino acid macromolecules that may he designed to bind tightly to specific targets. Targets include structures from proteins to small organic dyes. In solution, a chain of nucleotides forms intramolecular interactions that fold the molecule into a complex three-dimensional shape that allows it to bind tightly against the surface of its target molecule.
Peptide aptamers are short peptides of random amino acid sequences. As commonly used, these peptides are generally 15-20 amino acids-long. This length provides enough flexibility for the peptide to assume various conformations, while reducing the probability of randomly creating a stop codon in the aptamer coding sequence.
Aptamers can be “free” (i.e., as a “tag” on the end of another protein) or “constrained” (i.e., inserted between two other polypeptides). Constraining aptamers apparently lowers their free energy of folding and allows them more easily to take on conformations conducive to interactions with other proteins.
Peptide aptamers have a number of biological uses: as “tags” to identify interacting proteins (e.g., using BiFC, bimolecular fluorescence complementation), as “mutagens” to disrupt functionality of proteins by binding and thereby inhibiting activity (e.g., acting as competitive inhibitors for substrate or peptide binding), as a way to identify specific domains of proteins by binding to and inhibiting certain protein functions without necessarily inhibiting all functions of the target protein, and as a bioinformatics tool to identify interacting partners of target proteins (e.g., the sequence or structure of an interacting peptide aptamer may give clues as to what proteins may also interact with the target protein).
A peptide aptamer technology to investigate and manipulate protein function in vivo was developed. Aptamer expression “cassettes” were developed in which aptamers are constrained between components of autoflourescent proteins that allow detection of target proteins and may affect their function. An advantage of the methods and compositions disclosed herein is not only to determine when aptamers enter cells, but also when they interact with target proteins. Strong promoters (for example in plants, the CaMV double 35S promoter, in animals, the CMV promoter) may drive expression of the novel “polyprotein” cassettes containing the aptamers. A suitable polyprotein includes an autofluorescent protein-aptamer (e.g. a 20 amino acid peptide)-complementary autofluorescent protein fragment. A polyA addition signal may follow. A suitable autofluorescent protein is mCherry, a suitable complementary autofluorescent protein fragment is nVenus. Alternatively, a suitable polyprotein is cYFP-aptamer-mCherry. In this situation, the interaction of the aptamer can be detected using BiFC, with a protein tagged with nYFP or nVenus. Expression of the aptamer in cells results in fluorescence (e.g. red from mCherry).
The system can be set up for bimolecular fluorescence complementation (BiFC) if a known target protein is tagged with cCFP or cYFP (if nYFP or nVenus is part of the polyprotein), or if a known target protein is tagged with nYFP or nVenus (if cYFP or cCFP is part of the polyprotein). Interaction of the aptamer with the target protein can generate yellow fluorescence resulting from correct folding of nVenus/nYFP and eCFP/cYFP. A series of vectors contain restriction endonuclease sites between mCherry and nVenus, into which the aptamer can be cloned. The system is adopted for Gateway® Recombination Cloning Technology use, or a suitable cloning method, achieving the same goals, as known to those of skill in the art.
Separately, a Gateway®-compatible library of 2×108 random aptamer sequences was generated for incorporation into the final aptamer expression vectors.
As proof of concept, >10 aptamers were generated that were directed against the Agrobacterium virulence effector protein, VirE2. When introduced into plant cells with VirE2-cCFP, many aptamers generate yellow fluorescence, indicating aptamer interaction with the VirE2 target protein. Hundreds of transgenic Arabidopsis plants were generated expressing various aptamers targeted against VirE2. When roots of these transgenic plants were challenged with infection by Agrobacterium, plants containing some (but not all) aptamers were more resistant to infection than were wild-type plants.
Uses of peptide aptamers includes generation of a phenotype (aptamer “mutagenesis”) without mutating the genome. This is done by aptamers interacting with target proteins and inhibiting protein function. Thus, they are used to inhibit any protein or function for which there is a detection assay, providing wide applications. Aptamers in cassettes as disclosed herein can inhibit Agrobacterium-mediated plant transformation. They may inhibit aggressive mobility of cancer cells, targeting suitable proteins. Expression of toxic compounds may be inhibited. The system is not limited to plants.
The aptamer expression system disclosed herein is unique. There are no reports of autofluorescence and BiFC used with aptamer or Gateway® technology. Thus, the system is uniquely set up to maximize ease of use and multiple applications.
An “expression cassette” was developed to express peptide aptamers in cells. This cassette constrains aptamers between proteins, e.g., an autofluorescent protein, such as mCherry (GenBank Accession Number AY678264) which will generate red fluorescence in transformed cells, and a complementing autofluorescent protein fragment, e.g. nVenus (
Ten different 20-mer aptamers (and one 80-mer) that target potential protein interaction sites of VirE2 were inserted into cassettes.
An aptamer polyprotein interacts with VirE2 in transiently transfected tobacco BY-2 protoplasts and generates yellow fluorescence in the cytoplasm. A control polyprotein (“empty aptamer vector”) does not interact with VirE2.
Several hundred transgenic Arabidopsis plants that express the aptamer polyproteins were generated. Assays of T1 and T2 generation plants for transformation susceptibility indicate that expression of aptamers designated 2, 5, 6, and 7 inhibit transformation. Aptamer 6 is especially inhibitory.
BiFC approach is based on complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are brought together by interactions between proteins fused to each fragment.
nYFP: 1-174 a.a. or nYFP: 1-154 a.a.
cYFP: 175-239 a.a. cYFP: 155-239 a.a.
For these experiments, nVenus1-174 and cCFP155-239 were used.
As proof of concept, VirE2 was chosen as a target protein because: VirE2 is important for Agrobacterium-mediated transformation of plants, and defining its functions provides useful information on the mechanisms of transformation. VirE2 is known to interact with both plant and Agrobacterium proteins in vivo, including other VirE2 molecules, VirE1, VirD4, VIP1, VIP2, and various importin α proteins. In addition, VirE2 presumably interacts with T-strands in plants. Protein interacting domains of VirE2 have been mapped in yeast. A crystal structure of VirE2 in a complex with VirE1 (Dym et al., 2008) serves as a guide for VirE2-VirE2 and VirE2-DNA interactions.
Suitable target proteins are those for which a detection assay is available.
In
Examples are provided for illustrative purposes and are not intended to limit the scope of the disclosure.
BiFC was developed to study protein-protein interactions in plant cells. In this approach, a molecule of yellow spectral variant of GFP (YFP) is separated into two portions, N-terminal (nYFP) and C-terminal (cYFP), neither of which fluoresces when expressed alone. Fluorescence is restored when nYFP and cYFP refold, as they are brought together as fusions with interacting proteins. BiFC allows detection of protein-protein interactions in planta and simultaneously determination of the sub-cellular localization of the interacting proteins. BiFC is the basis for detecting peptide aptamer-protein interactions.
mCherry (Shaner et al., 2004) and Venus (Nagai et al., 2002) are enhanced, monomeric forms of dsRed and GFP, respectively. As well as “constraining” the aptamer at its N-terminus, mCherry serves as a red fluorescent marker for those cells that have received the aptamer expression cassette. nVenus serves both to “constrain” the aptamer at its C-terminus, and as a non-fluorescent peptide that, when correctly folded with a C-terminal fragment of another GFP derivative, will fluoresce yellow in BiFC. Amino acids 1-174 of nVenus are “paired” with the C-terminal portion of CFP (cCFP, amino acids 155-239). Complementation by this combination of fragments results in the strongest fluorescence signals.
As proof of concept, Agrobacterium VirE2 protein was chosen as the target for aptamer interaction and mutagenesis because: 1) VirE2 is important for defining transformation functions; 2) VirE2 is known to interact with both plant and Agrobacterium proteins in vivo, including itself, VirE1, VirD4, VIP1, VIP2, and various important α proteins. In addition, VirE2 interacts with T-strands; 3) Some of the interacting domains of VirE2 have been mapped in yeast.
The aptamer expression cassette was co-electroporated (either lacking or containing the various aptamers; see
Transgenic Arabidopsis expressing each aptamer polyprotein were generated, and hundreds of plants were assayed for susceptibility to Agrobacterium. The results of these assays (
Gateway® Recombination Cloning Technology, (Invitrogen by Life Technologies) was used. The typical cloning workflow involves many steps, particularly traditional restriction enzyme cloning. Gateway® recombination cloning uses a one hour, 99%-efficient, reversible recombination reaction, without using restriction enzymes, ligase, subcloning steps, or screening of countless colonies and makes expression-ready clones. Gateway® technology facilitates cloning of genes, into and back out of, multiple vectors via site-specific recombination.
These publications are incorporated by reference to the extent they relate materials and methods disclosed herein.
Dym et al. (2008) Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners. Proc. Natl. Acad. Sci. USA 105: 11170-11175.
Hu et al., 2002. Mol. Cell 9:789-798 Visualization of protein-protein interactions in living cells.
Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnol. 20, 87-90.
Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., and Tsien, R. Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567-1572.
This patent application claims priority from copending U.S. Provisional Application 61/432,944 filed Jan. 14, 2011, the content of which is herein incorporated by reference in its entirety.
The United States Government has rights in this invention pursuant to National Science Foundation (NSF) Grant Contract No. 103735 and CBPR Grant No. GO12026-318, between the United States Government and Purdue Research Foundation.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US12/20806 | 1/10/2012 | WO | 00 | 3/15/2013 |
Number | Date | Country | |
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61432944 | Jan 2011 | US |