The authors would like to acknowledge funding from Japan Science and Technology Agency Exploratory Research for Advanced Technology (JST-ERATO), the New Energy and Industrial Technology Development Organization (NEDO), and the Cross-ministerial Strategic Innovation Promotion Program (SIP), Japan.

1. Preparation of Peptide-Based Formulations
<ol>
	<li>Prepare stock solutions of each peptide as follows: (KH)9-BP100 (1 mg/ml or 800 nM), Cytcox-(KH)9 (1 mg/ml), BP100 (1 mg/ml) and (BP100)2K8 (70 &#181;M). Weigh the required amount of each peptide in a 1.5 ml microcentrifuge tube and add autoclaved ultrapure water to dissolve the peptide. Mix well by repeated pipetting until a clear solution is obtained.</li>
	<li>Amplify and purify the plasmid DNA (pDNA), double-stranded DNA (dsDNA) and double-stranded RNA (dsRNA) according to standard molecular methodologies. In 1.5 ml microcentrifuge tubes, make stock solutions with a concentration of 1 mg/ml (pDNA and dsDNA) or 400 nM (dsRNA).</li>
	<li>Prepare the protein stock solution with a concentration of 7 &#181;M, by dissolving 1 mg of protein (e.g., alcohol dehydrogenase, ADH) powder in 1 ml of sodium carbonate solution (0.1 M, pH 9). Label the protein with fluorescent probes such as rhodamine B (RhB) according to standard protocols to enable microscopic visualization of the protein delivered into cells.</li>
	<li>Combine the respective components in a 1.5 ml microcentrifuge tube.
	<ol>
		<li>For peptide-pDNA formulations targeting the cytoplasm, add 6.4 &#181;l of (KH)9-BP100 (1 mg/ml) to 20 &#181;l of pDNA (1 mg/ml) and mix well by pipetting. Allow the mixture to stabilize for 15 min at 25 &#176;C. Add 773.6 &#181;l of autoclaved ultrapure water to dilute the solution to a final volume of 800 &#181;l. Use the pDNA construct designed for nuclear expression (P35S-RLuc-TNOS, Table 1).</li>
		<li>For peptide-pDNA formulations targeting the mitochondria, add 6.6 &#181;l of Cytcox-(KH)9 (1 mg/ml) to 20 &#181;l of pDNA (1 mg/ml) and mix well by pipetting. Allow the mixture to stabilize for 15 min at 25 &#176;C and then add 2.4 &#181;l of BP100 (1 mg/ml). Allow the mixture to stabilize for 15 min at 25 &#176;C for another 15 min. Add 771 &#181;l of autoclaved ultrapure water to dilute the solution to a final volume of 800 &#181;l. Use the pDNA construct designed for mitochondrial expression (pDONR-cox2:rluc, Table 1).</li>
		<li>For peptide-dsDNA formulations, add 5.1 &#181;l of (KH)9-BP100 (1 mg/ml) to 8 &#181;l of dsDNA (1 mg/ml) and mix well by pipetting. Allow the mixture to stabilize for 15 min at 25 &#176;C. Add 786.9 &#181;l of autoclaved ultrapure water to dilute the solution to a final volume of 800 &#181;l.</li>
		<li>For peptide-dsRNA formulations, add 50 &#181;l of (KH)9-BP100 (800 nM) to 50 &#181;l of dsRNA (400 nM) and mix well by pipetting. Allow the mixture to stabilize for 15 min at 25 &#176;C. Add 700 &#181;l of RNase-free water to dilute the solution to a final volume of 800 &#181;l.</li>
		<li>For peptide-protein formulations, add 16 &#181;l of (BP100)2K8 (70 &#181;M) to 16 &#181;l of ADH (7 &#181;M) and mix well by pipetting. Allow the mixture to stabilize for 15 min at 25 &#176;C. Add 768 &#181;l of autoclaved ultrapure water to dilute the solution to a final volume of 800 &#181;l.</li>
	</ol>
	</li>
	<li>Allow the formulations to stabilize for 15 min at 25 &#176;C.</li>
</ol>
2. Characterization of Peptide-Based Formulations
<ol>
	<li>Transfer each solution (800 &#181;l) into a cuvette for dynamic light scattering (DLS) analysis. Determine the hydrodynamic diameter and polydispersity index of formed complexes with a zeta nanosizer, using a 633 nm He-Ne laser at 25 &#176;C with a backscatter detection angle of 173&#176;.</li>
	<li>Following size measurements, transfer each solution (800 &#181;l) into a folded capillary cell for zeta potential measurements at default parameters of dielectric constant, refractive index and viscosity of water at 25 &#176;C.</li>
	<li>Observe a small volume of complex solution, used for DLS analysis, by atomic force microscopy (AFM). Deposit 10 &#181;l of complex solution onto the freshly exposed cleaved surface of a mica sheet and leave the mica to air dry overnight in a covered plastic petri dish.</li>
	<li>Acquire an image of the complexes in air at 25 &#176;C using a silicon cantilever with a spring constant of 1.3 N/m in tapping mode27,28.</li>
</ol>
3. Infiltration of Plant Leaves
<ol>
	<li>Use 3 week old soil-grown plants (Arabidopsis thaliana24, Nicotiana benthamiana24 or poplar26). Transfect at least 3 leaves to serve as triplicate for the quantification of gene expression or protein delivery.</li>
	<li>Load a 1 ml needleless plastic syringe with 100 &#181;l of complex solution for the transfection of one leaf. Position the tip of the syringe on the abaxial surface of the leaf.</li>
	<li>Press the syringe tip against the leaf slightly and depress the syringe plunger slowly while exerting a counter-pressure with the index finger of a latex-gloved hand from the opposite side. Successful infiltration can be observed as the spreading of a water-soaked area in the leaf. Label the infiltrated leaves for ease of identification.</li>
	<li>Incubate the transfected leaf for optimized durations following infiltration with peptide-pDNA (12 hr), peptide-dsDNA (12 hr), peptide-dsRNA (9 - 48 hr) and peptide-protein (6 hr) formulations under the following conditions: 16 hr light/8 hr dark at 22 &#176;C for A. thaliana and poplar, or 24 hr constant light at 29 &#176;C for N. benthamiana.</li>
</ol>
4. Evaluation of Transfection Efficiency
<ol>
	<li>Excise the whole transfected leaf of smaller plants (e.g., A. thaliana) or a 1 cm2 section of the infiltrated region for larger plants (e.g., N. benthamiana).</li>
	<li>For transfection experiments using Renilla luciferase (Rluc) reporter vector, determine the transfection efficiency quantitatively using an Rluc Assay Kit.
	<ol>
		<li>Place each excised leaf or leaf section in a 1.5 ml microcentrifuge tube. Add 100 &#181;l of 1&#215; Rluc Assay Lysis Buffer per tube. In the same manner, prepare lysates of non-transfected control leaves (triplicate).</li>
		<li>Grind the leaf using a homogenization pestle and incubate the resultant lysate at 25 &#176;C for 6 - 10 hr.</li>
		<li>Centrifuge the lysate at 12,470 &#215; g in a microcentrifuge for 1 min. Transfer 20 &#181;l of the cleared lysate to a well in a 96-well microplate, and use the remaining volume for quantification of total protein concentration using a BCA Protein Assay Kit according to the manufacturer&#39;s protocol.</li>
		<li>Add 100 &#181;l of 1&#215; Rluc Assay Substrate (diluted using the Rluc Assay Buffer) into the well and mix by slow pipetting. Place the microplate in a multimode microplate reader and initiate measurement.</li>
		<li>Subtract the background luminescence (mean of the non-transfected triplicate) from each experimental sample&#39;s luminescence. Calculate the ratio of photoluminescence (relative light units, RLU) to the amount of protein (mg).</li>
	</ol>
	</li>
	<li>For transfection experiments using green fluorescent protein (GFP) reporter vector or fluorescently labeled protein (e.g., ADH-RhB), observe the fluorescence using a confocal laser scanning microscope.
	<ol>
		<li>Cut the edges of a whole leaf (to aid the removal of air spaces), while a sectioned leaf can be used as is. Remove the plunger from a 10 ml syringe and place each excised leaf or leaf section in the syringe.</li>
		<li>Replace the plunger and push it gently to the bottom of the syringe without crushing the leaf. Draw water into the syringe until it is approximately half filled.</li>
		<li>Point the syringe upwards and push in the plunger to remove air from the syringe through the tip. Cover the tip of the syringe and pull the plunger down slowly to expel air from the leaf. Repeat this process several times until the leaf appears translucent.</li>
		<li>Cover the surface of a microscope slide with adhesive tape. Cut a square area on the tape large enough to accommodate the leaf sample using a blade, and peel the square piece of tape off with forceps to create a specimen chamber. The tape will serve as a spacer between the slide and coverslip.</li>
		<li>Place the leaf in the chamber with the abaxial surface facing upward and fill the remaining chamber area with water. Seal the leaf within the chamber with a glass coverslip and secure the edges of the coverslip with adhesive tape.</li>
		<li>Examine the leaf sample using the confocal laser scanning microscope under a 40X objective or a 63X water immersion objective. GFP or RhB fluorescence can be visualized at excitation wavelengths of 488 nm or 555 nm, respectively.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Critical steps within the protocol that have been identified empirically are discussed. By syringe infiltration, the peptide-based formulations are introduced into the airspaces inside the plant leaf through the stomata. To ensure maximal uptake of the solution, the infiltration process should be carried out when the plants are under conditions that are conducive to stomatal opening i.e., provided with sufficient water and during the light period. With regards to the preparation of transfection complexes, for formulations that involve a combination of two peptides (for mitochondrial-targeted DNA delivery), the sequence for addition of each component is crucial and should not be inverted.
There are many plausible modifications to the procedure. Another option for the infiltration of plant cells is the use of vacuum infiltration, which is able to introduce the complex solution into whole plants and/or partial tissues including apical meristems. For this alternative procedure, seedlings are submerged in the transfection solution, and based on the pressure generated by the vacuum, the peptide-cargo complexes are forced through the stomata and into the plant cells (Yoshizumi, T., unpublished data).
Transient gene expression, based on studies using animal cells, has been shown to increase with higher DNA concentrations29-31. It is important to note that the ratio of peptide to DNA affects the biophysical properties (size, surface charge) of complexes (Figure 4), which influences transfection efficiency; hence, the optimal ratio needs to be maintained even with increased DNA concentration.
One of the main advantages in using peptides as a gene/protein delivery agent is that its sequence is amenable to tuning to fulfill a desired function. For example, the mitochondrial-targeting domain of the carrier peptide described in this study can be replaced with chloroplastic or peroxisomal-targeting sequences for localization to these organelles. While chloroplast transformation is possible in several plants using biolistics32-34, neither the Agrobacterium nor the biolistic method could introduce genes into the mitochondria or other organelles besides the nucleus (Table 2).
There are no rigid host-range limitations for peptide-based transfection, unlike the Agrobacterium-based method (Table 2). So far, formulations for the transfection of A. thaliana, N. benthamiana and poplar have been optimized (Table 1), but the method can also be applied for Nicotiana tabacum, tomato cultivar Micro-Tom, rice (based on preliminary experiments), and other mono- and dicotyledonous plants.
As yet, there is no known limitation in transgene size when peptides are used as transfection vectors. In contrast, large DNA molecules have been found to reduce the transformation efficiency of Agrobacterium-mediated methods35,36, and an upper size limit for transgenes of approximately 200 kb has been reported37-39. Using biolistics, on the other hand, large DNA fragments may be sheared during preparation or delivery into plants38. Although no upper limit has been determined for biolistic transformation so far, physical constrains have been found to restrict the size of DNA that can be transferred to much less than 150 kb40. The major benefits of using the peptide-based system for gene transfer compared to existing approaches employing either Agrobacterium or microprojectile bombardment, discussed above, are summarized in Table 2.
A few limitations exist for this method as it stands. Firstly, targeted delivery of pDNA to specific organelles, such as the mitochondria, has been proven possible by a simple combination of DNA-binding, cell-penetrating and organelle-transit sequences although the efficiency was low. Transgene expression could be detected, by confocal microscopy, only in a small population of mitochondria within cells. Hence, further modifications are necessary to: (i) enhance the translocation of more complexes across the cell/organellar membrane, and (ii) improve the dissociation and transfer of pDNA from the carrier peptide into the target organelle for expression. Secondly, using this DNA delivery system, the transient expression of exogenous reporter genes was successfully achieved in the cytosolic and mitochondrial compartment of cells. Stable incorporation and expression of the introduced genes in the plant nuclear/organellar genome have not been established yet, however, due to the absence of suitable selection strategies.
While acknowledging that there are areas for improvement or further development, the peptide-derived strategy described here remains a simple and versatile technique that has paved the way for the delivery of various cargoes into diverse plant types.

Plant genetic engineering is conventionally used for transferring beneficial traits to plants. In recent years, this technology has been applied to convert plants into bio-factories for the production of pharmaceutically important and commercially valuable proteins, many of which cannot be chemically synthesized and are very costly to produce using animal or microbial systems1. By the introduction of new genes, the plant's own metabolism can be manipulated for the production of various biopharmaceuticals like antibodies, metabolic enzymes, hormones, antigens or vaccine2.
Established gene transfer technologies for plants are the Agrobacterium-mediated delivery3, bombardment with DNA-coated microprojectiles (biolistics)4, and electroporation5 or polyethylene glycol6 treatment of protoplasts. Techniques requiring protoplasts are generally avoided because they are time-consuming, cumbersome and yield inconsistent results7. As a result, virtually all plant modification work at present utilizes either Agrobacterium or microprojectiles for gene transfer. The Agrobacterium method is more extensively used but not applicable to many economically important plant species. Meanwhile, microprojectile bombardment is more versatile due to a broad range of susceptible plants but requires specialized equipment and often causes severe tissue damage. Furthermore, these methods involve either a random (biolistics) or complex (Agrobacterium) delivery mechanism and have variable transformation rates8. Hence, a novel plant transformation technology that is simple yet effective, and applicable to different plant types is required.
Currently, plants are subjected to genetic modification primarily by the delivery of exogenous DNA encoding a desired trait, rather than by direct delivery of a target protein. The higher stability of DNA over proteins is a prime advantage; nevertheless, potential problems associated with DNA delivery include the random insertion of exogenous DNA into the plant genome and unintended transmission of antibiotic resistance genes to pathogenic bacteria via horizontal gene transfer9. For genome-editing purposes, the ability to edit plant genomes without introducing foreign DNA into cells may circumvent regulatory concerns related to genetically modified plants. Thus, an alternative DNA-free strategy for the modification of plants by direct delivery of protein will be able to cater to these needs.
Here we introduce a peptide-based system, originally developed for human gene therapy10-14, for the targeted delivery of exogenous genes or proteins in intact plants. Peptides are able to protect DNA from nuclease degradation and can mediate gene transfer across cell as well as organellar membranes15-17. They also have diverse and tunable properties besides being non-cytotoxic18-20. More importantly, with the use of peptides, genes can be precisely targeted to intracellular organelles such as the mitochondria21 or plastids (chloroplasts)22 for expression-a task not achievable by biolistic or Agrobacterium-mediated transformation. Depending on the cargo type, this new plant modification technology can be exploited to deliver proteins23 and either express (plasmid21,24 or double-stranded DNA25) or down-regulate (double-stranded RNA26) specific genes within the plant, throughout its cytosolic space24-26 or within a specific organellar compartment21. The designed carrier peptides consist of a cationic domain in the form of either polylysine (K8) or polylysine-co-histidine (KH)9 for binding and/or condensation of negatively-charged cargoes, which is conjugated to cell penetrating (BP100 peptide) or mitochondria transit (Cytcox peptide) sequences.

<table border="0" cellpadding="0" cellspacing="0" width="2358">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">(KH)9-BP100 peptide</td>
			<td style="width:355px;">Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Sequence: KHKHKHKHKHKHKHKH- 
			KHKKLFKKILKYL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">(BP100)2-K8 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: KKLFKKILKYLKKLFKKIL- 
			KYLKKKKKKKK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BP100 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: KKLFKKILKYL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cytcox-(KH)9 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: MLSLRQSIRFFKKHKHKH- 
			KHKHKHKHKHKH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">P35S-GFP(S65T)-TNOS and P35S-RLuc-TNOS</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Biomacromolecules 2013, 14, 10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pDONR-cox2:gfp and pDONR-cox2:rluc</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of cox2 mitochondrial-specific promoter (Ref: Chuah et al. Sci Rep 2015, 5, 7751)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">dsDNA (PCR-amplified from pBI221-P35S-Rluc-TNOS)</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Plant Biotechnol 2015, 32, 39)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP5 siRNA</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">For RNA interference-mediated silencing of green fluorescent protein (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHS siRNA</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">For RNA interference-mediated silencing of chalcone synthase (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1 mL and 10 mL Plastic Syringes</td>
			<td>TERUMO Corporation</td>
			<td>SS-01T, SS-10ESZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1.5 mL Microcentrifuge Tube</td>
			<td>AS ONE Corporation</td>
			<td>151212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">96-Well Flat-Bottom Plate</td>
			<td>Asahi Glass Co., Ltd.</td>
			<td>3860-096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Adhesive Tape</td>
			<td>Sekisui Chemical Co., Ltd.</td>
			<td>No. 835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Alcohol Dehydrogenase</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td style="width:315px;">A-7011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Atomic Force Microscope</td>
			<td>Seiko Instruments Inc.</td>
			<td>SPI3800, SPA 300HV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Atomic Force Microscope</td>
			<td>Hitachi High-Tech Science Corporation</td>
			<td>AFM5300E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cantilever</td>
			<td>Hitachi High-Tech Science Corporation</td>
			<td>K-A102001593</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Confocal Laser Scanning Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cork Borer</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td>Z165220</td>
			<td>For excision of leaves into 1-cm diameter disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coverslip</td>
			<td style="width:355px;">Matsunami Glass Ind., Ltd.</td>
			<td style="width:315px;">C02261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cuvette</td>
			<td style="width:355px;">Sarstedt</td>
			<td style="width:315px;">759116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Folded Capillary Cell</td>
			<td style="width:355px;">Malvern Instruments, Ltd.</td>
			<td style="width:315px;">DTS1070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Forceps</td>
			<td style="width:355px;">Shimizu Akira Inc.</td>
			<td style="width:315px;">Stainless pincet 150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Homogenization Pestle</td>
			<td>Ieda Trading Corp.</td>
			<td style="width:315px;">9993</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mica</td>
			<td>Nisshin EM Co., Ltd.</td>
			<td style="width:315px;">LC23Z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Microcentrifuge</td>
			<td>Beckman Coulter</td>
			<td style="width:315px;">BKA46472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate Reader</td>
			<td>Molecular Devices Corporation</td>
			<td style="width:315px;">Spectra MAX M3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Microscope Slide</td>
			<td style="width:355px;">Matsunami Glass Ind., Ltd.</td>
			<td>S011120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Pipette</td>
			<td style="width:355px;">Eppendorf Research&#174; plus</td>
			<td>3120000909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Pipette Tips</td>
			<td>AS ONE Corporation</td>
			<td>2-5138-01, 2-5138-02, 2-5138-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Plastic Petri Dish</td>
			<td>AS ONE Corporation</td>
			<td>1-7484-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Renilla Luciferase Assay Kit</td>
			<td>Promega corporation</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Rhodamine B Isothiocyanate</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td style="width:315px;">283924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNase-Free Water</td>
			<td style="width:355px;">Qiagen</td>
			<td style="width:315px;">129112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Sodium Carbonate</td>
			<td>Wako Pure Chemical Industries, Ltd.</td>
			<td>199-01585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Surgical Blade and Handle</td>
			<td>FEATHER Safety Razor Co., Ltd.</td>
			<td>Stainless steel No. 14 (blade), No. 3L (handle)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Syringe Tip Cap</td>
			<td>Musashi Engineering Inc.</td>
			<td>NC-3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Weighing Balance</td>
			<td>Sartorius</td>
			<td>CPA225D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Zeta Potentiometer</td>
			<td>Malvern Instruments, Ltd.</td>
			<td>Zetasizer Nano-ZS</td>
			<td style="width:1235px;"></td>
		</tr>
	</tbody>
</table>

peptide-derived method to transport genes and proteins across cellular and organellar barriers in plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

An array of nucleic acid and protein cargoes were successfully introduced into various plants using the designed peptides as delivery vectors. Electrostatic interactions between cationic peptides and negatively-charged cargoes resulted in the formation of transfection complexes that can be directly infiltrated into plant leaves using a needleless syringe (Figure 1). Optimized formulations (empirically determined in these studies21,23-26) for the transfection of plant cells are listed in Table 1, where each type of the wide range of delivered cargoes (pDNA, dsDNA, dsRNA and protein) is represented. The mean diameters of all peptide-based formulations were in the approximate range of 150 - 300 nm. Based on DLS analysis, all formulations displayed relatively low size polydispersities, indicating that the formed peptide-cargo aggregates have a uniform size distribution. The morphologies of complexes between peptide and pDNA (Figure 2A) or protein (Figure 2B), on mica, were imaged by AFM. Homogeneous globular complexes were observed for both peptide-pDNA and peptide-protein combinations, in agreement with the data from DLS measurements. In terms of the zeta potential of complexes (Table 1), pDNA- and dsDNA-derived complexes had net negative surface charges while dsRNA-based complexes had a near-neutral surface charge. Peptide-protein complexes, on the other hand, were positively charged.
The efficiencies of peptide-pDNA and peptide-dsDNA formulations in mediating the transfection of A. thaliana or N. benthamiana as model plant systems were evaluated quantitatively as well as qualitatively. The RLuc gene expression assay was employed for quantification of gene expression levels (Table 1), therefore, for this experiment pDNA or dsDNA encoding the RLuc gene must be used for complexation with the respective carrier peptides. Using the (KH)9-BP100/pDNA formulation, nuclear-targeted delivery and expression of pDNA can be achieved, following an incubation period of 12 hr, with an estimated RLU/mg value of approximately 1 &#215; 105. For mitochondrial-targeted delivery and expression of pDNA, a combination of peptides, Cytcox-(KH)9 and BP100, is required for complex formation. With the same optimized incubation period of 12 hr, however, a much lower level of transfection (approximately 1 &#215; 103 RLU/mg) was attained. Meanwhile, similar incubation period (12 hr) and gene expression level (approximately 1 &#215; 103 RLU/mg) was required/recorded for dsDNA-based complexes, also formulated using the (KH)9-BP100 peptide. Qualitative assessments of gene expression were carried out by direct microscopic observation of leaves treated with complexes prepared using pDNA or dsDNA encoding the GFP reporter gene. In cells transfected with non-targeted peptide-pDNA complexes, diffuse green fluorescence corresponding with GFP expression was clearly observed and found to localize in the cytosol (Figure 3A). Distinct differences in the localization pattern of GFP fluorescence were evident in cells infiltrated with mitochondrial-targeted peptide-pDNA complexes. Here, punctate green fluorescence that colocalize with the mitochondrial stain was visible, confirming the specificity of gene expression exclusively in the mitochondrial compartment of cells (Figure 3B).
In the case of peptide-protein formulations, conjugation of the protein cargo (ADH) to a fluorophore (RhB) will enable visualization of the delivered protein in the intracellular compartment. Within a short incubation period of 6 hr, ADH-RhB protein (blue) was found to be distributed throughout the cytosol and vacuole of infiltrated cells (Figure 3C). Meanwhile, rapid and efficient down-regulation of gene expression could be accomplished in various plants using peptide-dsRNA formulations. In the first experiment, A. thaliana leaf was infiltrated with peptide-dsRNA complexes to silence the chalcone synthase gene (CHS) responsible for anthocyanin (red pigment) biosynthesis under drought conditions. The difference in appearance of A. thaliana leaves under normal (Figure 3D, a) and drought conditions (Figure 3D, b) provided an easy means to evaluate CHS silencing using the optimized peptide-dsRNA formulation (arrow 1 indicates the infiltrated region). In the second experiment, peptide-dsRNA complexes were infiltrated into the leaves of transgenic A. thaliana expressing yellow fluorescent protein (YFP). An apparent reduction in YFP expression could be seen in the epidermal cells 9 hr post-transfection (Figure 3E, F). The effectiveness of the formulation in down-regulating gene expression in a different plant system (poplar, 12 hr post-transfection) was also verified (Figure 3G, H).
<img alt="Figure 1" src="/files/ftp_upload/54972/54972fig1.jpg" /> 
Figure 1: Peptide-based Formulations for the Delivery of Nucleic Acid and Protein Cargoes into Living Plants. 
The designed carrier peptides consist of polycations conjugated to cell penetrating or organellar transit sequences. Polycations enable binding and/or condensation of negatively-charged cargoes as well as escape from the endosomal compartment following internalization into cells. Delivery of cargoes into cells and subsequently to specific organelles is mediated by cell penetrating sequences and organellar transit sequences, respectively. Various cargoes that could be successfully delivered into the plant include nucleic acids such as pDNA, dsDNA and dsRNA, as well as model proteins like bovine serum albumin (BSA), alcohol dehydrogenase (ADH) and citrine (a variant of yellow fluorescent protein). Bioactive molecules are able to form transfection complexes with peptide conjugates via electrostatic interactions, which are introduced into plant leaves by syringe infiltration. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54972/54972fig2.jpg" /> 
Figure 2: Morphologies of the Peptide-based Formulations. 
(A) AFM amplitude image of (KH)9-BP100/pDNA formulation at N/P 0.5. (B) AFM height image of (BP100)2K8/ADH formulation at molar ratio 10. Reproduced with permission from published sources23,24. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54972/54972fig3.jpg" /> 
Figure 3: Microscopic Evaluation of Transfection Efficiencies using Optimized Peptide-based Formulations. 
(A) Cytosolic GFP expression (green), clearly distinguished from chloroplast autofluorescence (red), was observed in the spongy mesophyll cells of A. thaliana leaves infiltrated with (KH)9-BP100/pDNA formulation (N/P 0.5; 12 hr). (B) GFP expression (green) was detected in the mitochondria (red) in the epidermal cells of A. thaliana leaf infiltrated with Cytcox-(KH)9/BP100/pDNA formulation (N/P 0.5 for each peptide; 12 hr). Enlarged images of mitochondria with GFP expression are shown in the extreme right panel. (C) Delivery of ADH-RhB (blue) into the spongy mesophyll cells of A. thaliana leaves mediated by (BP100)2K8 at a peptide/protein molar ratio of 10, visualized 6 hr post-infiltration. (D) A. thaliana leaf before (a) and after (b) treatment with (KH)9-BP100/dsRNA formulation (molar ratio 2; 48 hr), which resulted in the suppression of anthocyanin biosynthesis pathway. A similar formulation containing GFP5 dsRNA was infiltrated into the leaf as negative control (c). Arrows 1 and 3 indicate the infiltrated area while arrows 2 and 4 indicate the non-infiltrated area within the leaf. (E) A. thaliana leaves expressing yellow fluorescent protein (YFP) and (F) the diminished YFP fluorescence following infiltration with (KH)9-BP100/dsRNA formulation (molar ratio 2; 9 hr). (G) Transgenic poplar leaves expressing yellow fluorescent protein (YFP) and (H) the diminished YFP fluorescence following infiltration with (KH)9-BP100/dsRNA formulation (molar ratio 2; 12 hr). Reproduced with permission from published sources21,23,24,26. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54972/54972fig4.jpg" /> 
Figure 4: Variation in Peptide-to-DNA (N/P) Ratio and the Effect on Biophysical Properties of Complexes. 
With increasing peptide to DNA ratio, peptide-based formulations decrease in size while their zeta-potential values transition from negative to positive. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54972/54972table1.jpg" /> 
Table 1: Characterization and Evaluation of Various Peptide-based Formulations. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972table1large.jpg" target="_blank">Please click here to view a larger version of this table.</a>
<img alt="Table 2" src="/files/ftp_upload/54972/54972table2.jpg" /> 
Table 2: A Comparison of Peptide-based and other Existing DNA Delivery Technologies for Intact Plants. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54972/54972table2large.jpg" target="_blank">Please click here to view a larger version of this table.</a>

Watch this Scientific Journal Video about Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants at JoVE.com

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

peptide-derived-method-to-transport-genes-proteins-across-cellular

Nanyang Technological University

Research

JoVE Journal

Genetics

10.5K Views.  RIKEN Center for Sustainable Resource Science. Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

Video: Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...