Name: high-density dna and rna microarrays - photolithographic synthesis, hybridization and preparation of large nucleic acid libraries
Uploaded: 2019-08-12T14:00:00.000+00:00
Duration: 11 min 22 s
Description: Watch this Scientific Journal Video about High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries at JoVE.com

This work was supported by the Austrian Science Fund (FWF grants P23797, P27275 and P30596) and the Swiss National Science Foundation (Grant #PBBEP2_146174).

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Solid-phase DNA and RNA synthesis is the bread and butter of every nucleic acid chemistry lab, and although the addition of the photolithography component is admittedly a complex operation, microarray fabrication mediated by UV light is also a very reliable process. It is, in addition, the only available method for in situ RNA synthesis on microarrays. Still, as in any multi-stage experimental procedure, there is ample room for human error.
Perhaps the most critical step is the coupling of a phosphoramidite, as it needs to be a constantly high-yielding chemical reaction in order to afford oligonucleotides with few synthetic errors. In our microarray synthesis protocol, phosphoramidite coupling is even more pivotal to overall synthesis quality since the fabrication process bypasses capping and prevents oligonucleotide purification. Stepwise coupling efficiencies above 99% have been calculated for all photosensitive DNA and RNA phosphoramidites, even for very short coupling times (15 s)24 but lower coupling yields can occasionally occur, particularly in the case of dG amidites. The stability of the solubilized phosphoramidites at room temperature has been investigated before and was shown to depend on the nature of the nucleobase, with guanosine phosphoramidites prone to extensive degradation in only a matter of days29,30. But when stored at -25 &#176;C, dG phosphoramidites dissolved in ACN as 30 mM solution were found to be stable for several weeks. The relative instability of dG phosphoramidite solutions at room temperature does however mean that they should not be kept attached to the DNA synthesizer for several days.
For RNA phosphoramidites, the coupling yield is very dependent on phosphoramidite quality (which can be assessed by 31P NMR spectroscopy) and coupling time. Coupling times of 5 minutes for rA, rG, rC and 2 min for rU appear necessary. Indeed, we found that shortening the condensation time to 2 min for all RNA phosphoramidites led to significantly lower hybridization signals.
The DNA synthesizer itself, as well as the reagents and solvents, certainly needs to be as clean as possible in order to achieve the highest yield of oligonucleotide synthesis. However, insoluble material, salts or particles, can accumulate over time in the lines and tubing of the delivery system, leading to a gradual decrease in consumption of reagents and reactants. Where a general cleaning of the synthesizer does not resolve a low output volume, an increase in the number of pulses can be an alternative solution. Particularly useful in the case of low phosphoramidite consumption, the line in the coupling protocol corresponding to the pumping of a mix of phosphoramidite and activator (third line of the coupling subsection in Table 1 and Table 2) can be modified, from 6 to 9 pulses without any appreciable negative effect on synthesis quality. Furthermore, the number of pulses of activator needed to bring the amidite/activator mix to the synthesis substrate (currently 6, fourth line in the coupling subsection, see Table 1 and Table 2) depends on the DNA synthesizer itself as well as on tubing length in the synthesis cell. This number can be adjusted after replacing the phosphoramidite with a colored solution and counting the number of pulses needed to push the colored mix to the glass substrate for coupling.
The method described herein allows for DNA and RNA synthesis to proceed simultaneously, on the same microarray. Hybrids of DNA and RNA may also be prepared without any change to the array fabrication protocols, and as long as the three-step deprotection protocol is followed. However, it should be noted that RNA-only microarrays only require a two-step deprotection: a decyanoethylation first with Et3N followed by hydroxyl and base deprotection with hydrazine. DNA nucleobases were found to be incompletely deprotected under those conditions, and need the additional step in EDA in order to effect the complete removal of phenoxyacetyl (Pac) groups. This extra treatment with EDA is shorter (5 min) than for the standard deprotection of DNA microarrays31, but it is sufficient to drive it to completion after the triethylamine and hydrazine treatments. In addition, a short reaction time with EDA limits the exposure of a fully-deprotected RNA oligonucleotide to basic conditions.&#160;&#160;
An advantage of in situ RNA array synthesis over alternative methods like spotting or DNA transcription32,33,34 is the ability to store the synthesized RNA microchip in protected form until use, thus avoiding the risk of potential RNA degradation. Post-synthetic procedures for RNA does, on the other hand, imply that consumables and reagents are kept sterile and that handling is performed under RNase-free conditions. Of note, we found that the addition of RNase inhibitor to the hybridization mix did not yield stronger hybridization signals for the RNA features.
The synthesis of DNA libraries on a base-sensitive monomer is more complex than the synthesis of a few control sequences on a surface, and as such is certainly more prone to design errors. Yet, assuming that the sequence design (i.e., the nature and the number of sequences) is correct, transforming this list into a collection of exposure masks and an ordered series of coupling cycles remains a straightforward process. However, important variations from standard microarray synthesis exist and are critical to a successful fabrication of a high-density library array.
First, a base-sensitive dT monomer is coupled as the first phosphoramidite after synthesis of the linker. The coupling yield of this monomer (Figure 1) was found to be relatively low, around 85%28, which is why efforts are made to improve its incorporation rate, either by increasing its concentration in ACN from 30 mM to 50 mM, or by repeating the coupling step: two consecutive coupling reactions using fresh monomers, or two separate but consecutive coupling cycles.
The second change is the addition of a &#946;-carotene solution in the back chamber of the synthesis cell, which conveniently absorbs 365 nm light. This is an important modification of the photolithography setup as it prevents UV light from reflecting back onto the array substrate. Indeed, after traversing the interstitial medium between the substrates, incoming UV light exits through the drilled slide and reaches the quartz block of the cell. The Fresnel equations predict that ~4% of perpendicularly incident UV light will reflect from each of the three downstream air-glass interfaces (exit side of the 2nd substrate and both sides of the quartz block) and back onto the synthesis substrate, leading to unintended exposure of photoprotected oligonucleotides. Diffraction and scattering also contribute to &#34;off-target&#34;&#160;photodeprotection and, therefore, to nucleotide insertion, which directly affects the error rate of synthesis, but these contributions are much smaller than reflections and can mainly be addressed by reducing synthesis density (leaving gaps between features). We have found that the level of &#946;-carotene solution in the lower chamber of the cell tends to slightly decrease only during the first minutes of array synthesis, and therefore needs to be monitored and readjusted.
Finally, the third change is the deprotection solution, replacing EtOH for toluene, which keeps the cleaved DNA library bound to the surface, presumably through electrostatic interactions. Applying a small amount of water to the synthesis area after ACN washing allows for the library to be conveniently collected. The process is however only successful if the water content in EDA and toluene is minimal, rendering the nucleic acid entirely insoluble in the deprotection cocktail. Alternatively, DNA libraries may be cleaved off the chip using ammonia9,10,14,35, then further deprotected by heating the DNA-containing aqueous ammonia solution to 55 &#176;C overnight. The recovery of DNA libraries using ammonia is however not compatible with RNA. RNA oligonucleotides on a base-cleavable&#160;substrate can be eluted from the surface using the same EDA/toluene procedure described above, but only at the penultimate stage after the Et3N and hydrazine two-step deprotection strategy28.
Alternative strategies to recover oligonucleotide pools from microarrays without the need for a specific basic treatment exist, are in principle compatible with photolithography and rely on the use of enzymes. For instance, a single deoxyuracil nucleotide can be the target of the uracil-DNA glycosylase (UDG) and excised from the rest of the DNA sequence, or a single RNA unit can be recognized by RNase H type 2 enzymes and the phosphodiester bond 5&#39;&#160;to the RNA cleaved, releasing the 5&#39;&#160;DNA part23.
We now have a powerful, reliable and high-density method for the synthesis of DNA, RNA, and hybrid DNA/RNA microarrays. These can not only serve as platforms for hybridization or binding assays36, but they also represent a fast and inexpensive way to produce complex nucleic acid libraries. For DNA-based digital data storage, microarray photolithography may become a potential solution to the &#34;writing&#34;&#160;bottleneck (i.e., to the encoding of information by synthesis). The success in digital encoding on DNA and in de novo gene assembly depends on sequence fidelity which, at the synthesis level, translates into the error rate. Synthetic and optical errors in our current array fabrication protocols will be discussed and reported on elsewhere. In parallel, efforts are now underway to further increase fabrication scale and throughput.

The practical use of DNA microarrays have traditionally been in the study of the variations in the gene expression levels between two cell populations, using complementary strands and fluorescence as a detection method1. Occasionally, DNA microarrays venture into binding events with non-nucleic acid ligands, such as proteins, with a strategy of systematic sequence permutation that offers a comprehensive overview of the binding landscape2,3,4,5. This approach effectively transforms microarrays from mere hybridization surfaces into platforms with broad sequence coverage, which would be an asset for the study of the richer and more complex world of RNA structure and function. Supported by the extremely efficient phosphoramidite coupling reaction6, in situ synthesized DNA arrays can now also be regarded as a cheap source of DNA7, which is becoming particularly relevant considering the ever-increasing demand for nucleic acid material for gene assembly8,9, DNA-based nanostructures10, information storage or sequencing11,12. Likewise, sequencing technologies are likely to benefit from the development of methods that yield very complex mixtures of RNA oligonucleotides13. In this context, array fabrication protocols that allow for oligonucleotides to be synthesized in situ and at high density are ideally placed to meet the needs of the rapidly expanding field of nucleic acid biotechnology. However, with a field as diverse as biotechnology, the purpose of each application may require that DNA on microarray be produced either at high-throughput or with a very low amount of synthetic errors14,15, or both, requiring a closer look at the synthesis protocols of DNA microarrays which, historically, have been primarily optimized for hybridization assays. Meanwhile, in situ synthesis of RNA microarrays has been found to be a challenging endeavor, with most of the difficulty associated with the protecting group for the 2&#39;-OH function, usually a silyl moiety in standard solid-phase synthesis that is removed with fluorine-based reagents, chemicals that are incompatible with glass or silicon surfaces. Those issues and challenges in DNA and RNA microarray synthesis have lately been the subject of a large body of work, in particular with the photolithography approach16.
Photolithography uses UV light to unblock oligonucleotides before coupling and requires masks to construct a pattern of UV exposure, thereby spatially organizing and controlling the growth of oligonucleotides. Physical masks have been replaced by computer-controlled micromirrors&#160; whose tilting selectively reflects UV light onto the microarray substrate17,18,19. As a UV source, we use 365 nm light from a high power LED source20. Current photolithographic setups are equipped with micromirror arrays containing 1024 &#215; 768 mirrors, corresponding to more than 780,000 individually addressable spots (&#34;features&#34;) on a small area of just 1.4 cm2, or 1080p with arrays of 1920 &#215; 1080, or &#62;2 million mirrors. Each of the mirrors in the device therefore has direct control over the sequence grown on the corresponding feature. With the exception of UV light, photolithography functions like a solid-phase synthesis technique and adopts the cycle-based phosphoramidite chemistry. Only it requires an entirely different protection strategy for RNA synthesis to succeed. We developed a new series of light-sensitive RNA phosphoramidites bearing hydrazine-labile protecting groups21. These monomers allow for the RNA to be deprotected under mild conditions that do not affect the integrity of the surface. A first deprotection step uses triethylamine to remove the cyanoethyl phosphodiester protecting groups, while hydrazine is used in a second, separate step to remove those at the 2&#39;-OH and exocyclic amine functions. In so doing, RNA oligonucleotides ~30-nt in length and of any sequence can now be synthesized in situ on microarrays22,23. In parallel, we also recently started to address the questions of throughput, quality and speed in DNA and RNA photolithography. We measured coupling efficiencies &#62;99% for all DNA and RNA amidites (Figure 1) and investigated each individual step in the oligonucleotide elongation cycle, from oxidation time, to choice of activator and to optimal UV exposure24,25. We have brought in new light-sensitive 5&#39; protecting groups that can be removed in seconds only, transforming the synthesis of hundreds of thousands of 100mers into a few hours-long process26. We have also doubled the throughput of array fabrication by exposing two substrates simultaneously27. Finally, we have introduced a dT phosphoramidite containing a base-sensitive succinyl group as a convenient way to cleave, collect and analyze DNA and RNA oligonucleotides, which is central to library preparation28.
In spite of the relatively mundane aspect of DNA and RNA solid-phase synthesis, especially for nucleic acid chemists, microarray photolithography remains a non-trivial upgrade requiring a complex setup, careful control and supervision of the process, and separate instructions for post-synthetic handling depending on the nature of the oligonucleotide and the type of application. In this article, we wish to provide a detailed presentation of the entire step-by-step procedure of in situ synthesis of DNA and RNA microarrays by photolithography, from experimental design to data analysis, with an emphasis on the preparation of instruments and consumables. We then describe the post-synthetic deprotection methods that correspond to the intended purpose of microarray fabrication (i.e., either hybridization or the recovery of nucleic acid libraries).&#160;

<table><tbody><tr><td>&lt;u&gt;&lt;em&gt;Slide functionalization&lt;/em&gt;&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetic acid &gt;99.8%</td><td>Sigma</td><td>33209</td><td>For RNA deprotection</td></tr><tr><td>CNC router</td><td>Stepcraft</td><td>300 CK</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td>1.07017.2511</td><td>For deprotection and functionalization</td></tr><tr><td>N-(3-triethoxysilylpropyl)-4-hydroxybutyramide</td><td>Gelest</td><td>SIT8189.5</td><td>Silanizing reagent</td></tr><tr><td>Nexterion Glass D microscope slides</td><td>Schott</td><td>1095568</td><td></td></tr><tr><td>Polymax 1040</td><td>Heidolph</td><td></td><td>Orbital shaker</td></tr><tr><td>Proclean 507 Ultrasonic water bath</td><td>Ulsonix</td><td></td><td>To clean slides after drilling</td></tr><tr><td>Tickopur RW 77 Special Purpose Cleaner</td><td>Sigma</td><td>Z860086</td><td>To clean slides after drilling</td></tr><tr><td>&lt;u&gt;&lt;em&gt;Microarray synthesis&lt;/em&gt;&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>0.25 M dicyanoimidazole in ACN</td><td>Biosolve</td><td>0004712402BS</td><td>Activator</td></tr><tr><td>0.7 XGA DMD</td><td>Texas Instruments</td><td></td><td>Digital Micromirror Device</td></tr><tr><td>20 mM I&lt;sub&gt;2&lt;/sub&gt; in pyridine/H&lt;sub&gt;2&lt;/sub&gt;O/THF</td><td>Sigma</td><td>L860020-06</td><td>Oxidizer</td></tr><tr><td>250 &amp;mu;m thick Chemraz 584 perfluoroelastomer</td><td>FFKM</td><td></td><td>Lower teflon gasket</td></tr><tr><td>2&#039;-O-ALE RNA phosphoramidites</td><td>ChemGenes</td><td></td><td></td></tr><tr><td>365 nm high-power UV-LED</td><td>Nichia</td><td>NVSU333A</td><td></td></tr><tr><td>5&#039;BzNPPOC DNA phosphoramidites</td><td>Orgentis</td><td></td><td></td></tr><tr><td>5&#039;NPPOC DNA phosphoramidites</td><td>FlexGen</td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Biosolve</td><td>0001205402BS</td><td>For DNA synthesis</td></tr><tr><td>Amidite Diluent for DNA synthesis</td><td>Sigma</td><td>L010010</td><td>For dissolving phosphoramidites</td></tr><tr><td>Cleavable dT</td><td>ChemGenes</td><td></td><td>Base-sensitive monomer for library preparation</td></tr><tr><td>DMSO</td><td>Biosolve</td><td>0004474701BS</td><td>As exposure solvent</td></tr><tr><td>&lt;u&gt;&lt;em&gt;DNA and RNA microarray deprotection&lt;/em&gt;&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethylenediamine &gt;99.5%</td><td>Sigma</td><td>3550</td><td>For deprotection</td></tr><tr><td>Expedite 8909</td><td>PerSeptive Biosystems</td><td></td><td>DNA synthesizer</td></tr><tr><td>Hydrazine hydrate 50-60% hydrazine</td><td>Sigma</td><td>225819</td><td>For RNA deprotection</td></tr><tr><td>Imidazole</td><td>Sigma</td><td>56750</td><td></td></tr><tr><td>Industrial Strength lower-density PTFE tape</td><td>Gasoila</td><td></td><td>Thin, upper teflon gasket</td></tr><tr><td>Pyridine &gt;99%</td><td>Sigma</td><td>P57506</td><td>For RNA deprotection</td></tr><tr><td>Triethylamine &gt;99%</td><td>Sigma</td><td>T0886</td><td>For RNA deprotection</td></tr><tr><td>&amp;beta;-carotene</td><td>Sigma</td><td>C9750</td><td>For library preparation</td></tr><tr><td>&lt;u&gt;&lt;em&gt;Hybridization and scanning&lt;/em&gt;&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>20x Sodium Saline Citrate</td><td>Roth</td><td>1054.1</td><td></td></tr><tr><td>5&#039;Cy3-labelled complementary strand</td><td>Eurogentec</td><td></td><td>For duplex hybridization</td></tr><tr><td>Biopur Safe-Lock microcentrifuge tube</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>BSA (10 mg/mL)</td><td>Promega</td><td>R3961</td><td></td></tr><tr><td>EDTA molecular biology grade</td><td>Promega</td><td>H5031</td><td></td></tr><tr><td>GenePix 4100A</td><td>Molecular Devices</td><td></td><td>Microarray scanner</td></tr><tr><td>Hybridization oven</td><td>Boekel Scientific</td><td>230500</td><td></td></tr><tr><td>MES monohydrate</td><td>Sigma</td><td>69889</td><td></td></tr><tr><td>MES sodium</td><td>Sigma</td><td>M3058</td><td></td></tr><tr><td>NaCl &gt;99.5%</td><td>Sigma</td><td>71376</td><td></td></tr><tr><td>SecureSeal SA200 hybridization chamber</td><td>Grace BioLabs</td><td>623503</td><td></td></tr><tr><td>Spectrafuge mini</td><td>Labnet</td><td>C1301</td><td>Microarray centrifuge</td></tr><tr><td>Tween-20 molecular biology grade</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>&lt;u&gt;&lt;em&gt;Data extraction&lt;/em&gt;&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td>For data extraction</td></tr><tr><td>MatLab</td><td>MathWorks</td><td></td><td>Microarray design</td></tr><tr><td>NimbleScan 2.1</td><td>Roche NimbleGen</td><td></td><td></td></tr><tr><td>&lt;u&gt;&lt;em&gt;Desalting and quantification&lt;/em&gt;&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>NanoDrop One Spectrophotometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Toluene</td><td>Merck</td><td></td><td></td></tr><tr><td>ZipTip C18</td><td>Millipore</td><td>ZTC18s008</td><td>Desalting pipet tips</td></tr></tbody></table>

high-density dna and rna microarrays - photolithographic synthesis, hybridization and preparation of large nucleic acid libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

DNA and RNA microarray hybridization 
Figure 5 shows the results of a hybridization assay performed on a microarray containing the DNA and RNA versions of a 25mer sequence (5&#39;-GTCATCATCATGAACCACCCTGGTC-3&#39;&#160;in DNA form). The scan in Figure 5A appears in a greenscale format corresponding to the excitation/emission spectrum of Cy3 fluorescence, with fluorescence intensity recorded in arbitrary units between 0 and 65536. The array design followed the 25:36 feature layout described in the protocol section. The scan is shown after proper orientation of the array, with the top left corner populated with the longest chain of fiducial features. Here, fiducial features contain the DNA version of the 25mer and should, in principle, always give a positive fluorescence signal in order to perform scan alignment and data extraction. The hybridized microarray should appear uniformly bright, with the edges of the synthesis area being however usually brighter than the center (up to 50% brighter). The large amount of sequence replicates, randomly distributed throughout the area, reduces the impact of spatial artefacts. Here, each sequence (DNA and RNA) was synthesized at 2,000 random locations. There is typically low fluorescence noise (background), &#60;50 a.u., which leads to a signal/noise ratio in the order of 200:1 to 800:1 in hybridization assays. After data extraction, fluorescence intensities are averaged out and plotted &#177;SD.
There is significant variability in absolute fluorescence values between experiments. Here, we show the results for three independent syntheses using the same fabrication parameters and the same post-synthetic handling. The 25mer DNA, when hybridized to its complementary Cy3-labelled DNA strand, will yield fluorescence signals ranging anywhere from 20,000 to 30,000, very rarely above or below. The 25mer RNA, when hybridized to the same Cy3-labelled DNA complement, will give fluorescence intensities on the corresponding features ranging from 15,000 to 20,000. However, fluorescence intensity of the RNA/DNA duplexes will occasionally drop below 8,000, when the corresponding DNA/DNA duplexes will still fluoresce within the 20,000-30,000 range. In such cases, the results for RNA may be regarded as sub-optimal. A synthesis or hybridization failure, either for DNA or RNA, will be immediately noticeable during scanning from the obvious lack of fluorescence. There are multiple opportunities for the RNA synthesis to either fail or partially succeed and they will be outlined in the discussion part.
Library deprotection, cleavage and recovery 
Depending on the complexity and the density of the library, the shape and outline of the synthesized array can be seen without magnification but under proper lighting (Figure 4), with the DNA still in protected form. After deprotection with EDA/toluene, and before the addition of water in order to collect the cleaved library, the synthesis area containing the now deprotected oligonucleotides may stand out as a hydrophilic zone, when the rest of the glass slide will appear covered with a turbid hydrophobic layer. The direct observation of the synthesis area depends on the total area used to synthesize oligonucleotides: greater use of the synthesis area will correspond to a greater chance of clear distinction between hydrophilic and hydrophobic regions on the surface. Conversely, libraries synthesized using fewer mirrors and with smaller features may not be immediately observable.
Similarly, the amount of recovered DNA after desalting is directly proportional to the total area used for synthesis. If all features are used for oligonucleotide synthesis, the cleavage and recovery procedure should yield between 25 and 30 pmol of DNA. A 10% use of the synthesis area will therefore afford only around 3 pmol of DNA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59936/59936fig1v2.jpg" /> 
Figure 1. Chemical structures of the DNA and RNA phosphoramidites used in oligonucleotide synthesis by microarray photolithography. Standard nitrophenylpropyloxycarbonyl (NPPOC) photosensitive protecting groups at the 5&#39;-OH are used in regular DNA and RNA microarray synthesis for hybridization purposes. For the synthesis of complex DNA libraries, the more photolabile benzyl-NPPOC (BzNPPOC) are preferred at the 5&#39;-OH, as BzNPPOC is removed twice as fast as NPPOC, which significantly reduces total microarray synthesis time. DNA oligonucleotides for libraries also require the coupling of a cleavable dT monomer at the 3&#39;&#160;end. This monomer, which carries a succinyl ester function, will be cleaved during deprotection, allowing for the DNA to be collected from the microchip. <a href="https://www.jove.com/files/ftp_upload/59936/59936fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59936/59936fig2v2.jpg" /> 
Figure 2. Example of a mask as an image file sent to the micromirror device during UV exposure. The white pixels correspond to mirrors which will be tilted in the &#34;ON&#34;&#160;position, reflecting UV light onto the synthesis cell. Black pixels correspond to &#34;OFF&#34;&#160;mirrors, where the UV light will be reflected away from the cell. White pixels will therefore allow for the coupling of the next incoming phosphoramidite on the oligonucleotides found at the corresponding features on the glass substrates. Oligonucleotides synthesized on the features whose corresponding mirrors are, in this mask file, black pixels will however remain inert during the next coupling event. <a href="https://www.jove.com/files/ftp_upload/59936/59936fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59936/59936fig3v2.jpg" /> 
Figure 3. Photographs of the microarray photolithography optical and synthesis setup. (A) Optical circuit for UV exposure. UV light from the UV-LED is first homogenized through a rectangular-cross-section light-pipe then reflects onto the micromirrors. Micromirrors which have been tilted into an &#34;OFF&#34;&#160;position will reflect UV-light away from the synthesis cell, but micromirrors in the &#34;ON&#34;&#160;position will reflect light onto the synthesis cell, situated at the focal plane, by first passing through a 1:1 Offner Relay imaging system. (B) The synthesis cell, once assembled, consists of a drilled slide placed first onto the quartz block of the cell, separated by a thick PTFE gasket (not shown). A second, non-drilled slide is then positioned over the drilled slide, separated by a thin PTFE gasket. A metal frame (not shown) holds the assembly together. (C). For library preparation, once the synthesis cell is attached at the focal plane of incoming UV light, the chamber located between the quartz block and the drilled slide is filled with a 1% solution of &#946;-carotene in CH2Cl2. To do so, an additional inlet and outlet tubing is attached to the quartz block and the orange solution flows from the rightmost to the leftmost position. The flow of reagents and solvents for the synthesis is shown in white arrows. <a href="https://www.jove.com/files/ftp_upload/59936/59936fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59936/59936fig4.jpg" /> 
Figure 4. The synthesis area is usually visible to the naked eye. Here, a DNA library can be seen on the glass surface right after synthesis, with the DNA still in protected form. <a href="https://www.jove.com/files/ftp_upload/59936/59936fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59936/59936fig5v2.jpg" /> 
Figure 5. Hybridization assays to the 25mer DNA and RNA sequences synthesized in situ on microarrays. (A) Fluorescence scan of the entire hybridized DNA and RNA microarray. 25mer DNA and RNA oligonucleotides are hybridized to their Cy3-labelled complementary strands. The array was scanned with a laser at a 532 nm excitation wavelength, at 5 &#181;m resolution. (B) Fluorescence intensities (arbitrary units) of the DNA:DNA and RNA:DNA duplexes in three separate experiments. The light green data for in situ synthesized RNA oligonucleotides can be considered suboptimal, when compared to the fluorescence intensity of the corresponding DNA sequences. Error bars are SD. <a href="https://www.jove.com/files/ftp_upload/59936/59936fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59936/59936fig6v2.jpg" /> 
Figure 6. Schematic representation of the deprotection, cleavage and recovery procedure for DNA libraries synthesized on microarrays. DNA sequences are grown on a base-sensitive cleavable dT nucleoside (shown in the zoomed area). After synthesis, deprotection of the DNA oligonucleotides (base protecting groups are represented as red spheres) in EDA/toluene leaves the deprotected material electrostatically bound to the surface and can then be pipetted out by applying a small amount of water onto the synthesized area. <a href="https://www.jove.com/files/ftp_upload/59936/59936fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59936/59936fig7v3.jpg" /> 
Figure 7. Representative absorbance spectrum (220 -&#160;350 nm) of a cleaved, desalted DNA library containing 4,000 different sequences, 100-nt in length. A total of 940 ng of DNA was isolated from a single array synthesis, corresponding to 30 pmol of DNA total, or 15 pmol per glass substrate. <a href="https://www.jove.com/files/ftp_upload/59936/59936fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/59936/59936table1.jpg" /> 
Table 1. Representative cycle protocol for coupling/oxidation/photodeprotection of 5&#39;-BzNPPOC-dA, assuming the corresponding phosphoramidite was loaded on port &#34;A&#34;. Coupling time (in seconds) is shown at the &#34;couple monomer&#34;&#160;line. The UV photodeprotection time, here corresponding to a radiant energy of 3 J/cm2 (BzNPPOC photochemistry), is calculated as the time elapsed between the two &#34;Event 2 Out&#34;&#160;communication signals.
<img alt="Table 2" src="/files/ftp_upload/59936/59936table2.jpg" /> 
Table 2. Representative cycle protocol for coupling/oxidation/photodeprotection of 5&#39;-NPPOC-rA, assuming the corresponding RNA phosphoramidite was loaded on port &#34;A&#34;. Coupling time (in seconds) is shown at the &#34;couple monomer&#34;&#160;line. The UV photodeprotection time, here corresponding to a radiant energy of 6 J/cm2 (NPPOC photochemistry), is calculated as the time elapsed between the two &#34;Event 2 Out&#34;&#160;communication signals.

Watch this Scientific Journal Video about High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries at JoVE.com

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

high-density-dna-rna-microarrays-photolithographic-synthesis

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Unit 2

Studying DNA and RNA

SCIENTISTS IN ACTION

17.9K Views.  University of Vienna. In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Video: High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Biology

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Immunology and Infection

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Biochemistry

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

Bioengineering

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Performing Custom MicroRNA Microarray Experiments

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1. Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally 2, 3. miRNAs control a broad range of biological processes 1. In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis 4, 5. It is important, therefore, to understand the expression and functions of miRNAs under many different conditions.
Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.

A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

Expression Profiling with Microarrays

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and nucleic acids derived from samples. Using these arrays, scientists can simultaneously evaluate the expression of thousands of genes. In addition, the expression profiles of different cells or tissue types can be compared, allowing researchers to deduce how the expression of different genes change during biological processes, and thus gain insight into how the genes may function in pathways or networks.Here, JoVE explains the principles behind microarrays. This is followed by a general protocol for performing a microarray experiment, and a brief introduction to analyzing microarray data. We end on a discussion of how scientists are currently using microarrays, for example to compare gene expression between different cell types derived from cancerous and non-cancerous tissues, to study important biological problems.

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and ...