CONSTRUYENDO – NOS

Library diversity

The process of making large amounts of single-stranded DNA libraries from microarrays consists of 4 steps, i) microarray synthesis, ii) emulsion PCR, iii) in vitro transcription and iv) reverse transcription. The latter 3 steps use polymerase enzymes with multiple purification steps. There is possibility that some percentage of the oligonucleotides maybe lost during each of these steps. The presence or absence of an individual oligonucleotide strand can be determined at each stage of the process on microarray. Further the products after each of the steps can be sequenced by ‘next-generation’ sequencing to determine preferential hybridization of signal over others.

Alternative amplification methods

One promising technique to make large quantities of single-stranded DNA is rolling circling amplification [255]. Here, the DNA molecules are circularized by ligase enzyme. They are amplified using a common primer to produce a long single- strand polymer chain. The primer sequences can be removed binding complementary primer sequence and nicking endonuclease. Any product biases in the above reactions (RNA amplification, reverse transcription, rolling circling amplification) can be minimized by performing them in an emulsion reaction.

144 Another method is to perform on-chip amplification. This would entail reducing the steric hindrance from the array surface and neighboring oligonucleotides for polymerase accessibility and primer extension. Also, the PCR reaction has to be enclosed to avoid evaporation of small volume during thermocycling. After 1-2 rounds of solid-phase PCR, the product could be collected for emulsion PCR (tube or chip). Also, chip based emulsion PCR has been developed by RainDance Technologies [256].

Process automation

Another option is to automate the entire process from emulsion PCR to single-strand generation. For now, the use flammable solvents (diethyl ether and ethyl acetate) to break the emulsions make the process unsafe for bench-top automation. Methods can be explored to change the oil-surfactant formulation to use solutions compatible with purification kits (isobutanol, isopropanol) or one can use magnetic beads in the emulsion reaction [257]. There are many robotic systems compatible with 96 well plates to do nucleic acid purification and magnetic bead selection. One such method is outlined here. Once the oil-surfactant and aqueous phase are pooled into the well, the emulsion can be formed by sonication. This followed thermal cycling and breaking of emulsion with isopropanol washes compatible with most 96 well purification systems yields dsDNA in the wells. To this is added the in vitro transcription reaction components, followed by purification on plates. The reverse transcription is done with biotinylated primer which is then captured on beads, followed by sequential on-bead digestions to first cut and remove the 3’ end primer binding sequence (PBS) and then the desired ssDNA molecule.

145 Other Application of diverse oligonucleotide libraries

DNA nanotechnology – Here nucleic acids are used as structural materials as opposed to carriers of genetic information. The field of DNA nanotechnology first conceptualized by Seeman N in 1982 [258] has fostered many subareas such as DNA origami, DNA computing and DNA robotics. The areas are based on Watson-Crick base-pairing rules.

In DNA origami, the single-stranded genomic DNA is used as a scaffold to create and build wide variety of nanostructures. Multiple short DNA fragments are bound along the length of the scaffold to create bends and curves to build 2D and 3D shapes such as tiles, basic geometric shapes, octahedron, hollow nanodevices, balls, boxes and polyhedral structures as reviewed by Li et al [259]. Similar to DNA, RNA strands can be used as building materials, although the latter employs tertiary interactions between structural motifs rather than annealing of DNA strands. Also complex and hybrid structures with multiple properties can be made by combining multiple materials (DNA, RNA, proteins, molecules) [259]. The use of RNA scaffolds have been described to control spatial organization of enzymes, protein, molecules within the cell [260]. ssDNA scaffolds have been used for ordered distribution of proteins, biomolecules to increase biological and chemical reactions.

In 2000, DNA strands were used to make molecular tweezers based on the principle of sequence complementarity. This mechanism along with DNA nanocages can be used to target-specific delivery of drugs [261]. Since then other environmental stimuli such as pH change, ionic strength, electric fields, light, nucleic acid hydrolysis and polymerization can be used to control DNA nanomachines [259].

DNA sensors – Here, DNA and RNA oligonucleotides can be bound to biological (nucleic acids, peptides, antibodies) and non-biological (biotin, gold,

146 silver, other metals and fluorescent dyes) to realize electrochemical [262], optical [263]and fluorescent sensors [264]. These sensors can be used for numerous applications ranging from detection of infectious pathogens , harmful metal ions [265] to screening of genetic disorders and cancer. Combined with microfluidic technology, they can be used to make handheld field testing devices [266, 267].

DNA and Computers – Currently computer chips are made using a ‘top down’ approach that involves etching of electronic components from large structures. Researches at IBM describe the use of lithography and etching techniques to control the formation of DNA origami structures. Here, DNA strands are used to guide integration of silicon nanowires and carbon nanotubes with conventional microcircuit fabrication [268]. This ‘bottom up’ approach will enable 6 nm distance between components (current 45 nm) making devices smaller, faster and power efficient.

DNA vaccines – Here, DNA oligonucleotide libraries can be used to engineer plasmid constructs to encode pathogen (viral, bacterial or parasitic) antigens. The libraries provide flexibility to enhance immunogenicity from codon optimization. The plasmid constructs (DNA vaccines), when administered produce non-functional pathogen proteins within host cells leading to a range of immunological responses [269, 270].