(Initial Paper) Received February 22, 2005: http://wbabin.net/saba/saba24.htm

In a prior paper "Lysogenic Bacteria, Capable of Therapeutic Virus Liberation Subsequent to Tumor Cell Targeting", an interesting apparently patentably novel functional screening utilizing arrays of cells was disclosed. Figure 1 is a recap of the previously described process.

It is to be appreciated that target binders produced by the arrayed library of cells would commonly be peptides or proteins, naked nucleic acids, or nucleic acid complexes (including viruses and nucleic acid-conjugated particles). Such target binders may be continuously expressed or inducible, and may be secreted, expressed on the cell surface, or liberated upon cell lysis.

It is also to be appreciated that the order in which the different cells are manipulated can vary. For example, one could cocultivate the target cells with the library member cells such as by having both types being present in the test tube of Figure 1, and layered simultaneously. In an alternative cocultivation process, a dilute mixture of target cells could be first layered on a dish, slide or biochip wafer, and subsequently the library cells applied as shown in Figure 2.

A nonramdom surface array format is also possible, such as by utilizing an array of precisely positioned agar gel pads. Figure 3 exemplifies the use thereof wherein the library cells are first arrayed, then expanded, and then contacted with target cells or molecules.

A more elaborate screen would be if the targets were viruses. Herein the library of arrayed cells express potential viruses inhibitors, viral targets are applied, followed by host cells. All sorts of such variations using different kinds and combinations of cells, viruses, and/or molecules (including antibodies viruses and molecules) can be conceived.

Furthermore it is conceivable that, utilizing sufficiently sensitive detection equipment, a single library cell expressing a clonal population of target binding viruses or molecules could effect a detectable functional alteration in a target, particularly a cell.

While Figures 2 and 3 exemplify support-affixed arrays, liquid based arrays are perhaps even more useful. Such a liquid-based array is sketched out in Figure 4.

As an alternative to clonally expanding cells of a library as in the above figures, one could 'clonally expand' a library of viruses or nucleic acids (including their complexes). Herein the library members are applied to a clonal population of host cells. Subsequent to penetrating host cells, the library members are replicated, transcribed, and/or translated. The products thereof may be naturally liberated from the host cells or could be liberated by host cell lysis. These free expansion products then contact, or are contacted with targets with the anticipation that some thereof will affect the target. Note that unlike the common viral assay using susceptible host cells, library cell-expressed viruses or molecules would not infectiously replicate within target cells.

Finally and importantly, the general process of first arraying individual library members, followed by their clonal expansion, need not require cells. For example, one could initially array a library of nucleic acids (including complexes thereof), perhaps by spraying. Then 'clonally expand' these arrayed nucleic acids via an amplification, followed perhaps by transcription and/or translation. Such an in vitro process would allow for easy incorporation of unnatural monomers, such as unnatural amino acids, greatly expanding the size of combinatorial libraries possible.

The following provisional claims attempt to encompass important aspects of the invention.

Addendum 4/14/05: Highly relevant prior art

High-throughput, cloning-independent protein library construction by combining single-molecule DNA amplification with in vitro expression. Rungpragayphan S, et al J Mol Biol. 2002 Apr 26;318(2):395-405
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Addendum 4/24/05

In a prior paper: Targeted Therapeutic Nucleic Acids, Derived From Libraries of Chimeric Nucleic Acids Which Encode Cis-Binding Proteins
Constructs comprising a cis-displayed polypeptide fused with a cell-affecting nucleic acid were disclosed. Also previously described was the use of unnatural nucleotides to position the puromycin during RNA synthesis.

Considering these teachings, along with the teaching of Rungpragaypan, et al, the following has be conceived. It is a means of forming an array of polypeptide-conjugated cell-affecting nucleic acids, involving arraying individual nucleic acids (prefereably DNA), optionally amplifying these nucleic acids, and then synthesizing multiple copes of the cis-encoding cell-effecting mRNA terminated in an unnatrual puromycin-conjugated nucleotide. Utilizing in vitro translation an array wherein each loci comprises multiple copies of a polypeptide-conjugated cell-affecting nucleic acid is fabricated.
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Addendum 4/27/05

Gel pads (1) are valuable in that they can be considered microvessels useful for multiple simultaneous analyses. Herein is described a simple alternative to gel pads, especially useful is fabricating arrays via the processes described above.

Essentially, instead of the gel being superior or above the surface, the gel resides in a recess. An example of such recesses would be an array of microwells within a ‘block’. Preferably the surface of the block is hydrophobic, while the interior walls of the microwells (perhaps formed with a laser) are hydrophilic.

An alternative to microwells would be to use a bundle of microtubules, each microtubule having a core of gel. It has also been recognized that an array of gel surfaces can be created by covering a gel surface with a membrane (optionally nonpermeable) wherein holes are arrayed.

1) Gel pad arrays and methods and systems for making them.
Taylor, et al US Patent Appl 20040253613
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Addendum 4/29/05

Herein is an interesting apparently novel array and fabrication method, especially suited for combinatorial assays such as those which involve cell library distribution and growth as described above. Consider a sheet of material for example plastic, perhaps with hydrophobic surfaces. As an example the dimensions are 1cm x 1cm x 0.3mm. Now consider that we perforate an array 10,000 or more small holes with a diameter 0.1 mm or less in this sheet, such as by using a laser.

Consider next that we fill the holes of this perforated sheet (perhaps by dipping) in a presolidified gel or solution. The result is a sheet wherein each locus contains a spot of gel or media. Appreciate that the presolidified gel or solution could contain a library of cells, viruses, or nucleic acids, and that after filling holes each thereof could contain one or a small number of library members. As described above such arrayed library members could be clonally expanded.

With two accessible surfaces, different reagents can be to each surface, perhaps by straying as described above. Furthermore, a perforated sheet comprising an arrayed library could be stacked, such as upon a homogenous gel surface or onto another perforated sheet comprising an arrayed library.
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