Herein is disclosed a novel method of fabricating polypeptide microarrays, particularly arrayed mRNAs are simultaneously translated in vitro in such a way as to form mRNA-polypeptide conjugates wherein the polypeptide translation product is associated with it encoding mRNA. Also disclosed are means of enhancing cis display technologies in general.

Figure 1 depicts the process, wherein at three loci of a biochip are affixed three different mRNAs via their 5' termini. The short blue sections represent translation start sites; the thick colored sections represent open reading frames, and the 3' terminal "*" represents a terminating moiety (e.g. puromycin), such as described by Szostak, et al.

Upon addition of translation machinery, these mRNAs are simultaneously translated into different polypeptides.

Supports other than a biochip can be utilized. For example, each different mRNA could be conjugated to an optionally encoded particle such as bead.

Various means producing arrays of RNA are know, including stepwise synthesis starting from the support, and affixing preformed terminally modified RNAs. Also one could design RNAs with distinct encoded terminal sequences allowing them to be addressed to, and perhaps ligated with, complementary arrayed oligonucleotide probes.

It is to be appreciated that the concept of affixing one or more cis protein coding nucleic acids (DNA or RNA) to a support is novel and embodied in the present invention (for example see reference 12). Besides the analytical advantages of utilizing a biochip or encoded particle supports, affixing cis protein coding nucleic acids to a support allows rapid purification of the nucleic acid-polypeptide conjugates. Magnetic beads may be particularly useful for this purpose.

Figure 2 is a modification wherein at each microarray loci is affixed two different mRNAs.

It is to be appreciated that even nonconjugated complementary universal nucleotide-containing polynucleotides have utility in enhancing the capabilities of both soluble and support-affixed mRNA-polypeptide conjugates. In that secondary structure in the mRNA is eliminated, the the duplex functions as a rigid scaffold in displaying the polypeptide.

Finally, recognize that the ability to expand the genetic code and thereby incorporate unnatural amino acids into a polypeptide product, greatly expands one's capacity to produce novel polypeptides. Furthermore, select chemical modifications can be advantageous.

In that this invention has not been explored in the laboratory, I'm seeking collaborators in its development.

Claims.

Here is a modification of this invention utilizing the cis-polypeptide encoding process of Lohse, et al (21). In their process they utilize a construct as shown in Figure 4, wherein the 3' terminus of the mRNA is capped with a puromycin-conjugated DNA hairpin. Subsequent to translation the DNA hairpin functions to prime DNA synthesis utilizing the mRNA as template.

As seen in the figure, what is now suggested is that the RNA of these hybrids be enzymatically removed, and the ssDNA-polypeptides be subsequently addressed to support-affixed oligonucleotides. So as to encode the ssDNA 3' terminal sufficient for addressing, the 5' end untranslated end of the initial mRNA is itself encoded.

Subsequent to addressing, hybrids could be stabilized by crosslinking or ligation (perhaps to hairpin capture probes).

Furthermore note that arrayed polypeptide-conjugated polynucleotides could be subsequently hybridized with ligand-conjugated polynucleotides to form ligand groups as shown in Figure 3.

It is suggested that exceptionally high density arrays of polypeptides could be designed in this way.

Addendum 1/29/05

Figures 5 and 6 are yet other derivations of this highly promising polypeptide array technology.

Figure 5, polynucleotide-conjugated polynucleotides are affixed noncovalently via addressing to arrayed complementary encoded probes.

While in Figure 6, the constructs prior to translation are covalently or noncovalently affixed to the array support.

Other derivations can be conceived.

The following claims attempt to consolidate certain important aspects of the above disclosed invention.

A support-affixed library, each member comprising a polypeptide-conjugated nucleic acid, which was derived via a cis in vitro translation process wherein each member's polypeptide was encoded by and binds to that member's nucleic acid.

Addendum 1/31/05

Figure 7 exemplifies an interesting modification of the cis-translation process for synthesizing soluble or support-affixed polypeptide-conjugates, especially libraries thereof. Instead of polypeptide-capturing puromycin being conjugated to the mRNAs, they are conjugated to the termini of encoded probes, to which different mRNAs can be addressed.

Claims

Claims

Addendum 2/1/05

Figure 9 represents a modification of the technique depicted in Figure 8 wherein identical arrayed capture molecules, positioned adjacent to different support-affixed transcription/translation nucleic acids, are utilized to form a polypeptide array (or alternatively a library of beads each of which has a distinct polypeptide). This is similar yet distinct from the CIS Display described by Odegrip, et al (12). Other means of affixing the nucleic acids are possible (15,17,18). Furthermore, this also applies to Figure 8; mRNA rather than transcription/translation dsDNA could be utilized.

Note: Contemplating the synthesis 3' terminal puromycin mRNAs, I recalled the following recent report.

Site-specific incorporation of a photo-crosslinking component into RNA by T7 transcription mediated by unnatural base pairs.
Kimoto, et al Chem Biol. 2004 Jan;11(1):47-55

This appears to be an exceptionally enabling technology, since the unnatural base-pairing nucleotide could be fitted with any kind of modification; fluorescent, translation terminating, affinity, etc. Furthermore, such a modified nucleotide could be precisely placed anywhere within a product polynucleotide; and each polynucleotide could contain multiple modified unnatural base-pairing nucleotides.

References