RNA array compositions and methods
Described herein are RNA arrays, and compositions and methods for generating RNA arrays, particularly high density RNA arrays. The disclosed methods for generating RNA arrays utilize template DNA arrays and RNA polymerase to generate RNA arrays. In some embodiments, the disclosed methods use an RNA polymerase and modified ribonucleosides to generate modified RNA arrays for various applications, e.g. RNA arrays having higher nuclease resistance, more conformationally stable RNA arrays, and higher binding affinity RNA aptamer arrays. In some embodiments, the disclosed methods are used to generate RNA bead arrays.
1. A method for generating a template array, comprising
(i) providing a solid support comprising a layer of protected deoxyribonucleosides that comprise a 5′-photolabile protecting group and are covalently linked at their 3′ end to a spacer layer bound to the solid support;
(ii) irradiating the layer of protected deoxyribonucleosides with ultraviolet energy sufficient to deprotect about half of the protected deoxyribonucleosides;
(iii) coupling the deprotected deoxyribonucleosides with a ribonucleoside phosphoramidite comprising a 5′acid-labile protecting group;
(iv) irradiating the remaining protected deoxyribonucleosides with ultraviolet irradiation sufficient to deprotect all of the remaining protected deoxyribonucleosides;
(v) extending the deprotected deoxyribonucleosides, at one or more locations, by light-directed 3′ to 5′ photolithographic synthesis to generate template DNA oligonucleotides of the deprotected deoxyribonucleosides;
(vi) coupling a protecting group to the 5′ ends of the template DNA oligonucleotides;
(vii) removing the 5′ acid-labile protecting group on the protected ribonucleosides by acid treatment; and
(viii) extending the deprotected ribonucleosides at one or more locations, by 5′ to 3′ chemical synthesis of RNA primers comprising a sequence that is complementary to a sequence at the 3′ end of the template DNA strands to obtain a template array.
2. The method of claim 1 , wherein in step (iii) the 5′ acid-labile protecting group comprises a 4,4′-dimethoxytrityl (DMT) group.
3. The method of claim 1 , wherein in step (vi) the protecting group coupled to the 5′-ends of the template DNA strands is an acetyl group or phenoxyacetyl group.
4. The method of claim 1 , wherein in step (viii) RNase-resistant modified ribonucleoside phosphoramidites are used in the extension of the deprotected phosphoramidites to obtain RNase-resistant RNA primers.
5. The method of claim 4 , wherein the RNase-resistant modified ribonucleoside phosphoramidites are 2′-fluoro ribonucleoside phosphoramidites or 2′-methoxy ribonucleoside phosphoramidites.