Compositions and methods relating to synthetic RNA polynucleotides created from synthetic DNA oligonucleotides

A method, the method comprising forming an RNA chimera from a plurality of DNA oligonucleotides in a single reaction chamber in a single reaction step, wherein the RNA chimera is encoded by a chimeric coding sequence and wherein the single step consists of contacting in the reaction chamber:

• (a) the plurality of DNA oligonucleotides, wherein the plurality of DNA oligonucleotides comprises:
• a first synthetic single-stranded DNA oligonucleotide having a size of up to 200 nucleotides and comprising, in a 5′ to 3′ direction, a 5′ end, a non-hybridizing sequence, a first hybridizing sequence, and a 3′ end, wherein the non-hybridizing sequence comprises, in a 5′ to 3′ direction, an RNA polymerase promoter sequence and at least a portion of the chimeric coding sequence;
• and
• a second synthetic single-stranded DNA oligonucleotide having a size of up to 150 nucleotides and comprising, in a 5′ to 3′ direction, a 5′ end, a second hybridizing sequence, and a 3′ end, wherein the second hybridizing sequence comprises at least a portion of the chimeric coding sequence and is complementary to the first hybridizing sequence;
• and
• (b) dNTPs;
• and
• (c) rNTPs;
• and
• (d) a DNA polymerase;
• and
• (e) an RNA polymerase,
• wherein
• the first and second hybridizing sequences hybridize;
• and
• the 3′ end of the first synthetic oligonucleotide is extended by the DNA polymerase to form a coding strand comprising, in a 5′ to 3′ direction, the RNA promoter and the chimeric coding sequence;
• and
• the 3′ end of the second synthetic oligonucleotide is extended by the DNA polymerase to form a template strand comprising, in a 5′ to 3′ direction, a sequence complementary to the chimeric coding sequence and a sequence complementary to the RNA promoter;
• and
• the chimeric RNA is formed by templated synthesis of RNA by the RNA polymerase using the template strand.