SINGLE-MOLECULE DETECTION OF RNA REPORTER PROBES BY AMPLIFICATION WITH Q-BETA REPLICASE

The addition of target-specific probe sequences within MDV RNA, an oth erwise efficient template for Q beta replicase, generally resulted in RNA molecules with inferior replication properties, including reduced replication rates and poor sensitivities. We have discovered that the replication characteristics of MDV RNA molecules with internally place d probe sequences are dramatically affected by short RNA sequences (sp acer elements) at the 5' and 3' ends of the probe sequence. For a give n probe sequence, the sequences of the flanking spacer elements effect ed replication sensitivity by six orders of magnitude and replication rate by three fold. By taking advantage of spacer elements, internal M DV probes were developed that permitted the reproducible, real time, f luorescence detection of a single RNA molecule in less than 25 min thr ough amplification with Q beta replicase. RNA structural analysis of s uch probes suggested that the spacer elements functioned by allowing t he RNA to fold in a way which substantially maintained the tertiary st ructure of the MDV domain. MDV reporter probes with suitable replicati on properties were obtained from libraries of RNA molecules in which t he probe sequence was flanked by many different spacer elements (gener ated by random nucleotide synthesis). We demonstrated that this is a g eneral method for developing RNA reporter molecules which are rapidly and reproducibly amplified by Q beta replicase, even from a single mol ecule. (C) 1996 Academic Press Limited