Transcription from the commonly used GTP- initiating T7 class III promoter phi6.5 frequently produces heterogeneous RNA at both 3' and 5' ends. We demonstrate here that RNA transcripts from the T7 class II promoter phi2.5 have superior 5' homogeneity over those from the phi6.5 promoter, with comparable total RNA yields. The overall homogeneity of RNA transcripts is improved to different degrees depending on RNA sequences, although transcription under phi2.5 does not affect the 3' heterogeneity of RNA. In combination with 3' RNA trimming by DNAzymes or ribozymes, this ATP- initiated transcription system based on the T7 phi2.5 promoter can provide excellent quality of RNA for applications requiring a high degree of RNA size homogeneity.

A series of efficient ribozymes with thioester synthetase activities have been isolated from CoA-linked RNA libraries containing four different lengths (30N, 60N, 100N, and 140N) of random nucleotide regions. Competitive evolution of these size-heterogeneous CoA-RNA libraries resulted in an RNA size population in the order of 30N > 60N >> 100N > 140N. From isolated clones in the 30N and 60N size groups, two predominant RNA sequences, TES1 (30N) and TES33 (60N), have been shown to catalyze the synthesis of different thioesters using various acyl adenylates as the substrates. Together with our previous findings, the current results demonstrate a CoA thioester synthetic pathway catalyzed by individual metabolic ribozymes, and suggest a likely mechanism for thioester synthesis and utilization in an RNA world.

Expanding our previous finding of an adenosine-initiated transcription system, we now demonstrate that either the 5 site or the N6 site of adenosine nucleotides can be modified extensively without abolishing their ability to initiate transcription under the T7 2.5 promoter. Two series of amino derivatives of adenosine nucleotides were synthesized. Fluorescein and biotin groups were coupled to AMP derivatives through linkers of different sizes and hydrophobicities. Both fluorescein-and biotin-conjugated (at either the 5 or N6 site) adenosine nucleotides can act as efficient transcription initiators, producing fluorescein-and biotin-labeled RNA at the specific 5 end by a one-step transcription procedure, eliminating posttranscriptional modification. Furthermore, N6-modified adenosine derivative-initiated transcription synthesizes 5 end modified RNA with a free phosphate group, providing the possibility for further derivatization. The current finding makes easily available a variety of site-specifically functionalized RNA, which may be used in nucleic acid detection, RNA structural and functional investigation, and generation and isolation of novel functional RNA.

virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8, are members of the human gammaherpesviruses and are associated with a variety of human malignancies. EBV is an important cause of lymphomas in severely immunocompromised persons, especially patients with AIDS and organ-transplant recipients (46,61,62,64,66). KSHV is believed to be the etiological agent of Kaposi's sarcoma (16,18,60) and is implicated in the pathogenesis of AIDS-associated primary effusion lymphoma (PEL), also called body cavity-based lymphoma, and multicentric Castleman's disease (23,60,86).Like other herpesviruses, EBV and KSHV go through both latency and lytic replication cycles. The KSHV replication and transcription activator (K-RTA) is necessary and sufficient for the switch from latency to lytic replication in KSHV (23,86,88). K-RTA is a sequence-specific DNA-binding protein that regulates many subsequently expressed viral genes (38,56,69,70,75,91,97). Also, K-RTA can interact with other factors to modulate its transcription potential (39,40,(51)(52)(53).EBV latent membrane protein 1 (LMP-1) is required for EBV transformation of primary B cells and establishment of EBV latency in vitro and is believed to play similar roles in EBV-associated tumor cells in vivo (45, 47). LMP-1 is an integral membrane protein and acts as a constitutively active receptor-like molecule that does not need a ligand (30,36,46,54). LMP-1 activates a variety of cellular genes that enhance cell survival and adhesive, invasive, angiogenic, and antiviral potential (31, 41, 59, 81-83, 87, 90, 94, 95).Interestingly, the majority of PELs are harboring both EBV and KSHV (13,14,42,68). In order to understand their contributions to the pathogenesis of PEL, it is important to address how EBV and KSHV interact with each other and affect biological properties of the cell and the viruses. PELs harboring two viruses have higher oncogenic potential (76), suggesting potential interactions between EBV and KSHV. This report describes a molecular interaction between EBV and KSHV, i.e., KSHV K-RTA potentiates EBV latency via induction of EBV LMP-1 and uses LMP-1 to curb KSHV lytic replication. These data suggest that the coinfection of EBV and KSHV in the majority of body cavity-based lymphomas might not be a coincidence: the presence of EBV is one of the strategies that KSHV uses for the maintenance of its latency. This report should be applicable to both KSHV and EBV studies in the majority of AIDS-associated PELs. MATERIALS AND METHODSCell culture, plasmids, adenoviruses, and antibodies. All cell lines used in this study and their properties are listed in Table 1. Expression plasmids for K-RTA (pCMV50) and the recombinant adenoviruses for green fluorescence protein (GFP) (AdGFP) and K-RTA (AdRTA) were a gift from Byrd Quinlivan (71).

The relationship between ribozyme size and catalytic activity is of fundamental importance for RNA catalysis and molecular evolution in the RNA world. We have performed a series of competitive in vitro selection experiments to probe the relationship using RNA libraries containing size-heterogeneous random regions. Our experiments have established an inverse correlation between RNA replication efficiency (the combined efficiency of PCR amplification, transcription, and reverse transcription) and RNA size. A number of ribozyme sequences have been isolated from different RNA size groups under competitive selection conditions. Comprehensive kinetic analysis on isolated ribozymes has revealed that large ribozymes do not confer a significant catalytic superiority over smaller ones under most selection conditions, and actually impose two significant problems of replication inefficiency and RNA misfolding into inactive conformations. The fraction of a misfolded ribozyme population is defined as alpha. Large ribozymes tend to possess high alpha values, which may significantly reduce ribozyme performance. Our results suggest that a random region of around 60 nucleotides represents the optimal balance between ribozyme catalytic activity, RNA misfolding (alpha), and replication efficiency, and may therefore constitute the most advantageous RNA libraries for successful isolation of functional RNA sequences.