Biomedical subjects

Faqing Huang

Publications and source records attributed to Faqing Huang.

7 recordsLinked to original sources

PubMed2005

Optimal random libraries for the isolation of catalytic RNA.

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.

Base Sequence↗

PubMed2005

Ribozyme-catalyzed aminoacylation from CoA thioesters.

Coenzyme A (CoA) thioesters play essential roles in modern metabolism. To demonstrate plausible biochemical functions of thioesters in the RNA world, we have isolated a new class of ribozymes (ACT) that catalyze self-aminoacylation from a number of CoA thioesters with catalytic efficiencies ranging from 7000 to 24 000 M(-1) x min(-1). Active thioester substrates are required to contain both a free alpha-amino group in the acyl moiety and a CoA as the thiol component. We hypothesize ribozyme-based aminoacylation systems using aminoacyl thioesters of CoA as the ancestors of modern aminoacyl tRNA synthetases. On the basis of our previous results [Huang et al. (2000) Biochemistry 39, 15548-15555; Coleman and Huang (2002) Chem. Biol. 9, 1227-1236], an extensive RNA-catalyzed "metabolic pathway" involving CoA and its thioesters is proposed. Complex contemporary metabolic systems could have evolved from the proposed ribozyme pathways.

3' Untranslated Regions↗

PubMed2005

Novel cyanine-AMP conjugates for efficient 5' RNA fluorescent labeling by one-step transcription and replacement of [gamma-32P]ATP in RNA structural investigation.

Two novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 phi2.5 promoter. Efficient fluorophore labeling of 5' RNA is achieved in a single transcription step by including F550/570 and F650/670 in the transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure, folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [gamma-32P]ATP to prepare 5' labeled RNA for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [gamma-32P]ATP.

Adenosine Monophosphate↗

PubMed2004

Superior 5' homogeneity of RNA from ATP-initiated transcription under the T7 phi 2.5 promoter.

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.

3' Untranslated Regions↗

PubMed2003

Efficient incorporation of CoA, NAD and FAD into RNA by in vitro transcription.

Protein enzymes frequently recruit small molecule coenzymes to perform a variety of biochemical reactions. While the catalytic activities of RNA have been expanding rapidly, a similar strategy for RNA to utilize coenzymes and to increase its functional capabilities has yet to be demonstrated. A general in vitro transcription procedure has been developed to efficiently prepare RNA with coenzymes CoA, NAD and FAD covalently attached to the 5' end. These adenosine-containing coenzymes initiate transcription under the T7 class II promoter by T7 RNA polymerase. In addition to the three coenzymes, other adenosine-containing molecules may be incorporated into the first nucleotide position of RNA as well. This method provides easy access to CoA-, NAD- and FAD-RNA, which may find broad applications in generating coenzyme- utilizing ribozymes. In addition, both oxidized FAD and reduced NADH are highly fluorescent. NADH-RNA and FAD-RNA can therefore be used as probes for DNA/RNA detection and for structural investigation of RNA function by fluorescence spectroscopy.

Adenosine↗

PubMed2003

Synthesis of adenosine derivatives as transcription initiators and preparation of 5' fluorescein- and biotin-labeled RNA through one-step in vitro transcription.

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 phi2.5 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.

Adenosine↗

PubMed2002

RNA-catalyzed thioester synthesis.

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.

Acyl Coenzyme A↗